Promotion - IWEVENTOS de...“Entendendo o microambiente cardíaco: caracterização do secreto-ma...

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1st Termis Americas Workshop4th International Meeting of Tissue Engineering and Regenerative MedicineJune 27th to July 1st, 2018.Porto Alegre, RS - Brazil.

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Livro de Resumos

ABSTRACT BOOK

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Summary

EDITORIAL...........................................................................................12COMMISSIONS..........................................................................19 Scientific Committee........................................................19 Organizing Committee ....................................................20GENERAL INFORMATION...................................................21 Location..............................................................21 Opening ............................................................................21 Happy Hour.....................................................................21 Exhibition..........................................................................21 Awards ..............................................................................21EVENT SCHEDULE..............................................................22 Presentations...........................................................................22 Courses..............................................................................23 1st Termis Americas Workshop........................................24ABSTRACT...................................................................................27 Bioartificial Tissue Organ.......................................27 “Acellular scaffold production for liver transplant”..........................................27 “An efficient protocol for porcine liver decellularization supports subse-quent recellularization”................................................................................28 “Decellularization and recellularization of liver in a perfusion bioreactor with mesenchymal stem cells”....................................................................29 “Modelo de cultura celular tridimensional para mimetizar tecidos nor-mais a tumorais”.........................................................................................30 “Production of an artificial skin substitute using tissue engineering princi-ples to be used for toxicological testes in vitro”............................................31 Biofabrication/Bioprinting .....................................32 “Computational simulation for a perfusion bioreactor project”............32 “Modeling and simulation in the histoarchitecture of the articular cartilage for biofabrication”........................................................................................33 “Nonadhesive micro-mold for enhancing cell alignment of myotubes”..34

“Optimization of 3D printing parameters for obtaining polyesterurethane scaffolds for tissue engineering”................................................................35 “Polymeric composites filaments for 3D printer based on poly-hydro-xybutyrate/bacterial cellulose for tissue engineering aplications”..............36 Biomaterials.........................................................37 “Biocompatibility evaluation of particulate PPy/p-TSA using zebrafish as a model organism”.......................................................................................37 “Bioglass 45S5: Structural characterization and analysis of biocom-patibility with adipose-derived mesenchymal stromal cells in vitro and in vivo”.....................................................................................................38 “Comparison of citotoxicity between 99.5 and 99.95% iron for use in cardiovascular stents produced by powder metallurgy”..........................39 “Conductive biocomposite with potential for medical applications”.....40 “Crosslinked electrospun membranes based on SPIMA for tissue en-gineering applications”................................................................................41 “Desenvolvimento de cerâmicas bactericidas e de elevada bioativida-de para aplicações odontológicas”............................................................42 “Development of a new biomaterial associated with human keratinocy-tes for use as a skin substitute”...................................................................43 “Enxerto de membrana biológica de intestino delgado de suíno na ci-catrização de córnea de cão: relato de experiência.”................................44 “Evaluation of biocompatibility of poly(lactic-co-glycolic acid) – galan-tamine microparticles in rat astrocytes”.......................................................45 “Evaluation of natural biomaterials made of chitosan for the regene-ration of tissues”.........................................................................................46 “Functional hybrid scaffold PVA-BG containing cobalt as a potential stra-tegy for tissue engineering”.........................................................................47 “Hidrogel de acetato de celulose e EDTAD com extrato etanólico de pró-polis para aplicações em tratamentos de queimaduras de segundo grau”.......48 “Hidrogel de quitosana reforçado com biovidro para aplicações or-topédicas”.............................................................................................49

Summary

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“Incorporation of the cobalt therapeutic ion into bioactive glass ne-twork and its implications for angiogenesis”..............................................50 “Influence of bioactive ceramics on keratinocytes’ viability”................51 “Membranas poliméricas microestruturadas e condutoras para aplica-ção na engenharia de tecidos”....................................................................52 “Porcine small intestinal submucosa sheets as a scaffold for human adipose-derived stromal cells for tissue engineering of soft tissues”...........53 “Production and characterization of nanocrystalline chittosan/buriti oil new biocomposite with potential in would healing”....................................54 “Superficially modified Mg-based alloy for biomedical applica-tions: interaction with cells in culture”..................................................55 “The viability of keratinocytes in adherent and non-adherent hy-drogels”.......................................................................................56 “Uso da latrunculina no controle de microorganismos: aplicação em engenharia celular”.....................................................................................57 Cell Therapy............................................................58 “Autologic implant of activated dermic cells in involutive skin treatment”............................................................................58 “Cell therapy in models of moderate and severe hemorrhagic strokes”..........................................................................................59 “Engineering the plasma membrane of pancreatic beta cells for a cell--based treatment in T1DM”........................................................................60 “Imunomodulação com plasma rico em plaquetas combinado ao Ba-cillus Calmette-Guérin para o tratamento do câncer de bexiga não-músculo invasivo”..................................................................................................61 “Recombinant human growth factors and mesenchymal stem cells de-rived exosomes for wound healing and skin regeneration”.....................62 “Recombinant human growth factors (PDGF, TGF-b, VEGFs, BMPs) as an effective alternative to platelet-rich plasma (PRP) for tis-sue regeneration”....................................................................................63 “Use of urine progenitors cells for in vivo renal repair in a new noninva-sive rat kidney injury model using high sodium oxalate-diet”......................64

Induced Pluripotent Stem Cells.............................65 “Entendendo o microambiente cardíaco: caracterização do secreto-ma de células-tronco embrionárias durante a diferenciação cardiomiogêni-ca in vitro”....................................................................................................65 “Resposta imune durante a diferenciação de células-tronco neu-rais embrionárias obtidas a partir de camundongos APPswe/PS1dE9, um modelo da doença de Alzheimer”....................................................66 “Electrical stimulus as a key componente for a maturation and characterizatin of human induced pluripotent stem cell-derived car-diomyocytes.”..........................................................................67 “Optimization of chondrocytes in 3D culture system for tissue en-gineering”....................................................................................68 “Performance of human pulp stem cells under 3D culture of hydro-gel modified by proteins”..........................................................................69 Mesenchymal Stem Cells.......................................70 “ADSC-derived exosomes improved sequels of fo-cal ischemia via intranasal administration 24 hours after the in-sult”..................................................................................................70 “Análise da contribuição do led nos comprimentos de onda de 630 e 850nm em diferentes fluências na cultura de célula-tronco me-senquimal”...........................................................................................71 “Analysis of limbal mesenchymal stem cells expanded on human denu-ded amniotic membrane porcine small intestine submucosa scaffolds”.......72 “Apical papilla stem cells: changes in protocol isolation and new dis-coveries”.........................................................................................73 “Avaliação clínica da aplicação de células-tronco mesenquimais de te-cido adiposo para o tratamento de úlcera de córnea persistente em cães: estudo piloto”..............................................................................................74 “BPA and TCDD action on rat adipose-derived stem cells(rASCs) differen-tiation potential”..........................................................................................75 “Células-tronco mesenquimais derivadas da medula óssea e osso esponjoso: Potenciais candidatas na formação de um nicho hematopoéti-co heterotópico”..........................................................................................76

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“Characterization of exosomes released by the ADSCs and their ef-fects on the motor symmetry of rats submitted to a focal ischemia mo-del”......................................................................................................77 “Desvendando os microambientes do tecido adiposo sub-cutâneo e a sua relevância em protocolos de engenharia de teci-dos”.............................................................................................78 “Dissociation and reconstitution of mesenchymal stem cell membra-nes to generate particles for cellular therapy”...........................................79 “Effect of angiotensin II and angiotensin-(1-7) in proliferation of stem cells from human dental apical papilla”....................................................80 “Effect of hepatitis C drugs sofosbuvir and daclatasvir treatment on me-senchymal stem cells viability, autophagy and migration capacity”.........81 “Estudo comparativo de H2AX em células-tronco mesenqui-mais derivadas do tecido adiposo abdominal e facial humano fren-te à RUVA e RUVB”.............................................................................82 “Evaluation of zoledronate and bioactive compounds in mesen-chymal stem cells extracted from deciduous teeth”...........................83 “GRX, a liver stem cell line, is not affected by hepatitis C sofosbuvir and daclatasvir drug treatment in vitro”....................................................84 “Imunossensor para identificação de células-tronco mesenqui-mais de coelho”.......................................................................................85 “Influence of amniotic membrane scaffold on the expression of adhe-sion molecules in stromal stem cells”..................................................86 “Influence of the source of mesenchymal stem cells for tissue engi-neering: Dermal versus adipose tissue in skin wound healing”................87 “Influência do diâmetro nas propriedades morfo-funcionais de es-feroides de células-tronco de tecido adiposo humano biofabricados de modo automatizado”..................................................................................88 “In vitro evaluation of mesenchymal stem cell behavior in scaffolds con-taining different concentrations of alginate and sodium chloride as a stra-tegy in regenerative medicine”....................................................................89 “Membrane particles of mesenchymal stem cells and contidio-ned medium polarizes macrophages in vitro to an anti-inflammatory phe-notype”.................................................................................................90

“Mesenchymal stem cell-derived factors protect the intestines from experimental colitis in organ culture”..........................................91 “Modulação da migração e morfologia celular de queratinócitos a partir de meio condicionado de células estromais mesenquimais deriva-das de adiposo humano.....................................................................92 “Neuroprotective effects of intravitreal injection of transgenic me-senchymal stem cells expressing hIGF-1 in mouse visual system af-ter optic nerve crush”............................................................................93 “Non-hypertrophic cartilage by scaffold-free tissues engineering va-lidated by comparative secretome analysis”......................................94 “Osteogenic potential of multicellular spheroids constitu-ted by human bursa subacromialis-derived mesenchymal stem cells”............................................................................................95 “Placenta-derived mesenchymal stem cells culture on pmma sca-ffolds as a 3D putative model of hematopoietic stem cells niche in vi-tro”......................................................................................................96 “Proliferação de células-tronco mesenquimais derivadas de pol-pa de dente permanente humano em polímeros termoplásticos ABS e PLA impressos em 3D”.....................................................................97 “Relato de caso de tratamento de ferida, por complicação pós-ope-ratória, com plasma rico em plaquetas (PRP)”..................................98 “The secretome of adipose stem cells human protects against ge-notoxicity generated by oxidative stress”..............................................99 “Using human adipose stem cells spheroids as novel approach for assessment of silver chloride nanoparticles chronic exposure”...........100 Scaffold.................................................................101 “Correlation between the softness of hyaluronic acid microparticulate scaffolds and the proliferation of human adipose mesenchymal cells”..........101 “Dynamic functionalization of vascular scaffolds”.............................102 “Estudo das propriedades adesivas e citotóxicas de membranas poli-méricas biosmart nanotechnology em culturas de células-tronco mesenqumais”.........................................................................................................103 “Liver bioscaffold as a new strategy for liver regeneration: A systematic re-view”........................................................................................................104

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“Properties of chitosan chloride modified with calcium nanophospha-te complex”..............................................................................................105 “Regenerated cellulose sponge as sacrificial template for the systhesis of three-dimensional porous alumina-silica scaffold for the engineering”..106 “Scaffold production of nanofibrous polycaprolactone and ascorbic acid 2-phosphate magnesium”..................................................................107 “Sterilization processes of natual scaffolds for tissues en-gineering”..........................................................................108 Tissue Engineering..............................................109 “Avaliação da cinética e das alterações morfológicas da fu-são de esferoides de células-tronco/estromais de tecido adiposo hu-mano”......................................................................................109 “Aplicação da engenharia de tecidos no reparo de cartilagem articu-lar em cães”.............................................................................................110 “Application of FGF-2/PLGA microfibers in spinal cord tissue en-gineering”...........................................................................................111 “Bone biofabrication from human adipose stem/stromal cell spheroids seeded into pla/cha 3D printed scaffold”.............................................112 “Caracterização de enxertos de válvula cardíaca porcina obtidos por dois processos de descelularização”..............................................113 “Development of a conduit of PLGA-gelatin aligned nanofibers produ-ced by electrospinning seeded with stem cells for nerve regeneration”....114 “Development of a red propolis-encapsulated nanocapsule hydrogel to be use in regenative medicine”..................................................................115 “Development of biphasic scaffolds of collagen type I for peripheral nerve regeneration”..................................................................................116 “Histological and biocompability evaluation of the bovine pericardium after soft descellularizarion process for the use in valve bioprothesis”.....117 “Human adipose stem/stromal cell spheroids as a model of osteogene-sis”............................................................................................................118 “Macrophage polarization in skin wounds treated with a dermal substitute associated with mesenchymal stem cells”.........................119 “Rastreabilidade nos resultados de medição do módulo de Young para esfroides celulares”..................................................................................120

““Scaffolds” de poliácido láctico (PLA) obtidos por manufatura aditiva funcionalizadas pelo plasma de oxigênio para aplicação na engenharia de tecidos”......................................................................................................121 “Thrombospondin-1 protein silencing in adipose stem cell sphe-roids”.......................................................................................122 “Transplante ortotópico de traquéias preparadas por técnicas de enge-nharia celular: modelo em coelhos”.........................................................123

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EDITORIALEditorial

Dear participants of the 1st. TERMIS-AM Workshop,

The Tissue Engineering and Regenerative Medicine International Society (TERMIS) is an important scientific society in the area of tissue engineering and regenerative medicine research worldwide. The society is represented throughout all the world. Every 3 years the Society hosts the World Congress. The location of the World Congress rotates between the three Chapters (Americas, Asia-Pacific and Europe). The 1st TERMIS World Congress occurred in 2006 in Pittsburgh, PA, USA. In the intermittent years between the World Congresses, the meeting occurs on the continents of the Americas, Asia Pacific and Europe. The 5th World TERMIS Congress will occur this year in Kyoto, Japan. In 2019, TERMIS-AM will host the 9th TERMIS-AM Conference in Orlando, FL, USA from December 2-5.

The TERMIS-AM Congresses have always occurred in North America. This year we organized the first TERMIS Americas Workshop, 2018, in South America. The Americas Chapter of TERMIS promotes education and research within the field of tissue engineering and regenerative medicine in the countries within North and South America. The first TERMIS Americas Workshop 2018 was an event dedicated to the dissemination of leading research in tissue engineering and regenerative medicine in all Latin America countries. The proposal was to discuss the challenges and therapeutic benefits in the application of technology that involve tissue engineering and regenerative medicine. This GLOBAL EVENT was brought for the FIRST TIME TO LATIN AMERICA thanks to the efforts and support of an important research group within the area. The meeting took place from June 27 to July 1, at the BarraShoppingSul Events Center, Porto Alegre, Brazil. The event was promoted and organized thanks to the combined efforts of 3 institutions: the International Society of Tissue Engineering and Regenerative Medicine (TERMIS), the Federal University of Rio Grande do Sul (Universidade Federal do Rio Grande do Sul – UFRGS) and the Stem Cell Research Instituto (Instituto de Pesquisa com Células-tronco- IPCT).

The event was initially organized thanks to the support of TERMIS-AMERICAS, represented by Dr. William Wagner, president of TERMIS-AM in 2017 and with the support of Dr. John Fisher, president of TERMIS-AM in 2018. We also had the support and approval of the World TERMIS, represented by its president, Dr. Rui Reis. With their support, we began to organize the first TERMIS-Americas Workshop, with the support of the celebrated researchers and members of the TERMIS Americas, Dr Martha Raquel Fontanilla, of Colombia and Dr Maria Margarita García Rizzo, of Uruguay. In Brazil, we received much appreciated assistance from the vice-president of the event, Dr. Jorge Vicente Lopes da Silva and the honorary president, Denise Crawshaw Pellin Silva, the British honorary consul in Porto Alegre. The programming presented themes of great impact for the medical community in the national and international scenario, such as 3D bioprinting and the development of technology focused on the area of organ

biofabrication; biomaterials with emphasis on regenerative medicine, using techniques such as nanotechnology, amongst others, with or without cells or stem cells, to promote the regeneration of different tissue types. We believe that all this technology will sooner or later become an integral part of people’s lives on a large scale.

The 1st TERMIS-AM WORKSHOP also included a scientific photography competition, with prizes for the three best photographs, judged by 2 scientists, both experts in scientific imaging, plus a professional photographer. Two poster sessions were presented and 9 prizes were offered, with the first 3 places being in the following three categories: Undergraduate student, postgraduate student and PhD/Professional. Prior to the principal conferences, there were seven theoretical-practical pre-courses, the subjects of which were molecular biology, bioinformatics, biofabrication and methodology for cellular evaluation and biomaterials production.

Last but not least, I cannot omit to offer my grateful thanks to all the members of the scientific and organizing commissions, represented by my personal colleagues, and all the team of students and researchers within the scientific and administrative sectors of the laboratory which I run. These people total some 25 members, who worked very hard and in great harmony, dedicating themselves with great commitment to guarantee the success of the event.

With the efforts and dedication of the entire team of the researchers and professors involved, we worked together to bring a high quality event to Latin America, the aim of which was to unite fellow researchers within the area of interest. We sincerely hope that this event has served as a means of meeting and exchange of knowledge between researchers from diverse countries. We hope that the event met the expectations of the participants and that this will be the first of many other workshops to take place in Latin America, bringing researchers together and divulging science of quality, thereby fulfilling the role of proliferators of knowledge and stimulating young researchers in their tireless quest for the optimum future of science in Latin America.

Finally, our thanks go to you, the participants, for the success of the first TERMIS -Americas workshop 2018, in Porto Alegre, Brazil.

Patricia Pranke,President.

EDITORIALEditorial

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EDITORIALEditorial

Dear 1st TERMIS-AM Workshop participants,

It has given me great pleasure to have accepted the invitation of Dr. Patricia Pranke to be the vice-president of the 1st TERMIS-AM Workshop. Firstly because of our partnership for many years, and secondly because of the fact that that the presence of a TERMIS in Latin America is a pioneering event. Dr. Patricia Pranke has worked very hard and has succeeded in making this become a reality. She also made an astute strategy by involving the topic biofabrication in the event, there by expanding the participant community beyond the biological field and involving people from material science, engineering and physics. During the more than one year working more closely with Dr. Patricia, I perceived her dynamism, entrepreneurship and catalyst qualities. It was reflected in the organizing committee, formed by a group of dynamic students and collaborators, in the venue, the referees and the quality of the lectures and posters presented. I have to express my sincere gratitude to be part of this pioneering and memorable event, fostering regenerative medicine, tissue engineering and biofabrication strategic topics in this part of the globe. My sincere gratitude is also extended to the many brilliant speakers both from Brazil and abroad who joined us in this mission, giving preference to us in their agendas from the first moment of contact with a positive response. Finally, I am very grateful to all the participants that, in the four days of the event in courses and lectures, showed an uncommon interest during high-level discussions and interactions.

Jorge Vicente Lopes da Silva, Vice President.

EDITORIALEditorial

Dear Colleagues,

It was a great honour for me to be chosen as Honorary President of the 1st TERMIS Latin America Workshop in Porto Alegre, where researchers and scientists in the tissue engineering and regenerative medicine areas from different parts of the US and South America had the opportunity to come together to share their work and experience and to present the advances and challenges in this crucial field. This prestigious and important event was conceived by Dr. Patricia Pranke last year and I was very proud to have taken part of several stages of the planning and also of lending a helping hand whenever necessary.

My sincere congratulations to Dr. Patricia Pranke and to her very dedicated staff, for putting together such a successful event, and look forward to welcoming subsequent editions in the coming years!

Denise Crawshaw Pellin,Honorary President.

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EDITORIALEditorial

Dear Colleagues,

On behalf of the Tissue Engineering and Regenerative Medicine International Society - Americas Council, welcome to Brazil! We are thrilled to be supporting a meeting in South Brazil and hope it is the first of many more within Latin America. We would like to extend a huge thanks to Dr. Pranke, and her colleagues, for her leadership in organizing this meeting. We hope that the meeting acts as a means to facilitate collaborations within the field, grow the impact of each individual’s research efforts, and spur the translation of technologies into clinical use. Finally, we encourage students and young investigators to participate in the various activities that are part of the meeting, as they grow their presence in the field.

We look forward to a fantastic meeting!

Best regards,

John Fisher,Chair, TERMIS-Americas Chapter.

EDITORIALEditorial

Dear Participants,

It is my pleasure to welcome you to the 1st TERMIS-AM Latin America Workshop in Porto Alegre, Brazil. The goal of TERMIS has been to foster ways to engage the membership within the emerging countries within the Chapters of the Society. Latin America, which united with the TERMIS-North America Chapter to form the Americas in 2012 has assisted with highlighting the tissue engineering and regenerative medicine research being conducted within the region.

We want to thank Dr. Patricia Pranke, Dr. Jorge Silva and their organizing team for planning a wonderful program that has attracted key leaders in the field. In addition, we would like to acknowledge Dr. Martha Fontanilla from Colombia and Dr. Maria Margarita Garcia Rizzo from Uruguay as well as Dr. Pranke for collaborating and reaching out to others within the Latin American community on behalf of TERMIS.

Representatives from TERMIS-Americas will be presenting at the workshop: Dr. William Wagner, Director for the McGowan Institute for Regenerative Medicine at the University of Pittsburgh. Dr. Wagner was the Chair of the TERMIS-AM from 2016-2017; Dr. James Yoo from the Wake Forest Institute for Regenerative Medicine.

Enjoy the science and hope the workshop encourages you to form new collaborations with your colleagues! If you have any questions about TERMIS, please do not hesitate to contact the Executive Administrator at [email protected].

Sincerely,

Rui Reis

TERMIS President

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COMMISSIONSCommissions

PresidentProfessor Dr. Patricia Pranke

Vice PresidentDr. Jorge Vicente Lopes da Silva

Honorary PresidentDenise Crawshaw Pellin

Scientific Committee

Professor Andréa TrentinProfessor Angela Cristina Malheiros LuzoProfessor Antônio Carlos Campos de CarvalhoPhD Daikelly Iglesias BraghirolliPhD Daniela SteffensProfessor Guilherme BaldoProfessor Gustavo Abel AbrahamProfessor Ionara Rodrigues SiqueiraProfessor Isabel Cristina TessaroProfessor José Ignácio Arias FernándezPhD Laura Elena SperlingProfessor Luciano Casagrande Professor Luiz Henrique CatalaniPhD Marco Antônio Sabino GutierrezProfessor Marco StefaniProfessor Marcus Vinicius Lia FookProfessor Maria Margarita García RizzoProfessor Martha Raquel Fontanilla PhD Natasha MaurmannProfessor Nilo Sergio Medeiros CardozoProfessor Regina GoldenbergPhD Rodrigo Alvarenga RezendePhD Silvia Ceré

EDITORIALEditorial

Dear Colleagues,

This first ever gathering in Latin America of the TERMIS Workshop will provide five days of intense information, from June 27 to July 1, in Porto Alegre, Brazil, sharing about the innovations and advances in tissue engineering and regenerative medicine. This is a wonderful opportunity for scientists and researchers from all over Latin America to come together to discuss challenges and successes, while exchanging information and finding inspiration about this important field.

Ultimately, our collective goal is to expand the number of conditions that can benefit from regenerative medicine by overcoming the scientific challenges of the field. I am encouraged every day by my colleagues in Latin America who are meeting these challenges and making crucial advances.

Our scientists from the Wake Forest Institute of Regenerative Medicine are looking forward to being present at this important conference. I am confident that workshop attendees will be motivated and inspired by the excellent program and productive discussions, while enjoying the local flavor of the venue.

Congratulations to Dr. Patricia Pranke and all the organizers of this conference for putting together such a great program!

With my very best wishes to all of you,

Anthony Atala,Director, Wake Forest Institute for Regenerative Medicine.

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Commissions

Organizing Committee

Andréia FerreiraBrendha Lang CamboimMD. Bruno José da Costa AlcantaraCarolina AlbrechtCassiano MendesCristian TeixeiraPhD Daikelly Iglesias BraghirolliDaniela BaglioniDaniela PavulackPhD Daniela SteffensMSc. Débora CzarnabayEduarda Peres CoutoMSc. Fernanda Stapenhorst FrançaGabriele Gulielmin DidóGuilherme Rodrigues MachadoJoão Vitor Barboza Cardoso MSc. Juliana Girón BastidasMSc. Karina Pires Reis PhD Laura Elena SperlingLaura Gonçalves PozzobonLuiz SommerMarcelo Garrido dos Santos Marina BriãoMauricio BorgesMorgganna BuenoPhD Natasha MaurmannNatascha MonteiroOranian dos AnjosPaola BotteziniPaula Martins FernandesPedro Marques da RosaPhD Rodrigo Alvarenga RezendeVictória PallaoroVictória TomazVitória Soares

General Information

General Information

LocationCourses: Theory classes: Anfiteatro – Av. Ipiranga, 2752.Salão nobre ICBS (Campus do Centro da UFRGS) - Sarmento Leite, 500Practice classes: Room 304g - Av. Ipiranga, 2752.Classroom 113 - Sarmento Leite, 500.1st TERMIS – AM WORKSHOP: Centro de Eventos do Barra Shopping Sul.

OpeningThe Congress Termis Americas Workshop congress started at 12h30pm at event cen-ter of Barra Shopping Sul in the city Porto Alegre, giving continuity through a cere-monial opening and a welcome happy hour to all of the congressmen at night, on the opening day, 06/29/2018.

Happy Hour Was performed on 06/29/2018 and 06/30/2018 at 6pm until 8pm, at the event center of Barra Shopping Sul.

ExhibitionExhibition of companies in stands, assembled in the event center, available every day during the Congress Termis Americas Workshop.

Awards Posters of undergraduate students, masters and PhD were evaluated for a commis-sion chosen by the direction of the scientific committee and the best posters of each category was awarded. The disclosure occurred at 5h30pm of on 07/01/2018.

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Event Schedule

Event SchedulePresentationPosters and pictures of the photo contest will be on the panels previously informed on the program. All the auditoriums will have computer, multimedia projector and sound system.

Courses

Course 1: Bioreactors and Biomaterials in Tissue Engineering.Duration: 4h: 8:00-12:00Date: 06/27/2018.Location: Av. Ipiranga, 2752.Instructors:Dr. Natasha Maurmann;Dr. Daikelly Iglesias Braghirolli.

Course 2: Basic flow cytometry course.Theory Class: 8:00-12:00Practice Class: Class 1: 13:00-15:00. / Class 2: 15:30-17:00.Date: 06/27/2018.Location: Campus Centro - Av. Paulo Gama,110.Instructors:Dr. Daniela Steffens;Dr. Laura Sperling.

Course 3: Basic Real-Time PCR Course.Theory Class: 8:00-12:00.Practice Class: 13:00-15:00.Date: 06/28/2018.Location: Campus Centro - Av. Paulo Gama,110.Instructors:Dr. Daniela Steffens;Dr. Laura Sperling.

Event Schedule

Event ScheduleCourse 4: The CRISPR-Cas9 system: Biological research tool.Duration: 8:00-12:00Date: 06/28/18.Location: Av. Ipiranga, 2752.Instructors: Prof. Dr. Guilherme Baldo.

COURSE 6: Theory-Practical Course: Basic Concepts in Stem Cells and Tissue Engi-neering.Theory Class: 06/28/2018- 13:00-17:00.Practice Class: Team 1: 06/28/2018-17:00-19:00. Team 2: 06/29/2018- 8:00-10:00. Location: Campus Centro - Av. Paulo Gama,110.Instructors:Dr. Daikelly Iglesias Braghirolli;MSc. Thayane Crestani.

Course 7: Basic Principles of Epigenetics.Duration: 8:00-12:00Date: 06/29/2018.Location: Campus Centro - Av. Paulo Gama,110.Instructors: Profa. Dra. Ionara Rodrigues Siqueira.

Course 8: Basic course in biofabrication and 3D bioprinter.Duration: 8:00-12:00.Date: 06/29/18.Location: Av. Ipiranga, 2752.Instructors: Dr. Jorge Vicente Lopes da Silva;Dr. Rodrigo Alvarenga Rezende;Dr. Janaína Dernowsek.

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Event Schedule

Event Schedule1st TERMIS AMERICAS WORKSHOPJune 29th, 30th, July 1st, 2018.Locate: Centro de Eventos do Barra Shopping Sul.

29/06/2018 (Friday)

12h30 - 14h: Handing of material and registration; 14h - 14h30: Opening ceremony

14h30: Ângela Cristina Malheiros Luzo (UNICAMP) Regenerative Medicine: Multidisciplinarity, interdisciplinarity or transdisciplinarity;Chair: Patricia Pranke (UFRGS)

15h20: Antônio Carlos Campos de Carvalho (UFRJ)Modeling diseases with iPS to improve health system challenges in the new millennium;Chair: Jorge Vicente Lopes da Silva (CTI)

16h10: Jorge Vicente Lopes da Silva (CTI)Biofabrication: state-of-the-art and future expectations;Chair: Marcus Vinícius Lia Fook (UCG) 17h - 17h15: Denise Rahal Lobato (Hospital Israelita Albert Einsten) Eretz.bio: support for regenerative medicine;

17h15 - 17h45: TERMIS-AM presentation;

18h - 20h: Guided visit with poster presentation (I) - Happy Hour.

Event Schedule

Event Schedule30/06/2018 (Saturday)

08h: José Ignacio Arias Fernández (Chile)Theme: Repair of diabetic wounds and calreticulin: multiple pathways for a single result;Chair: Maria Margarita García Rizzo (Uruguai) 08h50: Marcus Vinícius Lia Fook (UCG)Theme: Biomaterials: Integration, Conduction and Induction for Regenerative Medicine;Chair: Jorge Vicente Lopes da Silva (CTI)

09h40 - 10h10: Interval 10h10: Luiz Henrique Catalani (USP)Theme: Can layer-by-layer be a good reservoir model for protein release?;Chair: Rodrigo Rezende

11h: Gustavo Abel Abraham (Argentina)Theme: Small diameter bioresorbable vascular grafts;Chair: Martha Raquel Fontanilla (Colômbia)

11h50 - 14h: Lunch break

14h: Andréa Gonçalves Trentin (UFSC)Theme: Stem cells and skin wound healing;Chair: Patricia Pranke (UFRGS)

14h50: Regina Goldenberg (UFRJ)Theme: Bioenginnering strategies in liver regenerative medicine ;Chair: Natasha Maurmann (UFRGS)

15h40 - 16h10: Interval

16h10: Leandra Santos Baptista (UFRJ)Theme: Adult stem cells spheroids as potencial tools for regenerative medicine and toxicological tests;Chair: Rodrigo Rezende

17h: Patricia Pranke (UFRGS)Theme: Stem Cells and Nanotechnology in Tissue Engineering and Regenerative Medicine;Chair: Jorge Vicente Lopes da Silva (CTI)

18h00 - 20h: Guided visit with poster presentation (II) - Happy Hour.

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Event Schedule

Event Schedule01/07/2018 (Sunday)

08h: Martha Raquel Fontanilla (Colômbia)Theme: Differential delivery of healing factors induced by the directionality of collagen type I scaf-folds influences skin wound healingChair: Maria Margarita García Rizzo (Uruguai);

08h50: William Richard Wagner (USA)Theme: Approaching biomaterials design based on specific clinical needs Chair: Patricia Pranke (UFRGS);

09h40 - 10h10: Interval;

10h10: Francisco D. Benavides (USA)Theme: Neuroplasticity after Spinal Cord Injury Chair: Patricia Pranke (UFRGS);

11h: Marco A. Sabino Gutierrez (Venezuela)Theme: Basics on Hydrogels for 3D BioprintingChair: Jorge Vicente Lopes da Silva (CTI) ;

11h50 - 14h: Lunch Break ;

14h: Silvia Ceré (Argentina)Theme: Surface modification of metal implants to improve bone integrationChair: Laura Sperling (UFGRS);

14h50: Maria Margarita García Rizzo (Uruguai)Theme: A New Approach to Cell TherapyChair: Martha Raquel Fontanilla (Colômbia) ; 15h40 - 16h10: Interval

16h10: James Yoo (USA)Theme: Bioprinting 3D para Aplicações TranslacionaisChair: Jorge Vicente Lopes da Silva (CTI); 17h: Sabrina Ferri (Brasil)Theme: Case ReportsChair: Patricia Pranke (UFRGS);

17h30 - 18h30: Awards and closing ceremony.

Abstract - Bioartificial Tissue Organ

ACELLULAR SCAFFOLD PRODUCTION FOR LIVER TRANSPLANT.

Dias, M. L.¹; Da Silva, A. C.²; Julião, G.¹; Batista, C.¹; Monteiro, V.¹; Secomandi, V.¹; Faccioli, L.¹; Ramos, I.³; Christie, M.¹; Goldenberg, R.¹.

1Universidade Federal do Rio de Janeiro, Brasil. 2Hospital Universitário Clementino Fraga Filho, Brasil. 3Centro Nacional de Biologia Estrutural e Bioimagem, Brasil.

Liver transplantation is the only potentially curative treatment for patients facing acute or end-stage hepatic disease. The surgical procedures include (i) orthotopic liver transplantation (OLT), in which the native liver is removed and replaced by the donor organ in the same anatomic position as the original liver, (ii) heterotopic auxiliary liver transplantation (HALT) which involves implanting the new liver graft in a non-anatomical location and (iii) auxiliary partial orthotopic liver transplantation (APOLT) where a partial liver graft is implanted in an orthotopic position after leaving behind a part of the native liver. However, transplantation is mainly limited by the supply of transplantable donor organs, which is far exceeded by the demand resulting in increasing transplantation waiting lists. In this context, tissue engineering offers hope to overcome this shortage. Producing an acellular he-patic extracellular matrix (ECM) that may act as an inductive template for recellularization is a first step in bioengineering a liver. Objective: The aim of this work was to optimize a surgical technique to remove livers from rat donors isolating the main vessels and to improve matrix stiffness to produce transplantable acellular liver scaffolds. Methods: Donor animals received 100 µl of heparin (50 IU/ml) 15 min before surgery. Then under anesthesia (xylazine– 8mg/kg and ketamine-40 mg/kg) the animals were submitted to transverse abdominal incision and the portal vein (PV) was separated and cannulated. The infrahepatic vena cava (IVC) was dissected and a cuff Teflon tube was atta-ched to the IVC and fixed with 6-0 silk ligation. The superior vena cava (SVC) was clamped. After removal, the donor liver was perfused via PV with 5ml of custodiol solution (organ storage solution) supplemented with 10 µl of heparin (50 IU/ml) and placed in a cold saline bath for 5 minutes. Then, the livers were transferred to be perfused through portal vein using an infusion pump at 3 ml/min with water for 1 hour followed by 1% Triton X-100 for 3 hours and SDS 1% for 24h. After total de-cellularization, livers were washed with distilled H2O for 7 days to remove residual SDS and then were preserved at 4 ºC for up to 1 day. To analyze the ECM integrity post decellularization protocol, DAPI, H&E, Sirius red, DNA quantification, electronic scanning microscopy and immunohistoche-mistry assays against collagen type I, III, IV, laminin and fibronectin, were performed. Toluidine blue was used to examine the vasculature integrity. To improve the decellularized ECM shape the matrix was perfused with 10 ml of rat blood diluted in 40 ml of custodiol solution for 1 hour and washed with 3 ml of phosphate saline solution (PBS) to remove excess of blood. Then, the matrix was analyzed by H&E staining. Results: Toluidine blue showed that the vascular system was totally preserved. Macroscopy, microscopy and histological staining showed that the decellularization process preser-ves the structure and components of the ECM. After blood perfusion, the decellularized ECM shape resembled a native liver and H&E staining showed some cell retention on vessels of decellularized ECM. Conclusion: In the present study, we optimized a surgical technique isolating PV, IVC and SVC. Also, after blood perfusion, decellularized ECM shape resembled a native liver which may allow acellular rat liver scaffolds transplantable.

Keywords: Bio-Artificial Liver; Extracellular Matrix; Decellularization.

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AN EFFICIENT PROTOCOL FOR PORCINE LIVER DECELLULARIZATION SUPPORTS SUBSEQUENT

RECELLULARIZATION.

Dias, M. L.¹; Faccioli, L.¹; Suhett, G.²; Hoff, V.¹; Mendez, C.¹; Santiago, M.¹; Goldberg, A. C.²; Paren-te, D.³; De Carvalho, A. C. C.¹; Goldenberg, R.¹

1Universidade Federal do Rio de Janeiro, Brasil.2Hospital Albert Einstein, Brasil.3Instituto D’Or de Pesquisa e Educação, Brasil.

Decellularized porcine livers have a wide range of applications in regenerative medicine, however previously existing protocols for organ decellularization still need to be adjusted to a full size porcine liver. Objective: to establish a protocol that enables effective decellularization of the entire porcine liver in a short period. Method: Livers (n = 3) were perfused with decellularizing solutions through the portal vein during 3 days at room temperature. To analyze the extracellular matrix (ECM), after decellularization, histology and electron microscopy were performed. The vascular tree integrity was evaluated, by magnetic resonance and computed tomography, injecting gadolinium and iodi-ne, respectively. Toluidine blue dye was also used. The presence of residual cells were analyzed by DAPI and quantification of DNA by spectrophotometry. Collagen IV presence was detected by immunohistochemistry. To recellularize 1E6/ml of HEPG2 cells were cultured over the matrix. Cell presence in the recellularized matrix was visualized by DAPI and secretion of albumin was detected by ELISA. Results: After 3 days, this new protocol, based on the use of sodium-deoxycholate as de-tergent, was effective in preserving all ECM structures and the vascular system. DNA quantification indicated that >97% of cells were removed. Electron microscopy showed the presence of collagen in ECM and cells could not be detected. Cell seeding showed that ECM could be recellularized after 15 days.Conclusion: By enabling the achievement of liver scaffolds with preserved tissue integrity in 3 days at room temperature, this novel protocol brings new perspectives to the development of bioartificial livers.

Keywords: Decellularization; Porcine; Recellularization; Transplant.

Borges, M. F.¹,²; Maurmann, N.¹,²; Pranke, P.¹,²,³.

1Laboratório de Hematologia e Células-tronco, Universidade Federal do Rio Grande do Sul, Brasil.2Programa de Pós-graduação em Fisiologia, Universidade Federal do Rio Grande do Sul, Brasil.3Instituto de Pesquisa com Células-tronco, Brasil.

Organ transplantation is the preferred therapy for end-stage organ failure, but the demand is still higher than the number of donations. Recently, the technique for whole organ decellularization for posterior recellularization appeared as a promising option. With the appropriate cells, this solution could render organs inadequate for transplantation to become suitable free rejection surrogates. Re-search is still needed for what should be the best decellularization protocols and the type of cells to utilize. Perfusion is a method that can better distribute the solution and nutrients in tissue, mimicking the conditions of the tissue in vivo, and stem cells are a good option for recellularization as they can differentiate into the desired cell type and can be obtained from the patients themselves. Objectives: Utilize a perfusion bioreactor manufactured in the laboratory to decellularize and recellularize a rat liver with mesenchymal stem cells. Methods and Results: To strengthen the partnership of natio-nal industries with the academia, a partnership with the start-up Eva Scientific was established to construct a perfusion bioreactor. The bioreactor consists of a peristaltic pump connected to a glass chamber where the organs were placed. The decellularization was performed using distilled water for 24h, a 0.5% solution of sodium dodecyl sulfate for 24h followed by phosphate buffered saline with 1% penicillin, streptomycin and gentamicin for removal of residual SDS for 24h. Confirmation of decellularization was made by macroscopic observation, showing the discoloration of the organ. Histological analysis, using hematoxylin and eosin staining, showed absence of cellular nucleus. DNA quantification analysis showed less than 100ng/mg of wet tissue in the decellularized liver. Glycosaminoglycans were measured by the dimethylmethylene blue method as an indicator of ma-trix preservation, maintaining 50% of their total in the decellularized liver. Peracetic acid (PA) 0.1% with ethanol (EtOH) 4% and PSG 1% were utilized for 3h for sterilization. To verify the sterilization, supplemented culture media DMEM with 10% fetal bovine FBS and 1% PSG was perfused through the organs for 24h at 37°C and with 5% CO2. The nonexistence of turbidity in the media indicated the absence of microorganism growth. Liver recellularization was realized in a multistep seeding process with the total of 109 cells utilized, obtaining an efficiency > 97%. All the processes were carried out in a laminar flow hood. After 7 days, the live cells were shown marked with fluorescein diacetate and the dead cells marked with propidium iodide. Discussion: The perfusion method in the bioreactor guarantees a homogeneous distribution of the solutions, saving time and quantity of reagent. The organs rapidly acquired a white translucent color with the SDS solution, indicating loss of cellular mass. The histology confirmed the decellularized organs by the absence of nucleus colored with hematoxylin. According to data in the literature, a quantity of DNA lower than 100ng/mg of wet tissue indicates the absence of cells in the tissue. The association of PA, EtOH with anti-biotics was efficient to sterilize the decellularized organ. The multistep seeding was highly efficient, guaranteeing that the cells stayed inside the tissue. The presence of live cells after 7 days shows that the cells can survive the process.

Keywords: Tissue Engineering; Stem Cells; Decellularizaton; Recellularization; Bioreactor; Liver.

DECELLULARIZATION AND RECELLULARIZATION OF LIVER IN A PERFUSION BIOREACTOR WITH MESENCHYMAL STEM CELLS.


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MODELO DE CULTURA CELULAR TRIDIMENSIONAL PARA MIMETIZAR TECIDOS NORMAIS A TUMORAIS.

Matte, B. F.¹; Brand, L. M.¹; Ribeiro, F. P.¹; Diel, L. F.¹; Bernardi, L.¹; Lamers, M. L.¹.

1Universidade Federal Do Rio Grande Do Sul, Brasil.

O desenvolvimento de novos fármacos depende de testes realizados para avaliar sua eficácia ao objetivo pretendido e também a segurança para o organismo que entrará em contato. A maioria destes estudos ainda são realizados em cultura celular em monocamada que possui limitações em relação as reações observadas em organismos vivos. Além disso, tenta-se cada vez mais reduzir e eliminar uso de animais para estes tipos de testes. Desenvolver culturas celulares em modelo tridi-mensional que possa mimetizar tecidos sadios, como a pele, e tecidos alterados, como a displasia epitelial e o carcinoma espinocelular oral. Colágeno tipo I foi extraído de caudas de rato e utilizado como gel de matriz extracelular. A esta matriz foram adicionados fibroblastos e, na porção superior, células epiteliais e de diferentes graus de potencial invasivo tumoral. Quando estas células estão confluentes, elas são expostas a interface ar-líquido por 21 dias para analisar os efeitos deste es-tresse. As matrizes são fixadas e processadas para então serem analisadas em cortes histológicos corados por hematoxilina e eosina. A linhagem celular de queratinócitos e de carcinoma espinoce-lular oral de baixa agressividade formaram epitélio desorganizado com múltiplas camadas. Ainda que foi observada a estratificação, esta não acompanhou da mesma maneira que observamos nos tecidos humanos. Diferentemente, observou-se que a linhagem de carcinoma espinocelular oral altamente agressivo realiza a invasão da matriz extracelular de colágeno de uma maneira seme-lhante ao que observamos em espécimes humanas com a formação de pérola de queratina por exemplo. O comportamento fenotípico de linhagens celulares, quando expostas a materiais seme-lhantes ao que é presente em organismos vivos, depende do tecido de origem delas. As células são capazes, mesmo em experimentos in vitro, de reproduzir tecidos biomiméticos ao que observamos nos seres vivos. Portanto, pode-se utilizar este sistema para a triagem de potenciais fármacos. As observações deste modelo de cultura tridimensional demonstram que matrizes realizadas com o uso do colágeno e um sistema de interface ar-líquido apresenta grandes vantagens para o desen-volvimento de tecidos semelhantes ao que observamos nos seres humanos.

Keywords: Cultura Organotípica; Colágeno Tipo I; Matriz Extracelular.


PRODUCTION OF AN ARTIFICIAL SKIN SUBSTITUTE USING TISSUE ENGINEERING PRINCIPLES TO BE USED FOR TOXICOLOGICAL TESTS IN

VITRO.

Barbosa, J. L.¹; De Goes, A. M.¹; Gomes, D. A.¹.

1Universidade Federal de Minas Gerais, Brasil.

The EU launched in 2009 an effort to ban animal-tested cosmetics. Since then many models have emerged with the aim of replacing the use of animals in toxicological and cosmetic tests. Several have been pre-validated for this purpose, but they are limited in predicting whether or not a parti-cular molecule is toxic. Brazil follows the same trend; however, we do not have a national in vitro skin substitute technology for those tests. Chitosan, polyhydroxybutyrate (PHB) and gelatin are polymers widely used in tissue engineering due to their biodegradation, biocompatibility and non-to-xicity properties.We set out to produce a bilayer skin substitute - composed of a chitosan and gelatin membrane and a 3D matrix of PHB and chitosan, colonized with human keratinocytes (hKC) and fibroblasts (hFb), respectively - to be used in toxicological tests in vitro. Membranes were produced from the mixture of two 1.5% chitosan and gelatin solutions 2: 1 and left to dry. They were treated with alcohol and glycerin, washed, and then treated with water. The 3D matrix was obtained from the mixture of a 2.5% chitosan and PHB solution. The obtained solution was frozen at -20°C and lyophilized for 24h, followed by crosslinking with 0.005% glutaraldehyde and another lyophilization with the membrane. Subsequently, the materials were cut and sterilized at 15 kGy and used in phy-sical, chemical and biological tests. We observed that a bilayer structure was formed, consisting of a smooth surface and porous under layer, with pores of about 100 to 500 micrometers. The absorption rate showed that within 15 minutes the bilayers increased their weight by 1000% and thus remained for the rest of the analysis. We also treated the structure with a solution of lipase and lysozyme and after 8 weeks only 20% of the material remained. We tested then the viability of the composite by MTT assay, and we observed a similar viability between the control group and the membrane layer of the bilayer when cultured with keratinocytes. The same was seen when fibroblasts were grown on the porous structure of the bilayer. The viability of those cells was confirmed later by MEV analysis that showed adherence and growth of these primary cells on both layers. We conclude then that the bilayer structure constructed by our group consists of a skin substitute that pending further analysis can be used as an in vitro test platform for chemicals.

Keywords: Chitosan; PHB; Gelatin; Tissue Engineering; Skin Engineering; Bilayer Skin; Human Keratinocytes; Human Fibroblasts.


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Nonguera, J. A. ¹; Kemmoku, D. T. ¹; Noritomi, P. Y. ¹; da Silva, J. V. L. ¹; Dernowsek, J. ¹; Decarli, M. C. ²; Rezende, R. A. ³; Vilalba, F. ³.

1Centro de Tecnologia da Informação Renato Archer, CAMPINAS, SP.2Faculdade de Engenharia Quimica - Unicamp, CAMPINAS, SP, Brasil.3Centro de Tecnologia da Informação Renato Archer, CAMPINAS, SP, Brasil.

Bioprinting of tissues and organs can be defined as a computer-aided, layer-by-layer, additive biofa-brication of functional 3D tissue and organs constructs based on a digital model using tissues sphe-roids as self-assembling building blocks (Mironov et al., 2008). Tissue spheroids are cell agglomera-te that acquires the most stabilized form of a sphere. The diameter is limited since all mass transfer occurs by diffusion and the cells cannot be farther then 200μm of the flow of capillaries (Muschler et al., 2004). The product of the 3D printing process is not a fully operational organ and must be maturated. During the maturation the tissue spheroids will fuse and the cells can start a sequence of process to form the tissue. This process will occur in a bioreactor, a device in which biological and biochemical process develop under closely monitored and tightly controlled environmental and operating conditions (e.g. pH, temperature, pressure) (Martin et al., 2004). Because of the limitation of the diffusion range and the lack of circulatory system to improve the distribution of nutrients and oxygen for the agglomerate of cells, the size of tissue is limited. An alternative to help solve this problem is discussed in this project, the application of porous needles in a perfusion bioreactor to nourish the center of the tissue. The needles behave as an artificial circulatory system.The objective of the project is to simulate 3 parameters: temperature, shear stress and diffusion to compound a perfect environment for a tissue in a bioreactor. To validate the simulations the values will be compared with biological values. The cell produces energy (metabolism) which increase the temperature of the system. The needles will refrigerate the tissue, keeping it in a viable tempe-rature for the cell survival. With higher the velocity, more fluid flows through the tissue, increasing the quantity of nutrient and oxygen and the refrigerated area, but it also generates a higher shear stress in the cells. The software for the study were Rhinoceros 5.0® for design and Ansys/CFX® for simulation. The first model study the shear stress. The goal is to test different velocities of a flow through an agglomeration of spheres to find the maximum velocity that causes the maximum allowable shear stress. This velocity is applied to the second model consisting of a rectangular box (the tissue) and a needle (0.47mm of diameter and 0.04mm of diameter pores). This model will help determine the number of needles a tissue will need based on the volume a needle can refrigerate and nourish. The box is a porous model producing a metabolism, its cross-sectional area increase until the temperature reaches a value higher than allowed by the conditions imposed. The simu-lations concluded so far shows that different tissues need different numbers of needles since they depend on different conditions. The accuracy of the information of this conditions is essential for the precision of the simulations.In the future others parameters will be considerate to create a prototype. All the analyzes contribute to avoid large expenses with laboratory tests. References Martin, I., et al. The role of bioreactors in tissue engineering. TRENDS in Biotechnology ,22(2), 80, 2004 Mironov, V., et al. Organ printing: promises and challenges,2008 Muschler, G.F., et al. Engineering principles of clinical cell-based tissue engineering.JBJS, 86(7), 1541, 2004.

Keywords: Bioreactor; CFD; Bioprinting; Biofabrication.

COMPUTATIONAL SIMULATION FOR A PERFUSION BIOREACTOR PROJECT.

Abstract - Biofabrication/Bioprinting Abstract - Biofabrication/Bioprinting

MODELING AND SIMULATION IN THE HISTOARCHITECTURE OF THE ARTICULAR CARTILAGE FOR BIOFABRICATION.

Dernowsek, J. A.¹; Idogava, H. T.²; Decarli, M. C.²; Kemmoku, D. T.¹; Noritomi, P. Y.¹; Silva, J. V. L.¹.

1Centro De Tecnologia Da Informação Renato Archer, Brasil.2Universidade De Campinas, Brasil.

The biofabrication of engineered tissues is an essential area to reconstruct and regenerate tissues, and in the future, organs. The cartilage tissue is a complex biological system that needs to be more studied. To understand complex biological systems such as cells, tissues, and organs, it is not su-fficient to identify and characterize the individual molecules in the system. It also is necessary to obtain a thorough understanding of the interaction between biological components and its relation with biomechanical properties. Until now, to the best of our knowledge, it has not been possible to mimic the biological and biochemical properties of cartilage. New approaches to developing a new tissue become an attractive target for bioprinting, which is emerging as an essential tissue engi-neering strategy to recreate the histoarchitecture and the relationship between cells, matrix, and microenvironment of cartilage. With a layer-by-layer assembly, 3D tissues with complex structures can be bioprinted using images and simulations to study the biomechanical profile.The goal is to analyze the impact of stress on the hierarchical layers of the cartilage using finite element method. Methodology: We developed an anisotropic and microstructural finite element model of some com-ponents of articular cartilage to applied pressure. The variation of the physical behavior of the mo-del, an anisotropic characteristic, will be verified from the geometric configuration of the collagen fibers among the chondrocytes, in order to mimic the real microstructure of interstitial materials with isotropic properties. The microstructures of the articular cartilage layers modeled in the Rhinoce-ros® 5.0 (McNeel North America, Seattle, WA, USA) software, and the file in format .stp (step) was imported into Ansys 17.2 (ANSYS Inc, Houston, TX, USA) for the finite element analysis (FEA). The model (BioCAD) consisted of the extracellular matrix – type II collagen - and chondrocytes located in the deep zone. This zone was chosen because corresponds to a 45% of the ECM volume. Chon-drocytes were surrounded by a collagen matrix, a hydrogel and were assumed spherical before load application. Material properties of the chondrocyte and the ECM were obtained from the literature. The materials were considered isotropic, the BioCAD of cartilage components and the boundary conditions have been selected in literature. Contact regions between the cartilage components – cells, collagen, and interstitial fluid (~ water) – they were considered correctly bonded. The collagen fiber contribution can be observed by analyzing the deformation and minimum principal stress. The results suggest that elongation of the collagen fiber - the major cartilage´s component - may involve a change in the ECM structure, probably because the collagen fibers interact with several molecu-les. The deformation analysis, allows us to visualize which regions are moving in response to the applied pressure. It was verified that there was a gradient of displacement along the whole structure. The minimum main tension is to emphasize the areas that undergo significant compression efforts, allowing to evaluate how the chondrocytes and the collagen distribute the efforts in the system.The use of computer simulations in a microscale study is a crucial factor to understand specifics proper-ties of biological tissue for biofabrication of tissues.

Keywords: Modeling; Articular Cartilage; Biofabrication; Computer Simulations; Biomechanics.

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Dernowsek, J. D. A. ¹; Carvalho, R. A.²; Amaral, A. C.²; Silva, J. V. L.¹.

1Centro de Tecnologia da Informação Renato Archer, Brasil.2Universidade de Araraquara, Brasil.

Significant efforts have been made to develop various molds for application in tissue engi-neering and biofabrication. A range of factors affect the behaviors of cells on a substrate, including surface chemistry, feature geometry, and elastic modulus. The ability to control the position of cells in an organized pattern on a substrate has become increasingly crucial for the development of tissue engineering. With the advent of microfabrication techniques, the effects of micro-scale grooved models on cell alignment, elongation, migration, morphology, and orientation have been studied. When seeded into nonadhesive micro-molds (MM), cells self-assemble via the action of cytoskeletal-mediated contraction and cell-cell adhesion. The size and shape of the tissue is a function of the cell type and the size, shape, and obstacles of the MMs. In this context, an approach known as modular tissue engineering focuses on biofabricating tissues using cells or spheroids as building blocks with specific microarchi-tectural features. The objective of our study is to microfabricate microtissues with cellular alignment, homogenous size, and shape using MMs fabricated by 3D printing technology. Methodology: First of all, the molds were designed an array with micro patterns using Rhi-noceros software generating a Stereolithographic (STL) file. The patterns of MMs are 600 μm high and 400 μm wide. After that, the MMs were materialized by additive manufacturing (AM) or also 3D printing. The STL file was carried out into the 3D printer machine. The AM machine is the Polyjet Objet Connex 350 by Stratasys. The 3D printer jets and instantly UV-cures tiny droplets of liquid photopolymer commercially named VeroClear. Results: The MMs were designed to be used in both 6, 12 and 24-wells culture plates. When the gelled agarose already as a positive mold is removed from the negative MM, it is transferred to the standard 6, 12 or 24 wells tissue culture dishes and equilibrated with cell culture medium for cell seeding. C2C12 cell line - a subclone of myoblasts - was used to seed the MMs, and we have observed the morphology, migration, spheroid formation and alignment. The results suggested that cells can contribute to understanding the myotube formation (myogenesis), among other biological processes. The MMs produced during this study was successful in creating an environment to promote the cell alignment and growth of 3D micro-tissues, in a relatively inexpensive, user-friendly, and efficient nonadhesive MM as compared to other methods. This method enables the inclusion of topographical cues into 2D or 3D cultures to generate relevant physiological models to study differentiation processes of cells.

Keywords: Biofabrication; Micro-Molds; Tissue Spheroids; 3D Printing; Myotubes.

NONADHESIVE MICRO-MOLD FOR ENHANCING CELL ALIGNMENT OF MYOTUBES.

Abstract - Biofabrication/Bioprinting Abstract - Biofabrication/Bioprinting

OPTIMIZATION OF 3D PRINTING PARAMETERS FOR OBTAINING POLYESTERURETHANE SCAFFOLDS FOR TISSUE ENGINEERING.

Lores, N. J.¹; Hung, X.¹; Talou, M. H.¹; Abraham, G. A.¹; Caracciolo, P. C.¹.

1Instituto De Investigaciones Em Ciencia Y Tecnología De Materiales, Argentina.

In recent years 3D printing technologies have gained the attention of researchers specialized in tissue engineering (TE). The mean role of 3D printing in TE is to provide a microenvironment that mimics the intricate properties of the native extracellular matrix (ECM), favoring the infiltration of seeded cells and conducting to the generation of a specific tissue. Moreover, 3D printing techno-logies must be able to yield quality scaffolds with controlled pore size, interconnectivity, adequate mechanical strength, biodegradability, and the ability to support cellular growth. Fused deposition modelling (FDM) is one of the most common 3D printing methods to fabricate structures with con-trolled internal and external geometry layer-by-layer using computer-aided design (CAD). Fabricate bioresorbable scaffolds by FDM using filaments obtained by extrusion of segmented polyesteru-rethanes (SPU). Fabrication of SPU filaments Bioresorbable SPU based on poly(ε-caprolactone)diol (PCL diol), 1,6-hexamethylene diisocyanate (HDI) and 1,4-butanediol (BDO) (50% w/w hard segment) was synthetized by two-step polymerization using N,N-dimethylacetamide as solvent and dibutyltindilaurate as catalyst. The product was extruded by a twin-screw microextruder (Thermo Fisher Scientific Process 11) to prepare polymer filaments. FDM printing The effects of different FDM processing parameters such as nozzle size, printing speed, layer thickness, printing orien-tation, nozzle and build plate temperature on the scaffolds fabrication were evaluated. In order to optimize the pore size, different infill densities were studied. Morphology analysis The scaffolds were examined by optical microscopy and scanning electron microscopy (SEM). After setting pro-per conditions, SPU filament was easily obtained by extrusion of the powdered elastomer without additives. The SPU filament was evaluated for the fabrication of 3D printed scaffolds by FDM. The control of pore size is essential in the development of 3D scaffolds for tissue engineering. Particu-larly, it was reported that scaffolds with a mean pore size of 325 micrometer are optimal for bone tissue engineering. Thus, FDM parameters were explored to control pore size and porosity of the material. Depending on the infill density employed, 3D printed SPU scaffolds with controlled pore sizes between 120 micrometer and 1 millimeter were obtained. Moreover, the defect-free scaffolds were obtained. In order to evaluate their potential for bone tissue engineering, nanocomposites with bioactive nanoparticles are being incorporated and mechanical properties as well as in vitro assays will be studied. 3D printed scaffolds based on SPU were fabricated successfully. The printed struc-tures showed dimensional stability and high reproducibility. The desired geometry, pore size and porosity could be achieved by setting FDM parameters. Considering the rapid growth of the 3D prin-ting techniques, materials engineered to be compatible with these processes are likely to become a field of increased importance in the upcoming years. This is expected to be driven by increased industrial adoption of 3D printing processes and an increased demand for materials that meet their needs and expectations.

Keywords: Polyesterurethane; Filaments; Extrusion; Fused deposition modelling; Scaffolds; Tissue Engineering.

37 36

Barud, H. D. S.¹; Batista, I. T. S.¹; Finoccio, H. D. S. B.².

1Universidade De Araraquara, Brasil.2Afinko, Brasil.

Currently have been growing the interest in biopolymersto produce biomaterials using 3D printing, since they are biodegradable, biocompatible and non-toxic. Among all natural polymers, poly-hydro-xybutyrate (PHB), which is a polymer produced from bacteria Alcaligeneseutrophus, it is a renewab-le, linear, semi-crystalline and belonging to the class of polyhydroxyalkanoates. The main disadvan-tage of this biopolymer is excessive cost of production and some deficiencies in its properties, such as low mechanical resistance and thermal instability. In this work, the polymers composite filaments based on PHB and bacterial cellulose (BC) were prepared using a single-screw homemade extru-der. BC wastes from the dressing industry were used as a sustainable and low-cost alternative. Scanning electron microscopy (SEM) showed good compatibility between the materials. Thermal properties were evaluated by thermogravimetric and differential scanning calorimetry analysis. The dynamic mechanical analysis was used to determinate of the mechanical properties of the composi-tefilaments. PHB/BC composites filaments were used to printed 3D scaffolds with excellent quality.Our results demonstrated that the new filaments prepared are attractive candidates for use in 3D printing for tissue engineering applications.

Keywords: Polymeric Composites Filaments; 3D Printer; Poly-Hydroxybutyrate; Bacterial Cellulose; Tissue Engineering.

POLYMERIC COMPOSITES FILAMENTS FOR 3D PRINTER BASED ON POLY-HYDROXYBUTYRATE/BACTERIAL CELLULOSE FOR TIS-

SUE ENGINEERING APPLICATIONS.

Abstract - Biofabrication/Bioprinting Abstract - Biomaterials

BIOCOMPATIBILITY EVALUATION OF PARTICULATE PPY/P-TSA USING ZEBRAFISH AS A MODEL ORGANISM.

Valente, C.¹; Costa, K. M. D. C.¹, Pereira, T. C. B.¹; Soares, J. C.¹; Cruz, F. F.¹; Basso, N. R. D. S.¹; Bogo, M. R.¹.

1Pontifícia Universidade Católica Do Rio Grande Do Sul, Brasil.

Zebrafish (Danio rerio) is an animal model increasingly used in biomedical research including hu-man toxicology (Raldúa et al., 2012) and one of the most promising in vivo model systems for toxi-city screening (Bohnsack et al., 2012). Recently, zebrafish have been used to evaluate the toxicity of several nanomaterials (Wang et al., 2015) because is a facile model for the rapid evaluation of the potential toxicity and biodistribution of nanomaterials (Fako et al., 2009). After the discovery that electrical signals can regulate cell attachment, proliferation and differentiation (Rivers et al., 2002), researches sought to incorporate conducting polymers into biomaterials to take advantage of electrical stimuli, and the polypyrrole (PPy) has been studied in biomedical applications (Tian et al., 2012). The present study aimed at analyzing the biocompatibility of PPy doped with p-toluene-sulfonic acid ((p-TSA); PPy/p-TSA), after exposition of different concentration of nanomaterial in zebrafish larvae. PPy/p-TSA was synthesized by oxidative polymerization method. The morphology of PPy/p-TSA were measured using Field-Emission Scanning Electron Microscopy (FESEM) and Transmission Electronic Microscopy. The conductivity was determined by four-point probe method and particle size was estimated by FESEM. The Animal Ethics Committee (CEUA 15/00479) appro-ved all the protocols. The zebrafish embryos were exposed to the dispersion PPy/p-TSA from up to 4 hours post-fertilization (hpf) until 144 hpf. Survival Curve: Larvae mortality was evaluated in the groups (Naïve, Vehicle, 25; 100; 250 and 500 µg/mL PPy/p-TSA) in 24, 48, 72, 96, 120 and 144 hpf after particulate PPy/p-TSA exposure (30 larvae per group). Embryo toxicity test: The embryonic spontaneous movement (1 min) were monitored with the aid of a microscope at the time point of 24 hpf (10 embryo per group) and the frequency of the heart bates of embryo/larvae (1 min) were monitored with the aid of a microscope at the time point of 48 hpf (10 embryo/larvae per group). Statistical Analysis: We used Kaplan-Meier method for the survival curve and One-way Analysis of Variance (ANOVA) followed by Tukey’s test in the spontaneous movement and cardiac rate. Data were expressed as mean ± S.E. and p<0.05 was considered significant. The morphology of p-TSA-doped PPy showed a granular morphology with irregular spheres particulate. The statistical distribution from FESEM images shows that about 84% of particles ranging 178-518 nm in size, 321 ± 139 nm in average (mean ± S.D., n = 880) and minimum of 23 nm and maximum of 896 nm. The electrical conductivity of PPy/p-TSA is 4.4x 10 -6 S/cm, within the semiconductor range (10 -7 – 10 3 S/cm) (Balint et al., 2014). To evaluate the possible toxicity of the suspensions of PPy/p-TSA in diffe-rent concentrations to zebrafish, the mortality was analyzed and a significant reduction in the larvae survival in the doses of 500 µg/mL it was noted after 144 h exposure, featuring 70% mortality. The spontaneous movement analysis shows a trend of increasing movements of animals in the groups treated with higher concentrations of the nanomaterial. However, so far has not found any statistical difference in relation to the treated groups when analyzing the average heart beats. Additional ex-periments are in progress to evaluate the potential inflammatory or toxic of PPy/p-TSA, and will be useful to establish quality in the development of biomaterials in the field of regenerative medicine.

Keywords: Polypyrrole; Ppy/P-Tsa; Biocompatibility; Nanotoxicity; Zebrafish.

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Abstract - Biomaterials

BIOGLASS 45S5: STRUCTURAL CHARACTERIZATION AND ANALY-SIS OF BIOCOMPATIBILITY WITH ADIPOSE-DERIVED MESEN-

CHYMAL STROMAL CELLS IN VITRO AND IN VIVO.

Rodrigues, C.¹, Zanatelli, C.¹, Paim, T. C.¹, Naasani, L. S.¹, Azevedo, J. G.¹, Wink, M. R.¹, Buchner, S.².

1Universidade Federal de Ciências da Saúde de Porto Alegre, Brasil.2Universidade Federal do Rio Grande do Sul, Brasil.

Bioactive glasses have potential applications in the field of regenerative medicine due to their bioac-tivity that permits interaction with both hard and soft tissues. In the same way, mesenchymal stromal cells (MSCs) have been experimentally tested as part of engineered constructs considering their self-renewal and multipotent capacities. However, to design an association, it is crucial to investi-gate the physical properties of bioglass 45S5, as well as its biocompatibility. Therefore, we inves-tigated the structural short-range order of the stoichiometric 45S5, by obtaining its total structure factors (S(K)) and its radial distribution functions (RDF). The in vitro compatibility of human MSCs with 45S5 was verified by viability, morphometry and osteoinduction assays, and scanning electron (SEM) analysis. The compatibility outcome was verified through a subcutaneous implantation in a murine model by grafting the 45S5 as a scaffold for allogeneic MSCs. An increase of hydroxyapatite (HA) depositions around cells was observed. Cells showed satisfactory compatibility patterns when growing over 45S5 up to 90 days. The implant did not show any apparent toxicity for organs, or strong immunogenic reactions, and it was accompanied by a dense capsule formation around the graft. Our results indicate that MSCs can grow in the long term on the 45S5 while maintaining their characteristics. This fact, together with a non-toxicity to animals means that the 45S5 can be imple-mented in pre-clinical trials aiming MSC’s transplantation leading to further bone and tissue repair.

Keywords: Bioglass 45S5; Mesenchymal Stromal Cell; Bioactivity; Short Range Order; Radial Dis-tribution Functions.


COMPARISON OF CITOTOXICITY BETWEEN 99.5 AND 99.95% IRON FOR USE IN CARDIOVASCULAR STENTS PRODUCED BY POWDER

METALLURGY.

Paim, T. C.¹; Rodrigues, C.¹; Naasani, L. S.¹; Martins, V.²; Wermuth, D. P.³; Tavares, A. C.³; Wink, M. R.¹.

1Universidade Federal de Ciências da Saúde de Porto Alegre, Brasil.2Instituto Federal do Rio Grande do Sul, Brasil.3Universidade Fderal do Rio Grande do Sul, Brasil.

Biodegradable materials are promising candidates to be used in cardiovascular stents because they provide a provisional framework for the vessel and avoid long-term clinical problems such as vessel restenosis. Biodegradable metallic materials (BMMs) have been proposed nowadays as it is expected to corrode gradually in vivo after providing the structural support to the tissue during it regeneration and healing processes. Most of medical devices including BMMs are produced from metallic ingots fabricated using both casting and thermomechanical treatment. Recently, the use of new processes in the fabrication of biodegradable metallic stents, such as powder metallurgy, has been investigated. Among the metals, iron has been proposed as a material in the manufacture of stents because its mechanical properties are similar to Stain Steel 316L, the golden standard. However, it is still unclear what level of impurity concentration is to be considered tolerable, and thus may be left in the material to maintain the device properties. In the present study we investigated the biocompatibility in vitro of the iron in two in two different purities and conformations with ADSCs (Adipose Derived Stem Cells) to evaluate their biomedical applicability. For this purpose, samples with 99.5% and 99.95% iron were tested in the powdered state before and after the sintering pro-cess via powder metallurgy. The fabrication process included: Powder mixing (Fe and 1.5% wt % Zn-stearet), compacting (P=600 MPa,) and solid-state sintering (T=1150C; t=1h; atmosphere: Ar-5%). ADSCs were isolated from human adipose tissue (CEP-ISCMPA 882968), tested to verify their capacity to differentiate into adipogenic, chondrogenic and osteogenic cells and characterized by immunophenotyping on the flow cytometer using the antibodies anti CD44, CD105, CD45, CD14 and CD34 proteins. The samples of powdered and sintered iron were sterilized in an ultraviolet light irradiator. Sintered iron discs and their correspondent mass in powder were incubated, separately in accordance with the EN ISO 10993:12, for 48h. At end, it was collected and sterilized in 0.22 μm membrane for use in the indirect culture. After direct and indirect culture with 99.5 and 99.95% powdered and sintered iron during 48h, ADSCs viability was evaluated by MTT assay and Trypan exclusion method. The morphological analysis was perfomed by labeling the nucleus and actin cytoskeleton in order to verify the biocompatibility of the tested biomaterial with ADSCs. Genotoxic damages were identified by comet alkaline assay. It was observed that the cells maintained their viability when treated directly or indirectly with the iron powder, but decreased viability when expo-sed to high concentration of 99.5% iron powder. However, Fe after sintering by powder metallurgy proved to be cytotoxic, reducing the viability of the cells submitted to indirect contact and causing the death of the cells cultured in direct contact with the biomaterial. Genotoxic damages were ob-served in cells that were cultured indirectly with the powdered and sintered iro. The previous results showed that the sintering of the metal caused a reduction in the viability of the tested cells, reques-ting a readjustment in the process. Further studies are needed to determine the toxicity threshold of an orthosis of Fe, considering its biodegradability in the body.

Keywords: Adipose Derived Stem Cells; Powder Metallurgy; Biocompatibility; Iron Stent; Biodegra-dable Metallic Materials.

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CONDUCTIVE BIOCOMPOSITE WITH POTENTIAL FOR MEDICAL APPLICATIONS.

Sander, M. M.¹, Ferreira, C. A.¹.

1Universidade Federal do Rio Grande do Sul, Brasil.

The main objective of tissue engineering is to obtain materials similar to those found in the human body, and to use them to improve health and extend quality of life. In the case of cardiac applica-tions, a material with similar properties to myocardial tissue, like elasticity, tenacity and conductivity, could be an aid in medical intervention to remedy heart conditions. It can help to regenerate tissues by providing a site for cell growth and also offer protection to injured muscle tissue. Many biomate-rials have been developed in the last decades, including poly (glycerol sebacate) (PGS). This bio-degradable polyester is prepared by polycondensation of sebacic acid and glycerol, both obtained from castor oil. PGS exhibits good cell and tissue compatibility, and can be used in many biomedical applications, like heart patches, nerve guides, retinal transplantation, and cartilages. In the same way, PPy is a biocompatible and electrically conductive polymer, even in small amounts. Medical applications of PPy have been studied with success over the years. We hypothesized that PGS and PPy polymers, together in a conductive polymer biocomposite, can achieve similar mechanical and electrical properties to the myocardial tissue, with the goal of developing a material which could be used as a heart patch. An electrically conductive composite of poly(glycerol sebacate) (PGS) and polypyrrole (PPy) was prepared by solvent free synthesis. The PGS matrix was obtained with a 1:1 proportion of sebacic acid and glycerol. The PPy was prepared by chemical synthesis, using iron chloride as oxidant, and purified in water. Films containing 1, 3 and 5% PPy were prepared by ad-ding the conductive polymer, as a finely dispersed powder, into the PGS pre-polymer matrix. The composites were crosslinked in a vacuum oven, for 48 h at 130 °C. The films were characterized by dynamic mechanical analysis and electrical conductivity tests. In order to be used as a cardiac patch, a candidate material must present similar mechanical properties to those of myocardial tis-sue, in terms of elongation and stiffness. The Young’s modulus of a human heart muscle ranges between 0.02 – 0.5MPa. Under normal conditions, the maximum strain of dynamic loading required by soft tissue such as cardiac muscle is typically around 15%. Tensile tests are performed in PGS and PGS/PPy composites, indicating that the matrix behaved as an elastomer at room temperature. The PGS control had a Young’s modulus of 0.15 MPa and elongation at break of 126%. With 3% of PPy in the composite, the Young’s modulus was 0.26 MPa and elongation at break was 25%. With 5% the Young’s modulus was three times that of the PGS control, but the elongation at bre-ak decrease to 13%. The PPy acted as a filler in the composite, thereby increasing the Young’s modulus, achieving mechanical characteristics similar of heart muscle. The electrical conductivity of the composite films was 5x10-5 S/cm. That value is in the range of the electrical conductivity of myocardial tissue, of 1.6x10-3 S/cm (longitudinally) and 5x10-5 S/cm (transversally), indicating that the conductivity of the composites is similar to that of myocardial tissue. As both PGS and PPy are good candidates for biomedical applications, and the findings are in agreement with the mechanical and electrical characteristics of myocardial tissue, this novel biocomposite could potentially be used in heart reparation treatment.

Keywords: Biocomposite; Electrical Conductivity; Cardiac Patch.


CROSSLINKED ELECTROSPUN MEMBRANES BASED ON SPIMA FOR TISSUE ENGINEERING APPLICATIONS.

Cunha, M. D. P. P. D.¹; Aldana, A. A.¹; Abraham, G. A.¹.

1Instituto de Investigaciones en Ciencia y Tecnología de Materiales, Argentina.

In tissue engineering and regenerative medicine fields, the mimicking of the extracellular matrix (ECM) is being widely studied to improve the cell adhesion, proliferation and differentiation. Among the techniques used to reproduce the EMC’s morphology, electrospinning has been intensively employed to obtain nanofibrous structures from polymeric solutions, emulsions, suspensions or molten polymers. Soy protein isolate (SPI) electrospun mats have been studied for different biome-dical applications, such as wound dressing, tissue engineering, and drug delivery systems, among others. However, the high water solubility of electrospun proteins limits their use; therefore, several strategies to tailor the SPI stability, such as chain crosslinking or blending have been explored. Ob-jectives To increase the water stability of electrospun SPI nanofibrous mats by photocrosslinking of functionalized SPI (SPIMA) chains. Methodology SPIMA was synthesized in 2 steps. Initially, an alkaline pretreatment was carried out by adding a 1.6 M potassium hydroxide solution to a 14% w/w SPI aqueous solution, then stirred during 1.5 h at 70°C. Then a predetermined amount of me-thacrylic anhydride (Sigma Aldrich) was added into the previous solution and reacted during 2 h at 70°C under magnetic stirring. The mixture was neutralized and dialyzed in distilled water during 3 days at room temperature, and then freeze-dried for 72 h. The degree of methacrylation was deter-mined by Habeeb’s test, in which the variation in the absorption band of amino groups at 340 nm was measured. The solution for electrospinning was obtained by dissolving 3% w/v photoinitiator and 1% w/v SPIMA in 10 M acetic acid. Protein solution was electrospun using a Yflow equipment, and a 0.6 kV/cm electric field. The ambient temperature was maintained at 21°C and the humidity at 40%. Crosslinking was performed using UV light at different exposure times (0, 2.5, 5, and 10 min) and the mats named S1, S2, S3, and S4, respectively. Electrospun matrices were observed using a scanning electron microscope (JSM-6460 /LV, JEOL Inc.). Results and Discussion SPIMA was successfully synthesized with a methacrylation degree of 55%.SEM micrographs showed a fibrous morphology for all the membranes. S1 sample displayed a defectless fibrous structure with an ave-rage diameter of 219 nm. On the other hand, the fibers of crosslinked samples lost the perfect round shape and exhibited an increase in fiber diameter with the exposition time (284, 299, and 308 nm for S2, S3, and S4 samples, respectively). This morphological change can be due to the fact that UV--crosslinking procedure was carried out by immersing samples in ethanol. Conclusion The SPIMA electrospun mats were obtained by electrospinning. The optimization of electrospinning parameters allowed handling the morphology and diameter of fibers. The crosslinking process affected the mor-phology of fibers, but it was necessary to improve the mats stability in water. Moreover, additional tests are being carried out to further characterize the membranes, and to determine whether the process was able to reduce protein solubility.

Keywords: Soy Protein; Isolate Protein Functionalization; Crosslinking; Electrospinning; Tissue En-gineering.

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DESENVOLVIMENTO DE CERÂMICAS BACTERICIDAS E DE ELEVA-DA BIOATIVIDADE PARA APLICAÇÕES ODONTOLÓGICAS.

Siqueira, R. L.¹; Martins, C. H. G.²; Cintra, L. T. A.³; Peitl, O.¹; Zanotto, E. D.¹.

1Universidade Federal de São Carlos, Brasil.2Universidade de Franca, Brasil.3Universidade Estadual Paulista, Brasil.

Desde o seu desenvolvimento no final da década de 1960, as cerâmicas bioativas têm se mostra-do promissoras para várias finalidades por possuírem um conjunto excepcional de propriedades, como a capacidade de se degradarem a uma taxa controlável e serem convertidas em hidroxicar-bonatoapatita (HCA) para se ligarem ao tecido ósseo e tecidos moles. Além disso, ao liberarem certa quantidade de íons durante a degradação, induzem processos celulares que são favoráveis à regeneração in situ dos tecidos, como a migração, proliferação e diferenciação celular. Neste estudo, avaliamos a bioatividade in vitro e a atividade antibacteriana de cerâmicas cristalinas à base de sílica contendo até 3% em mol de Ag, Mg, Sr, Zn Ga e B, elementos que são atrativos para uso como agentes terapêuticos. No teste de bioatividade in vitro, realizado pelo novo método proposto pelo Comitê Técnico 4 (TC04) da Comissão Internacional de Vidros (ICG) para contornar as limitações que a ISO 23317 tem em relação à materiais particulados, os materiais exibiram for-mação de HCA em suas superfícies a partir de ensaios com duração de 24 horas — mesmo tempo observado para o Bioglass®, material considerado ʺgold standardʺ nesse segmento. Em relação a atividade antibacteriana, foram testadas 23 bactérias do meio oral relacionadas à cárie e infecções endodônticas empregando-se três métodos diferentes: diluição em ágar, capacidade de formação de biofilme e contato direto. Para o método de diluição em ágar, utilizando-se concentrações na faixa de 0,5 a 24 mg/mL, encontramos resultados suscetíveis para várias bactérias anaeróbias e aeróbias/microaerofílicas testadas, sendo os menores valores de concentração inibitória mínima (CIM) relativos as amostras dopadas, especialmente aquelas contendo Ag. No ensaio de capacida-de de formação de biofilme, não observamos nenhuma célula viável após 24 horas de incubação para todas as amostras testadas. Para finalizar, nos ensaios de contato direto todas as amostras apresentaram um efeito antibacteriano considerável, promovendo uma redução drástica no número de células viáveis após apenas 10 minutos de contato com os microrganismos. Assim, testando o novo método TC04 para avaliar a bioatividade in vitro dos materiais, constatamos que ele também é válido para materiais cristalinos. O método é de fácil implementação e rápido, além de necessitar de pouca amostra. Avaliando a atividade antibacteriana dos materiais, ficou evidente o expressivo efeito que apresentaram contra um grande número de bactérias do meio oral, relacionadas a cáries e infecções endodônticas. Por terem apresentado amplo espectro de atividade em baixas concen-trações e por terem sido extremamente efetivos no contato direto, os materiais aqui desenvolvidos são habilitados para utilização em diferentes aplicações, como tratamentos de defeitos ósseos orais, desinfecção do canal radicular e em procedimentos de profilaxia dentária.

Keywords: Biocerâmica; Cerâmica Bioativa; Bioatividade In Vitro; Atividade Antibacteriana; Profila-xia Dental.


DEVELOPMENT OF A NEW BIOMATERIAL ASSOCIATED WITH HUMAN KERATINOCYTES FOR USE AS A SKIN SUBSTITUTE.

Alcantara, B. J. C.¹,²; Steffens, D.²,³; Pranke, P.¹,²,⁴.

1Programa de Pós-graduação em Fisiologia, Universidade Federal do Rio Grande do Sul, Brasil.2Laboratório de Hematologia e Células-tronco, Universidade Federal do Rio Grande do Sul, Brasil.3Universidade de Caxias do Sul, Brasil.4Instituto de Pesquisa com Células-tronco, Brasil.

The replacement of large damaged skin areas, such as those resulting from major trauma, burns and skin cancer is an old challenge for medicine. Autografts are limited to the donor site and xeno-grafts and allografts are of limited use due to tissue immune reactions and the possibility of diseases transmission. Since the currently available treatments are insufficient to prevent scar formation and promote complete tissue regeneration, tissue engineering is an alternative for the development of a more efficient skin substitute. With the idea of encountering such a skin substitute, the aim of the current study has been to produce an EGF enriched biomaterial using PDLLA polymer and Type 1 Collagen for use as a scaffold for tissue engineered skin substitutes. For this proposal, scaffolds were constructed by the coaxial electrospinning technique and divided into 3 groups: 1) PDLLA – a coaxial PDLLA fiber, 2) PDLLA/EGF – a coaxial fiber with EGF/albumin solution core and 3) PDLLA/Collagen - a PDLLA/EGF scaffold with Type 1 collagen coating. Immortalized human keratinocytes (HaCaT) were seeded on top of the scaffolds for biological analysis. Random fibers were obtained for all the groups without beads, and characterization of the scaffolds was achieved by scanning electron microscopy (SEM) for morphology and pore size evaluation. Confocal microscopy was used to visualize core-shell structure and Fourier transform infrared spectroscopy (FTIR) to analyze the presence of collagen on the fibers. Contact angle measurements (WCA) were performed for hydrophilicity measurements. The fiber diameters for group 1 was 1.293±0.320 µm, 1.235±0.486 µm for group 2 and 1,219±0.423 µm for group 3. All the groups showed similar pore size, being 7.750 µm, 7.410 µm and 7.314 µm for groups 1, 2 and 3, respectively. Core-shell relation was confirmed by confocal microscopy and FTIR analysis showed a strong peak in 1,540 - 1,660 cm-1 region in group 3. This was probably due to stretching vibrations of the peptide C=O groups (Amid I), which suggests the presence of collagen in these fibers. Furthermore, the WCA for group 3 was 108.69º while for group 1 it was 118.21º and 116.58º for group 2. The groups were evaluated for cell viability and cytotoxicity on days 1, 7 and 14. As a result, on day 1, cell viability was greater in the PDLLA/Collagen scaffolds with an absorbance of 0.622±0.059 in comparison with 0.349±0.063 for the control (PDLLA scaffold) and 0.507±0.102 for the PDLLA/EGF scaffold. On day 7, the absor-bance for the PDLLA scaffold was 0.486±0.140, the PDLLA/EGF group was 0.616±0.004 and the PDLLA/Collagen was 0.793±0.175. On day 14, absorbance for groups 1, 2 and 3 were 0.474±0.081, 0.442±0.020 and 0.410±0.030, respectively. In terms of the biological analysis, the PDLLA/Collagen group showed the best results for cell viability tests up to day 7, with no significant difference on day 14. Cytotoxicity assays through LDH measurements performed on days 1, 7 and 14 showed sta-tistical significance (p<0.05) when comparing the PDLLA group with the PDLLA/EGF and PDLLA/Collagen groups on day 14. In conclusion, the PDLLA/EGF and mainly the PDLLA/Collagen groups, showed good results for cell growth, with the presence of viable cells. New in vitro studies are cur-rently being performed to evaluate the full potential of this new biomaterial, which could become an option for skin substitute studies.

Keywords: Skin Substitute; EGF; Collagen; Keratinocytes.

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ENXERTO DE MEMBRANA BIOLÓGICA DE INTESTINO DELGADO DE SUÍNO NA CICATRIZAÇÃO DE CÓRNEA DE CÃO: RELATO DE

EXPERIÊNCIA.

Malago, R.¹; Kawakami, H. P.¹; Pinto, L. R. D.¹; Silva, S.¹; Akamatsu, A.¹; Silva, W. G.¹; Resende, R. M.¹; Siqueira, L. J. R.¹; Sampaio, L. M.¹; Bittencourt, M. K. W.².

1Centro Universitário de Itajubá, Brasil.2Vetprime Especialidades Veterinárias, Brasil.

O enxerto de membranas biológicas tem sido uma alternativa viável utilizado para a reparação de lesões extensas e ou profundas da córnea em animais e seres humanos. Estas membranas podem ter diversas origem, como de pericárdio homólogo, membrana aminiótica e submucosa de intestino delgado de suínos, sendo conservadas refrigeradas, liofilizadas, congeladas ou em glicerina. A membrana de submucosa de intestino delgado de suíno implantada em úlceras de cór-nea profundas em cães e gatos se mostrou um tratamento viável quando comparado ao método tradicional, pois mantem a transparência corneana, sua integridade e a visão. Contudo, durante a reconstrução da superfície ocular pode haver a permanência de cicatriz, reabsorção parcial da membrana, pigmentação e visão prejudicada. Entre as doenças de córnea que necessitam de correção cirúrgica e reconstrução da córnea pode-se citar o dermóide, uma massa de crescimen-to normal em local ectópico, de caráter congênito, de origem embrionária, podendo conter tecido conjuntivo, epitélio escamoso queratinizado, folículos pilosos e glândulas. O tratamento consiste na realização da ceractetomia lamelar superficial e, dependendo da extensão e profundidade da lesão, a utilização de membrana biológica pode ser uma alternativa para auxiliar na cicatrização e na prevenção de possível perfuração de córnea. Com objetivo de avaliar a cicatrização da córnea mediante a utilização de membrana biológica de submucosa de intestino delgado de suíno (Vetrix® BioSIS ECM Technology), relata-se um caso clínico de dermóide límbico com extensão em córnea, no olho esquerdo de um cão da raça Shih Tzu, macho, com uma ano e meio de idade. O cão pas-sou por procedimento de ceratectomia lamelar superficial para correção de dermóide e recebeu o enxerto da membrana biológica, suturada com pontos simples separado e fio de nylon 9-0, além de uma tarsorrafia temporária. No pós-cirúrgico o paciente recebeu terapia antibiótica tópica a base de moxifloxacino e analgesia sistêmica, por via oral, a base de cloridrato de tramadol. Com 24 e 72 horas de pós-cirúrgico foi observada descarga conjuntival, lacrimejamento, blefaroespasmo, refle-xos visuais (ofuscamento e ameaça) presentes e visualização da membrana na superfície ocular. Sete dias após o procedimento, a membrana já apresentou sinais de transparência na superfície corneana, descarga conjuntival normal, lacrimejamento, redução do blefaroespasmo, reflexos visu-ais presentes, além de teste de fluoresceína positivo para área central do enxerto e intensa vascu-larização sobre o enxerto proveniente da região de limbo. Com quatorze dias, houve aumento da área de transparência, o lacrimejamento ainda estava presente, reflexos visuais presentes, além de área de vascularização mais concentrada em região periférica do enxerto. Até o referido momento, a cicatrização da córnea foi considerada satisfatória e o enxerto da membrana uma alternativa vi-ável após a exérese de dermóide límbico, principalmente com retorno da transparência corneana e manutenção da visão, sendo ainda necessário o acompanhamento do processo cicatricial até o restabelecimento da superfície ocular.

Keywords: Membrana Biológica; Biomateriais; Dermóide; Cicatrização; Córnea.


EVALUATION OF BIOCOMPATIBILITY OF POLY(LACTIC-CO-GLYCO-LIC ACID) – GALANTAMINE MICROPARTICLES IN RAT

ASTROCYTES.

Tomaz, V.¹,², Sperling, L. E.²,³, Prezzi, F.⁴, Wink, M. R.⁴, Pranke, P.²,³,⁵

1Universidade Federal de Ciências da Saúde de Porto Alegre, Brasil.2Laboratório de Hematologia e Células-tronco, Universidade Federal do Rio Grande do Sul,Brasil.3Programa de Pós-graduação em Fisiologia, Universidade Federal do Rio Grande do Sul, Brasil.4Laboratório de Biologia Celular, Universidade Federal de Ciências da Saúde de Porto Alegre, Bra-sil.5Instituto de Pesquisa com Células-tronco, Brasil.

Galantamine, an acetylcholinesterase inhibitor used in the treatment of patients with Alzheimer’s disease, increases neurogenesis and produces a neuroprotective effect. Previous studies from our group have indicated that galantamine improves recovery after spinal cord injury (SCI). However, the need of repeated dosing and cholinergic side effects of galantamine are the major hurdles for the optimum usage of this drug. Drug release characteristics are improved with the incorporation of biodegradable polymer carriers such as polyseters, which sustain the release of encapsulated drugs. Electrospraying is acknowledged to be an important technique for the preparation of nanoparticles. Astrocytes are important players in the formation of the scar that impedes regeneration after SCI, and they express cholinergic receptors, which can be indirectly modulated by galantamine.The ob-jective of this study has been to develop a drug delivery system using poly (lactic-co-glycolic acid) (PLGA) microparticles with galantamine and test their biocompatibility in astrocyte cultures from rat cortex. Galantamine containing microparticles were produced by electrospraying 4% PLGA polymer solutions with three different drug proportions (1%, 5% and 10%). The parameters used for the electrospraying were: flow rate 0.5 ml / h, voltage of 23-26kV and 9 cm of distance from the needle to the collector plate. The morphology of the particles was evaluated by Scanning Electron Microscopy (SEM) and the diameter and zeta potential of the particle suspension was measured by the Zetasizer. Astrocytes isolated from the cortex of Wistar rat pups were incubated with the different microparticles and the cellular viability and cytotoxicity were analyzed by the WST8 and LDH assay, respectively. For morphological analysis, the cell cultures were labeled with GFAP (glial fibrillary acidic protein) and DAPI (nuclear marker). The particle morphology varied according to the galan-tamine concentration used, as can be seen by the MEV analysis. The zeta potential of the particles was of -41.5 ± 4.9 mV for particles with only 4% PLGA and -28,03 ± 3,0 mV, -14 , 33 ± 0.15 mV and -33.78 ± 2.9 mV for particles containing 1%, 5%, and 10% of galantamine, respectively. The average particle diameter was 434.73 ± 49.67 nm for particles with 4% PLGA alone, and 835.1 ± 69.53 nm, 762 ± 338.03 nm and 576.45 ± 235.07 nm for the PLGA particles with 1%, 5% and 10% of galan-tamine, respectively. The WST8 assay showed that 4% PLGA increased astrocyte viability on day 1 (1.60 ± 0.3) as well as day 7 (2.261 ± 0.1) as compared to the controls (cells with no treatment) 0.770 ± 0.37 and 1.691 ± 0.16 on days 1 and 7, respectively. However, cell viability was lower in the 4% PLGA groups associated with 10% Galantamine on both day 1 (0.62 ± 0.14) and day 7 (1.04 ± 0.16) when compared to the groups treated with galantamine (1.15 ± 0.34 and 1.78 ± 0.14 on days 1 and 7, respectively). The LDH assay showed that the 4% PLGA particles are not cytotoxic for the cells (132.08 ± 2.5) when compared to the control (248.893 ± 21.9). With the present study, it can be concluded that 4% PLGA microparticles present good biological activity with astrocytes and are non-cytotoxic, and when associated with galantamine, decrease astrocyte viability, being, therefore, a possible strategy to reduce glial scaring in the future.

Keywords: Astrocytes; Galantamine; Electrospraying; Microparticles; Glial Scar.

47 46


EVALUATION OF NATURAL BIOMATERIALS MADE OF CHITOSAN FOR THE REGENERATION OF TISSUES.

Machado, G. M.¹; Kasper, R. H.¹; Maurmann, N.²,³; Bavaresco, C. S.¹; Pighinelli, L.¹; Pranke, P.²,³,⁴.

1Universidade Luterana do Brasil, Brasil.2Laboratório de Hematologia e Células-tronco, Universidade Federal do Rio Grande do Sul, Brasil.3Programa de Pós-graduação em Fisiologia, Universidade Federal do Rio Grande do Sul, Brasil.4Instituto de Pesquisa com Células-tronco, Brasil.

The use of natural polymers in the production of structures used to stimulate cell development and tissue regeneration is being a promising strategy in tissue engineering. In this study, chitosan and its derivatives were produced and evaluated alone or associated with Buriti oil and Salmon fish scale regarding their influence on the viability of human keratinocyte cell. Cells from the immortali-zed keratinocyte line (HaCaT) were seeded at a 5,000/well density, directly in 96 well tissue culture plates (TCPs). After 24 hours, the cells were treated with culture medium (control), 0.13% of chito-san, 0.13% of chitosan conjugated with Salmon fish scales or 0.30% of chitosan conjugated with Buriti oil. Cell viability was assessed based on (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay after four days cultivation. The results of cell viability showed statistical sig-nificance between the cells cultivated in the well-plate (control group) and chitosan tested alone. The mean and standard deviation values obtained from the normalized results of the cell viability (%) of the treated groups compared to the TCPs (used as a control group) were 100±10.1% and the chitosan only was 87.6±9.7% (p<0.01). In contrast, cultivation of both types of conjugated chitosan conjugated were higher than the chitosan alone and similar to the control. The value obtained for chitosan conjugated with Buriti oil was 100.3±6.8% (ns) and 95.9±15.2% (ns) with the fish scales. Therefore, it is possible to affirm that the association of chitosan with Buriti oil and Salmon scales was beneficial for the viability of the cultivated cells, being promising biomaterials for tissue rege-neration.

Keywords: Natural Polymers; Biopolymers; Biomaterials; Regenerative Medicine; Salmon Fish Sca-les.


FUNCTIONAL HYBRID SCAFFOLD PVA-BG CONTAINING COBALT AS A POTENTIAL STRATEGY FOR TISSUE ENGINEERING.

Laia, A. G. S. D.¹, Sá, M. A. D.¹, Pereira, M. D. M.¹.


Tissue Engineering consists of the development and manipulation of molecules, cells, tissues, or organs to replace or assist in the function of defective or damaged body parts. The possibility of de-veloping complex three-dimensional tissues, reproducing the design and function of human tissue is a recent development. The need for alternatives to repair or replace living tissues has generated a search for superior materials. One class of biomaterials studied for use in tissue engineering is hybrid materials. Functional organic-inorganic hybrids are gaining large relevance as materials for Tissue Engineering and Regenerative Medicine. Bioactive glasses (BGs) have a variable and highly flexible composition, allowing the incorporation of metallic ions with therapeutic effect, a propitious approach in the development of BGs with superior properties for tissue engineering. Cobalt (Co) presents great potential for incorporation into the structure of bioactive glasses, since it may influen-ce the formation of new blood vessels. Objective: The main objective of this work was to synthesize PVA and BG hybrid scaffolds with new compositions in the SiO2-CaO-P2O5-CoO system, by the sol-gel method, and to investigate the effects of Co incorporation on its structure, to obtain angio-genic properties in a material with controlled release capacity of this ion. Methodology: The hybrid scaffolds obtained were subjected to the strict characterization through ICP-OES, FTIR, MicroCT, XRD, SEM and EDS analyzes. Results and Discussion: Hybrid scaffolds with BGs containing 5% and 10% Co were obtained, and the composition was confirmed by chemical analysis (ICP-OES and FTIR). The porous three-dimensional structure was characterized by MicroCT. The porosities and interconnectivities obtained are within values favorable for the cellular environment. XRD analy-zes showed amorphous phase with some crystalline peaks compatible with calcium silicate. SEM images showed microporous and macroporous surface corroborating with structural MicroCT re-sults. Final considerations: The results indicate that functional hybrid scaffolds produced with cobalt incorporation into their structure presented favorable properties and it is potential strategies to ob-tain auxiliary materials for tissue engineering.

Keywords: Bioactive Glass; PVA; Cobalt; Scaffolds; Tissue Engineering.

49 48


HIDROGEL DE ACETATO DE CELULOSE E EDTAD COM EXTRATO ETANÓLICO DE PRÓPOLIS PARA APLICAÇÕES EM TRATAMENTOS

DE QUEIMADURAS DE SEGUNDO GRAU.

Duek, E. A. R.¹; Borges Gomes Da Silva, G.¹; Martins Ferreira, L.¹; Cesar De Azeredo Bissoli, M.¹; Ri-goni Marcato, V.¹; G. Melero, A. M.²; A. Hausen, M.¹; Maciel De Oliveira , N.¹; Rosalen, P. L.³; Matias De Alencar, S.⁴ .

1Pontifícia Universidade Católica de São Paulo, Brasil.2Universidade Federal de Ouro Preto, Brasil.3Universidade Estadual de Campinas, Brasil.4Escola Superior de Agricultura Luiz de Queiroz, Brasil.

Recentemente, o uso de polímeros biorreabsorvíveis vem se destacando na área médica uma vez que representam uma alternativa viável para casos em que a regeneração tecidual é fortemente comprome-tida. Estes são biocompatíveis e os produtos gerados na sua degradação não são tóxicos. Com base nesse contexto, utilizou-se neste trabalho uma membrana de Hidrogel de Acetato de Celulose (HAC) e Dianidrido de Ácido Etilenodiaminotetraácido (EDTAD) como matriz polimérica, associado à base de extrato etanólico de própolis OP6, objetivando-se analisar, através de técnicas histológicas, os efeitos antiinflamatórios e regenerativos do complexo HAC-EDTAD/OP6 sobre queimaduras induzidas de se-gundo grau. O estudo in vivo foi realizado em 60 ratos Wistar, divididos em 2 grupos conforme o tempo dos curativos (7 e 14 dias). Cada conjunto, por sua vez, foi subdividido em controle negativo, controle positivo e tratamentos HAC-EDTA/OP6 em concentrações 15, 30 e 60% (m/v) de Própolis. A lesão foi induzida por escaldo a 70ºC por 10 segundos, após analgesia e bloqueio neuromuscular, resultando uma queimadura de 2º grau, em uma área tricotomizada de aproximadamente 4cm², em região dorso cervical. Logo, ocorreu a fixação do biomaterial nas diferentes concentrações nos grupos de estudo. Associado a isso, foram adicionadas ataduras elásticas autoaderentes sobre a área das lesões e cola-res elisabetanos para evitar o auto traumatismo e possível remoção dos curativos. Por fim, as áreas de implante foram excisadas e processadas para análise histológica, sendo então seus resultados com-parados a um ensaio de liberação controlada de própolis, que foi executado através de espectrômetro UV-Vis. Nessa análise, feita em solução de PBS, a 35°C, o hidrogel apresentou uma taxa de liberação inicial de própolis rápida, cujos valores para as amostras com 15, 30 e 60% de própolis foram 7,5; 28,9 e 52,1mg/h, respectivamente. Portanto, houve uma correlação direta entre a concentração de própolis incorporada e a taxa de liberação, sendo a amostra de OP6 60% a que apresentou a maior quantidade liberada ao final da análise (484,3 mg). No que tange à análise histológica, o grupo controle negativo apresentou epiderme irregular com tecido de granulação e exsudato. Já o positivo, regeneração parcial e infiltrado inflamatório ausente. Ambos os controles demonstraram pouca ou nenhuma regeneração dos anexos cutâneos. Em contrapartida, todos os grupos tratamento mediaram melhor processo rege-nerativo. Em relação à 15%, ocorreu reepitelização mais acentuada do que o controle positivo, porém os grupos 30% e 60% apresentaram melhores resultados devido a reepitelização a partir dos anexos cutâneos remanescentes. Destaque para 30%, com modulação mais equilibrada do processo inflama-tório comparada a 15% e 60% e, consequentemente, melhor processo regenerativo. Não obstante, comparando-se o ensaio de liberação à análise histológica, notou-se que no tratamento 60% houve maior concentração da substância na lesão, possivelmente ocasionando um processo inflamatório mais acentuado em relação ao tratamento 30%, sendo necessário realização de estudos de dose-res-posta dessas concentrações. Desse modo, concluiu-se que a preservação dos anexos cutâneos com relação à baixa ação inflamatória e à concentração intermediária, auxiliou no processo regenerativo, representado pelos resultados do grupo tratamento 30% como a melhor concentração associada ao biomaterial aplicado nas queimaduras de segundo grau.

Keywords: Acetato De Celulose; Própolis; Queimaduras.


HIDROGEL DE QUITOSANA REFORÇADO COM BIOVIDRO PARA APLICAÇÕES ORTOPÉDICAS.

Spohr, D. L.¹ .

1Universidade do Vale Rio do Sinos, Brasil.

Os biomateriais disponíveis hoje no mercado são baseados em “scaffolds”, ou seja, poros interli-gados feitos de materiais biocompatíveis que permitem a passagem de células e a promoção da bioregeneração. Esse modelo funciona para problemas menos complexos, como preenchimen-tos ósseos. Porém a engenharia de tecido caminha para simular o ambiente do corpo humano, incorporando células, fatores de crescimento e nutrientes aos biomateriais. Para tanto, os “scaf-folds”, devido sua baixa permeabilidade, mostram-se insuficientes. Uma nova abordagem que as substituem são os hidrogéis que permitem facilmente imitar a matriz extracelular com excelente dispersão celular e fácil prototipagem por bioimpressoras, tendo como desvantagem baixa esta-bilidade mecânica. Por isso, é vantajosa a fabricação de um biocompósito. A seleção de material nesse caso é pensada de maneira a conferir algumas características importantes para aplicações médicas como ósseo indução, vascularização, pesticida e anti-inflamatória. Levando em conside-ração o presente cenário, o trabalho propõe a criação de um hidrogel de quitosana reforçado com biovidro para aplicações ortopédicas. Objetivo Geral. Confecção de um hidrogel de quitosana re-forçado com biovidro para aplicações ortopédicas.Objetivos Específicos.Avaliar a interação entre matriz e reforço;Avaliar a bioatividade do compósito.METODOLOGIA.Materiais.Quitosana, massa molecular de 2x10e5 Dalton e grau de desacetilação de 87%.Sal fosfato dissódico de glicerol – Re-ticulante físico e ajuste de pH. Genipina (nome IUPAC: metil-2-hidroxi-9-(hidroximetil)-3-oxabiciclo-nona-4,8-dieno-5-carboxilato)- Reticulante químico. • Bioglass® 45S5 em forma de pó produzido pela Vetra Brazil. Método. Dissolve-se 2,0g do polímero em 100 mL de solução aquosa de ácido acético (0,5%, v/v), e submete-se à agitação magnética, a temperatura ambiente, por um período de 24 horas para garantir a completa dissolução do polímero. Filtra-se a amostra para remoção de qualquer material insolúvel e mede-se o pH que deve estar em torno de 5,5. Adiciona-se 6g do pó de biovidro (Luz e Mano, 2012). Depois, dissolveu-se fosfato dissódico de glicerol em água destilada. Esse, é adicionada a solução de quitosana gota a gota, sob agitação magnética até ter 1,5g do sal na solução quitosana a um pH de 7,4. Caso houver necessidade, adiciona-se mais da solução até atingir o valor de pH. A essa solução, adiciona- se a genipina em pó na concentração de 0,15% em massa. Deixa-se a amostra maturando por 24 horas e então a mistura é aquecida a 37 ºC em estufa, originando o hidrogel fisicamente/quimicamente co-reticulado. Todos os hidro-géis são obtidos em molde de silicone, com poços cilíndricos de diâmetro de 15 mm e altura de 10 mm. 3.3 Caracterização. A morfologia e estrutura são avaliadas por microscopia eletrônica de varrimento (Scanning Electron Microscopy), microscopia eletrônica de varrimento ambiental (Envi-ronmental Scanning Electron Microscopy), porosimetria de intrusão de mercúrio e espectroscopia de infravermelho por transformada de Fourier (Fourier Transform Infrared Spectroscopy), FTIR. A quantificação da extensão da reação de reticulação é determinada por ensaio da ninidrina. Como o intuito é de aplicações biomédicas, são avaliadas a degradabilidade, capacidade de absorção de água e citotoxicidade. O trabalho esta em fase de desenvolvimento, devido a este fato, não se tem nenhum resultado conclusivo.

Keywords: Biomaterial; Hidrogel; Quitosana; Medicina Regenerativa; Ortopedia.

51 50


INCORPORATION OF THE COBALT THERAPEUTIC ION INTO BIOAC-TIVE GLASS NETWORK AND ITS IMPLICATIONS FOR

ANGIOGENESIS.

Laia, A. G. S. D.¹; Barrioni, B. R.¹; Sá, M. A. D.¹; Valverde, T. M.¹; Cunha, P. D. S.¹; Pereira, M. D. M.¹.


Bioactive glasses (BGs) are used mainly for the repair of bone tissues due to their high bioactivity and biocompatibility. BGs have a variable and highly flexible composition, allowing the incorpora-tion of metallic ions with therapeutic effect, a propitious approach in the development of BGs with superior properties for tissue engineering. These ions have the advantage of being released exactly at the implant site by optimizing the therapeutic effect and reducing side effects in the patient as they are released in a controlled manner during the process of material degradation. Cobalt (Co) presents great potential for incorporation into the structure of bioactive glasses, since it may influen-ce the formation of new blood vessels from the stabilization of the hypoxia induction factor (HIF) and vascular endothelial growth factor (VEGF), known to activate genes related to angiogenesis. Objective: In this context, this work aims to develop novel sol-gel bioactive glass compositions and evaluate the incorporation of cobalt on the glass network as a potential strategy to develop superior materials with sustained ion release for tissue engineering. Methodology: Microparticles of Co-con-taining bioactive glasses with up to 2,5% CoO was obtained and analyzed by ICP-OES assays, En-dothelial Cell Growth Curve, MTT, Immunohistochemistry. Results and discussion: The composition of the microparticles of Co-containing bioactive glasses was confirmed by chemical analysis. The influence of the incorporation of cobalt in partial substitution to the CaO content in the structural and biological properties of the bioactive glasses was studied. The construction of the endothelial cell growth curve consisted of analyzing the progress of cell monolayer density over time. This analysis allowed to obtain estimated data of the folding time of the cell lines for use in the experiments of cellular proliferation and immunofluorescence. Endothelial cell in vitro study in the MTT assay was performed. A cell-friendly environment was generated as observed through MTT assay, with no significant differences in cytotoxicity. Immunohistochemical assay to verify VEGF expression was performed through an in vivo study. Samples containing VB with 2.5% Co demonstrated VEGF expression. Final considerations: The microparticles of Co-containing bioactive glasses with up to 2,5% CoO had VEGF expression and adequate structural properties, producing a friendly cellular environment and indicating that Co-containing sol-gel derived glasses are a potential candidate for application in tissue engineering and regenerative medicine.

Keywords: Bioactive Glass; Cobalt; Angiogenesis.


INFLUENCE OF BIOACTIVE CERAMICS ON KERATINOCYTES’ VIABILITY.

Brião, M. M. M.¹,²; Pavulack, D.²,³; Siqueira, R.⁴,⁵; Maurmann, N.¹,²; Zanotto, E.⁴; Pranke, P.¹,²,⁶.

1Programa de Pós Graduação e Fisiologia, Univeridade Federal do Rio Grande do Sul, Brasil.2Laboratório de Hematologia e Células-tronco, Universidade Federal do Rio Grande do Sul, Brasil.3Universidade Federal de Ciências da Saúde de Porto Alegre, Brasil.4Universidade Federal de São Carlos, Brasil.5Centro Federal de Educação Tecnológica de Minas Gerais, Brasil.6Instituto de Pesquisa com Células-tronco, Brasil.

Bioactive ceramics (BC) are an important class of biomaterials used in tissue engineering because of their positive interactions with not only hard tissue (bone and teeth), but also soft tissue, such as skin. In this study, a BC of the SiO2–CaO–Na2O–P2O5 system was synthesized and tested regar-ding its influence on the viability of keratinocytes, which are a cellular type found in the outermost layer of the skin. For this purpose, the human cell lines HaCaT were seeded in 96-well tissue cul-ture plates (TCPs) and treated with BC at concentrations of 0.5, 1, 1.5, and 2 mg/mL. Cell viability was then evaluated by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (MTT) after four days of cultivation. The mean and standard deviation values obtained from the normalized results of the cell viability (%) of the treated groups compared to the TCPs (used as control group) were 100.0 ± 13.9 % for TCP, 140.2 ± 24.2% (p<0,02) for 0.5 mg/mL, 235.0 ± 31.6 % (p<0,00) for 1 mg/mL, 232.8 ± 31.8% (p<0,00) for 1.5 mg/mL and 296.8 ± 37.2 % (p<0,00) for 2 mg/mL of the BC. The composition and crystallinity of bioceramics are aspects which make them capable of affecting the concentration of ions in the cell culture medium and, consequently, influencing the cell viability. By varying the concentration of the bioactive ceramics in the cell culture, it was therefore possible to favor HaCaT proliferation for a particular application of interest, such as wound healing and skin regeneration. Comparisons between the tested BC showed that the cell viability was dose-depen-dent, with the maximum evaluated (2 mg/mL) being statistically better than the others and with the “p” value below 0.005 in all cases. In conclusion, the tested BC can be used to promote cell viability, thereby being a useful tool in tissue engineering with further application in regenerative medicine. As a following step, evaluation of the material will be made with a view to creating a product for skin regeneration in vivo.

Keywords: Bioactive Ceramics; Biomaterials; Tissue Engineering; Regeneration.

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MEMBRANAS POLIMÉRICAS MICROESTRUTURADAS E CONDUTO-RAS PARA APLICAÇÃO NA ENGENHARIA DE TECIDOS.

Valente, C. A.¹; Tondo, P. H. F.¹; Sgarioni, B.¹; Santos, S. A. M.¹; Malmonge, J. A.²; Papaléo, R. M.¹; Basso, N. R. D. S.¹.

1Pontifícia Universidade Católica do Rio Grande do Sul, Brasil.2Universidade Estadual Paulista, Brasil.

O controle morfológico e elétrico no desenvolvimento de biomateriais é fundamental para aplica-ções na Engenharia de Tecidos (ET). Destaca-se o uso dos polímeros biodegradáveis no desen-volvimento de novos suportes (scaffolds) para ET, pois permitem, principalmente, variar a taxa de degradação dos scaffolds. Os polímeros condutores, por exemplo, o polipirrol (PPy), estão sendo explorados na área médica, além de apresentar biocompatibilidade, devido à sua natureza condu-tora que permite que células ou tecidos cultivados sobre eles sejam estimulados através da apli-cação de um sinal elétrico. O objetivo é preparar membranas de policaprolactona (PCL), poli(ácido láctico-co-glicólico) (PLGA) e polipirrol (PPy) para atuarem como suportes na ET. Previamente, PPy é sintetizado via polimerização química oxidativa do pirrol (Py) na presença de agentes do-pante (ácido p-toluenossulfônico) e oxidante (cloreto férrico). As razões molares utilizadas foram de [1]:[4]:[1,7] entre [Py]:[dopante]:[oxidante], respectivamente, em solução aquosa. Membranas poliméricas com diferentes razões (m/m%) entre PCL/PLGA (100/0, 90/10, 80/20, 70/30, 0/100) foram preparadas por evaporação do solvente utilizando molde microestruturado com canais longi-tudinais de 5, 10, 15 e 20 µm de largura e altura constante (25 µm). Compósitos microestruturados (PCL/PLGA/PPy) foram preparados pela adição de 10% (m/m) do PPy. A morfologia e a condutivi-dade elétrica (CE) das amostras foram caracterizadas por microscopia eletrônica de varredura com emissão de campo (MEV-FEG) e método 4 ou 2 pontas. A molhabilidade das membranas foi deter-mina pelo ângulo de contato do espalhamento de uma gota de água sobre a superfície sólida com as microestruturas. O PPy apresentou morfologia de fibras com diâmetro de 391 ± 21 nm (média ± desvio padrão) e CE de semicondutor (10-2 S/cm). No geral obteve-se membranas com canais de 5,14±0,69, 9,79±1,04, 14,02±0,87 e 19,28±1,19 µm (média ± desvio padrão) e ângulos de contato com a água de 86º±6,8°, 89°±5,4°, 79°±4,4° e 90°±8,2° (média ± desvio padrão), respectivamente. As membranas puras de PCL e PLGA e suas blendas (PCL/PLGA) são isolantes (10-14 - 10-10 S/cm), enquanto seus compósitos apresentaram-se como semicondutores (10-7 - 10-3 S/cm). A adição do PPy nas membranas poliméricas favoreceu a obtenção de material com melhores pro-priedades elétricas, além de não alterar, significativamente, a formação estrutural dos canais mi-croestruturados e a hidrofilicidade da superfície das membranas, independentemente do tamanho da estrutura. Os compósitos microestruturados são sugestões de scaffolds para culturas celulares que precisam de aplicação de estímulos elétricos, pois além das microestruturas para orientar o crescimento das células, a presença do polímero condutor pode ajudar a modular as atividades celulares, como adesão celular, proliferação e diferenciação através do uso de estímulos elétricos. Keywords: Microestruturas; PLGA; PCL; Polipirrol; Estímulos.


PORCINE SMALL INTESTINAL SUBMUCOSA SHEETS AS A SCAF-FOLD FOR HUMAN ADIPOSE-DERIVED STROMAL CELLS FOR TIS-

SUE ENGINEERING OF SOFT TISSUES.

Rodrigues, C.¹, Zanatelli, C.¹, Naasani, L. S.¹, Azevedo, J. G.¹, Paim, T. C.¹, Buchner, S.², Wink, M. R.¹.

1Universidade Federal de Ciências da Saúde de Porto Alegre, Brasil.2Universidade Federal do Rio Grande do Sul, Brasil.

Mesenchymal stromal cells (MSCs) play an important role in tissue regeneration, and have been experimentally tested as part of grafts to regulate the host immune response. The decellularized porcine small intestinal submucosa (SIS) is a xenogeneic graft material able to induce specific tis-sue remodeling in the organ or tissue into which it is placed.In this work, the combination between human adipose-derived MSCs (ADSCs) and SIS was investigated as an alternative for recellulari-zed scaffolds for soft tissue engineering. The SIS membranes were tested by Fourier transformed infrared spectroscopy, and DTA analysis, aiming to elucidate part of its physical properties. The 96h-day ADSC cultures on SIS were investigated as to their viability and the morphological features, such as the F-actin cytoskeleton and nuclei searching parameters to evaluate biocompatibility. Both fluorescence and scanning electron (SEM) microscopies revealed that the ADSCs could adhere to SIS and grow on its surface normally. The cellular viability, F-actin organization, and nuclei were not affected by SIS, when compared to a coverslip surface. The ADSCs could differentiate into adi-pocytes when adhered to SIS as well as exhibit stemless maintenance up to 26 days. As a natural scaffold, SIS is compatible with ADSCs, thus providing a base for both autologous or allogeneic cell transplantations.

Keywords: Mesenchymal Stromal Cells; Small Intestine Submucosa; Xenogeneic Graft, Biocompa-tibility.

55 54


PRODUCTION AND CHARACTERIZATION OF NANOCRYSTALLINE CHITTOSAN/BURITI OIL NEW BIOCOMPOSITE WITH POTENTIAL IN

WOUND HEALING.

Broqua, J. S.¹; Pighinelli, D. L.¹; Figueiredo, M. J.¹; Lisboa, C. M.¹; Rockenbach, A.¹; Persson, P.¹; Pospichil, R.¹; Machado, L. E. L.¹.

1Universidade Luterana Do Brasil, Brasil.

The skin is a precisely organized extracellular matrix. By being in contact with the environment, it’s exposed to external factors capable of damaging its structure and functions. Wounds have a high impact both individually and economically, which makes this problem of enormous relevance to the health area and the scientific community. The wound healing is a biological process where cellular matrix and components act in combination to repair damage and replace lost tissue. Biopolymers has shown to provide desirable properties, such as biodegradability and low or non-toxicity for appli-cation in regenerative medicine and tissue engineering. A structural polysaccharide named chitin (β-(1-4)-N-acetyl-D-glucosamine) is the second most abundant biopolymer and has the highest bio-degradability present in nature. Chitin can be subjected in a process called deacetylation to obtain chitosan, the most important derivative of chitin and the only positive charged biopolymer in nature. Chemically, chitosan is defined when the degree of deacetylation is more than 50%, predominating the amino groups. Besides being biodegradable and absent of toxicity and bioactive, chitosan has antioxidant activity, antimicrobial and antifungal activity, due to its exclusive cationic character. In the process of obtaining chitosan nanoparticles (NCCH), the crystalline area is reduced and some characteristics are potentialized, such as reactivity, hemostatic and mucoahdesive properties. The complexation of nanocrystalline chitosan and essential oils provide an exclusive biocomposite with notable potential for regenerative medicine. Buriti oil is an essential amazonian oil with high con-centration of fatty acids, poly-phenols, ascorbic acid, antioxidant and antimicrobial activity, besides being rich in vitamins A, B1, B2, C and E. The objective of this work is to create a biocomposite of nanocrystalline chitosan and Buriti oil, to obtain a unique biomaterial with all the characteristics and properties required for tissue engineering. First, was dissolved chitosan powder (1% polymer content) initially homogenized with deionized water and slow addition of acetic acid solution 0,4%, in constant stirring. Then, the dissolution with 1L and pH 4.6 was purified. The complexation occur-red with gradual addition of NaOH 4% solution with stirring. In pH 5.0 was added the Buriti oil on the same polymer rate and the NaOH solution was proceeded until pH 7.4. Was prepared 10 sam-ples (25mL) in Petri dishes. In Fourier-transform Infrared Spectroscopy (FTIR-ATR) confirmed both structure of NCCH and Buriti oil. The Scanning Electron Microscope (SEM) analysis of the NCCH/B complex showed a homogeneous distribution of oil in the polymer matrix. Particle Size and Zeta Potential showed a minimal increase of the particle size on NCCH/B comparing to the standard NCCH. A swelling aspect was noted on the complex that suggests the oil encapsulation by NCCH, an expected property due to the amorphous area in the polymeric matrix. The biological tests with Escherichia coli e Staphylococcus aureus showed more antimicrobial activity of the NCCH/B than just the NCCH or Buriti oil. The final material proved itself as a complex in acidic medium with no phase segregation, maintaining the initial properties and increasing some characteristics, proving the potential bioproduct for treatment in regenerative medicine and wound healing.

Keywords: Biomaterials; Nanochitosan; Buriti Essencial Oil; Nanocomposite; Wound Healing.


SUPERFICIALLY MODIFIED MG-BASED ALLOY FOR BIOMEDICAL APPLICATIONS: INTERACTION WITH CELLS IN CULTURE.

Katunar, M. R.¹; Merlo, J. L.¹; Canizo, J.²; Gomez Sanchez, A.³; Cere, S. M.¹.

1Electroquímica Aplicada, Instituto de investigaciones en ciencia y tecnología de materiales, Con-sejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Mar del Plata, Argentina.2Laboratorio de Biotecnología de la Reproducción, Departamento de Producción Animal, Instituto Nacional de Tecnología Agropecuaria, Argentina.3CIT-Villa María, Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.

Development of new biodegradable materials for fracture repair implants is required in order to avoid a second surgery for their removal. Magnesium (Mg) and Mg-based alloys are promising can-didates for this application given their good biocompatibility and osteoinductive properties. However, the degradation rate of Mg in aqueous media is high and releases hydrogen gas, which causes pain and local swelling. Superficial treatments, such as anodization, emerge as a potential solution to this limitation. Objective. To evaluate the influence of surface modification achieved by anodization at low voltage on Mg-based implants on mammalian fibroblasts adhesion and growth. Metodology. AZ91 pieces were polished and anodized at low voltage for 40 min in potassium hydroxide solution, and sterilized by dry heat. As control material, non-treated AZ91 pieces were polished and sterilized in the same way. First, the evaluation of superficial microstructure of materials was carried on by scanning electron microscopy (SEM) and rugosimetry. Then, bovine fibroblasts were seeded on the materials (anodized AZ91 or control) for 24 h; as cellular control, cells were cultured without mate-rial (on plastic). Cellular adhesion and proliferation were evaluated after 24 h by Hoechst staining. Culture medium pH and osmolarity were also recorded. Surface roughness and weight of materials was assessed before and after cell culture. Results. The surface of AZ91 anodized pieces showed a homogeneously distributed oxide layer with cracks and rough (average roughness: Ra = 0.88 ± 0.005 µm; Rz = 1.23 ± 0 µm). AZ91 control surface was homogeneous and smooth, with scratches associated with polishing (Ra = 0.2 ± 0.05 µm; Rz = 1.43 ± 0.27 µm). Number of cells adhered and grown on the anodized AZ91 was significantly higher than those adhered on the AZ91 control and also to those grown without the material (on the dish surface; one way ANOVA, F = 118, p < 0.001). pH and osmolarity of culture medium were similarly increased for anodized AZ91 and AZ91 control, in relation with values from cell culture without material. Ra of anodized AZ91 and AZ91 control did not change after cell culture (RaAZ91 anodized = 0.81 ± 0.07 µm; RaAZ91 control = 0.22 ± 0.03 µm), but the depth of valleys on the surface increased markedly for anodized AZ91 (Rz = 5.16 ± 0.47 µm) and lightly for AZ91 control (Rz = 1.92 ± 0.27 µm). Weight loss of both materials was despicable after cell culture. Discussion. Anodization of Mg-based alloy at low voltage generated a homogeneous oxide layer on the material surface. This treatment increased the adhesion of bovine fibroblasts, probably due to a lower release of hydrogen gas from the surface or the topography of the oxide layer. At the time of evaluation, differences in the level of degradation (release of osmoly-tes, changes in pH and weight loss) were not detected between anodized AZ91 and AZ91control. Conclussions. Anodization of Mg-based AZ91 alloys at low voltage improves cellular surface adhe-sion. Therefore, this treated material is a promising candidate for future in vitro –e.g. in contact with osteoblasts and macrophages- and in vivo assessment.

Keywords: Anodization; Fibroblasts; Cellular Adhesion And Proliferation; Anodization Of Mg-Based AZ91 Alloys; Osteoblasts And Macrophages.

57 56


THE VIABILITY OF KERATINOCYTES IN ADHERENT AND NON-A-DHERENT HYDROGELS.

Fernandes, P. M.¹; Brião, M. M. M.1,²; Maurmann, N.¹,²; Pranke, P1,²,³.

1Laboratório de Hematologia e Células-tronco, Universidade Federal do Rio Grande do Sul, Brasil.2Programa de Pós-graduação em Fisiologia, Universidade Federal do Rio Grande do Sul, Brasil.3Instituto de Pesquisa com Células-tronco, Brasil.

Biopolymers, such as alginate and gelatin, have been widely used as hydrogel scaffolds for tissue engineering applications. Alginate is a non-adherent polymer, because it does not contain integrin--binding motifs. Gelatin is obtained by the hydrolysis of collagen which is the principal protein found in the skin. With the aim of producing tissue-engineered skin substitutes, in this study, gelatin and alginate (an adherent and a non-adherent hydrogel) were investigated regarding their influence on the viability of human keratinocyte cells. Cells from the immortalized keratinocyte line (HaCaT) were seeded directly at a 220,000/well density, in 48 well tissue culture plates (TCPs), a two dimensio-nal culture (2D), and in three-dimensional (3D) scaffolds composed of 0.1mL of 1% alginate, 1.5% gelatin or 1% alginate associated with 1.5% of gelatin. Cell viability was evaluated by (3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay after one week of cultivation. The results demonstrated that the cells remained viable in all treatments after seven days of cultivation. The average ± standard deviation (SD) values of absorbance obtained related to cell viability were 0.38 ± 0.06 for the TCP group (control); 0.52 ± 0.11 (p≤0.01) for the 1% alginate only; 0.43 ± 0.09 (p≤0.01) for the 1.5% gelatin only and 0.38 ± 0.08 (ns) for the combination of 1% alginate and 1.5% gelatin. These results indicate that cell viability was better in the alginate and gelatin when compa-red with the control. The lower cell viability in the TCPs can be attributed to the reduced space for cell proliferation at 2D. A statistical difference was not found between the alginate and gelatin tested, thus demonstrating that cell viability was not influenced by adherence to the proposed systems. The cells cultivated in 1% alginate associated with 1.5% gelatin showed lower cell viability than alginate only and gelatin only. The lower viability may be caused by the higher stiffness promoted by the combination of alginate and gelatin. Final considerations: The obtained scaffolds can be combined with other biomaterials, such as synthetic polymers obtained by electrospinning, with the aim of further use in regenerative medicine of soft tissues, such as skin.

Keywords: Natural Polymers; Biomaterials.


USO DA LATRUNCULINA NO CONTROLE DE MICROORGANISMOS: APLICAÇÃO EM ENGENHARIA CELULAR.

Kohori, N. A.¹; Prudenciatti, A.¹; Da Silva, M. D. O.¹; Bovolato, A. L. D. C.¹; Garces, H. G.²; Golim, M. D. A.³; Da Cunha, M. D. L. R. D. S.⁴; El Sayed, K. A.⁵; Bagagli, E.²; Deffune, E.¹.

1Laboratório de Engenharia Celular, Hemocentro, Faculdade de Medicina de Botucatu, Brasil.2Laboratório de Biologia de Fungos, Departamento de Microbiologia, Instituto de Biociências de Bo-tucatu, Brasil.3Laboratório de Citometria de Fluxo, Hemocentro, Hospital das Clinicas de Botucatu, Brasil.4Laboratório de Bacteriologia, Departamento de Microbiologia, Instituto de Biociências de Botucatu, Brasil.5Departamento de Farmacologia, Universidade de Louisiana, Monroe, Estados Unidos.

A contaminação biológica em estufas de laboratório de cultura celular é um problema universal. A consequência dessa adversidade vai desde o comprometimento parcial das pesquisas até a perda da credibilidade da instituição. Para evitá-la, existem alguns produtos no mercado. Contudo, o uso desses compostos é contraditório nas publicações científicas e seu custo pode ser proibitivo. O au-mento exponencial do uso das técnicas de cultivo de células animais em biomedicina impulsiona as pesquisas para solucionar o desafio de identificar e validar o uso de novas moléculas com ação sobre microrganismos. A Latrunculina é um composto com propriedades antibióticas extraído da Negomba-ta magnifica, um porífero que habita o Mar Vermelho. Objetivo: A proposta desta pesquisa é avaliar o desempenho da Latrunculina, estimando sua concentração inibitória mínima, frente a um painel de fungos e bactérias ambientais mais prevalentes em ambientes laboratoriais de cultura celular ana-lisando curvas de crescimento de diferentes tipos de células animais após a exposição à Latruncu-lina. Metodologia: Este projeto teve início após aprovação do comitê de Ética em Pesquisa (CAAE: 71393417.8.0000.5411). Transcorridos os trâmites de autorização legal e ética foi realizado o moni-toramento diário das culturas celulares como previsto nos Procedimentos Operacionais Padrão dos dois Laboratórios Engenharia Celular do Hemocentro de Botucatu e do Centro de Venenos e animais Peçochentos – Cevap, com a parceria com o Laboratório de Microbiologia do Instituto de Biociências da UNESP de Botucatu, com expertises nas áreas de fungos e bactérias. A coleta de material foi diferenciada. Após a observação e suspeita de contaminação, fúngica ou bacteriana, as placas e ou frascos de cultura foram isolados no laboratório de Quarentena e transferidos para o Lab. de Micro-biologia para a coleta específica de cada material. Os fungos contaminantes foram coletados levando em consideração os estágios intermediário e avançado de contaminação, provenientes de pesquisas desenvolvidas nos laboratórios alvo. A coleta ocorreu em dois laboratórios distintos: Laboratório de Engenharia Celular do Hemocentro de Botucatu (LEC) e no Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP) da UNESP. Resultados e Discussão: Foram identificados, caracterizados por métodos clássicos e por biologia molecular os seguintes fungos: Aspergillus niger, Penicillium cap-sulatum, Aspergillus versicolor e Candida parapsiloses. A Concentração Mínima Inibitória (MIC) foi realizada comparando-se com anfotericina B e fluconazol (antifúngicos tradicionalmente utilizados na cultura celular) com Latrunculina A, A sintética e B. Para a realização da citotoxicidade foram utiliza-dos os kits de anexina V e iodeto de propídeo pelo método de citometria de fluxo. As análises eviden-ciaram que não houve alteração do controle auto-fluorescente quando as células foram expostas às concentrações de 5,10 e 100nM. O controle basal evidenciou, como esperado, alta taxa de apoptose e necrose. Conclusão: Observou-se que a exposição à Latrunculina A, A sintética e B controlou os processos de necrose celular com uma diminuição de 3 X em relação ao controle. Estes resultados apontam que a Latrunculina natural ou sintética pode ser usada como antifúngico e no controle das metástases.

Keywords: Concentração Inibitória; Controle De Qualidade; Latrunculina A/B; Cultura Celular.

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Abstract - Cell Therapy

AUTOLOGIC IMPLANT OF ACTIVATED DERMIC CELLS IN INVOLUTI-VE SKIN TREATMENT.

Civila, E.¹; Gonzalez Vazquez, B.¹.

1Asociación Uruguay De Medicina Regenerativa E Ingeniería De Tejidos, Uruguai.

A great variety of procedures employed in esthetics medicine promote the tissular stimulation throu-gh an injury with the purpose of reverting the alterations of cutaneous ageing. One of the most notable and studied is the phenol chemical peeling, which generates a stimulus mainly fibroblastic with subsequently generation of collagen. It is a biological treatment which through a deep exfolia-tion, activates the cellular and plasmatic mechanisms with skin’s repair and regeneration. Based on the above-mentioned, we developed a new technic with the purpose of reverting the involutive cutaneous process through regeneration of tissue in the receptive areas, without producing the cli-nical alterations of dermic injury caused by a deep peeling or a resurfacing laser, nor the histologic following the dermic repair visible in these treatments. This procedure consists of generating an important biological stimulus “in vivo” at a dermical level, through a phenolic peeling in a pinpointed small retroauricural area (donor zone). Afterwards, in an arranged in series way once a week, lumps of cellular material are removed from the mentioned area through tap with fine cannula, in order to do each time autologic implants with the obtained elements plus platelets growth factors in a dermic level, mainly facial (receiver zones). After three implants, after 21 days of starting the treatment, the main histologycal changes are observed at a dermic level, highlightening: 1)the existence of a cellu-lar infiltration with increase of vascularization and collagen in relation to the non treated skin, with conservation of elastic fibres, and 2)regarding the phenolic peeling a minor cellular infiltration and less production of collagen even though with fibres of similar characteristics of ordinary skin. After several sessions, is clinically visible a progressive improvement of the skin, characterized by the de-crease of the wrinkles and dryness, increase of tonicity and elasticity, and filling effect. It is evident a structural and functional recovery of the skin of a regenerative kind without elements of repair, with a proper clinical-histological correlation. It is described a new therapeutic model with the application of already existing procedures, orientated towards regenerative medicine and based on foundations of cellular biology.Regarding the indicated results we attribute particular importance to the interac-tion among, the different cellular types coming from the implant and residents(in both cases dermic cells),the extracellular matrix and the platelets growth factors, which doubtless, also take part in the process of tissue reactivating with regeneration of cutaneous tissue without elements of repair.The activation “in vivo”,extraction and subsequent stimulation of dermic cells with PRP, as well as the implants technic, implies a work methodology with minimal manipulation applicable in clinic.It is an outpatient procedure, very simple, of quick execution, without toxicity, without risks inherent to the implantation of foreign materials, and without the participation of the laboratory, with the exception of the preparation of PRP.The fact that there are no laboratory manipulations with cultures “in vitro” to increase the cells populations and to differentiate stem cells, has the advantage of avoiding risks and not contravene current legal regulations in that regard.Besides, this treatment is tolerated in an excellent way with an important patients adhesion.

Keywords: Activated Dermic Cells; Autologic Implant.


CELL THERAPY IN MODELS OF MODERATE AND SEVERE HEMOR-RHAGIC STROKES.

Mello, T. G.¹; Rangel Junior, W. S.¹; Almeida, R. S.²; Nascimento, M. A.¹; Pijuan, T. P.¹; Gutfilen, B.¹; Souza, S. A. L.¹; Rosado-De-Castro, P. H.¹; Mendez-Otero, R.¹; Pimentel-Coelho, P. M.¹.

1Universidade Federal do Rio de Janeiro, Brasil.2Universidade Iguaçu, Brasil.

Stroke is the second leading cause of death in the world and the third leading cause of disability. The disease represents the first cause of death and incapacity in Brazil, which generates great economic and social impact. Stroke is a multicausal disease and can be classified as hemorrhagic or ischemic, with hemorrhagic stroke being the second most common type, accounting for 10% to 20% of cases. The objective of this study was to evaluate the therapeutic potential of human Wharton’s jelly-deri-ved mesenchymal stem cells (MSCs) in models of moderate and severe intracerebral hemorrhage (ICH) in rats. Male Wistar rats were anesthetized and positioned in the stereotaxic apparatus. An incision was made in the midline of the scalp, bregma was located and the point with the following stereotactic coordinates was found: 3 mm anteroposterior and 0.2 mm medial-lateral. A craniotomy was made around this point, the cerebral parenchyma was exposed, 6 mm of the dorsal-ventral axis coordinate was subtracted and 0.10U or 0.25U of collagenase (models of moderate and severe ICH, respectively) was injected in the striatum. The animals were randomized to receive an intra-venous injection of 3 million MSCs or vehicle 24 h after the injury. A group of false-operated animals (sham) received an intrastriatal injection of saline. In order to evaluate the functional alterations due to ICH, two behavioral tests were performed: elevated body swing test (EBST) and Rotarod test, before surgery and for a period of 21 days after injury. To evaluate lesion volume, the animals were anesthetized and perfused transcardially with saline and 4% paraformaldehyde on the 22nd day. Magnetic resonance imaging (MRI) of animal heads was acquired on a 7T scanner. 3D gradient echo images were performed and data was processed using OsiriX software. The biodistribution of MSCs was evaluated in a separate cohort of animals by labeling the cells with 99mTechnetium (99mTc) before injection into ICH, sham, and naive animals. After 24 hours, the remaining radioac-tivity in each organ was measured in a gamma counter. The results of the behavioral tests showed no significant differences between the saline and MSCs groups after moderate ICH. There was no differences between the sham group and the MSCs or saline groups at almost all time points, indi-cating that the moderate injury did not cause significant functional impairments. However, compared to the saline group, the MSCs group exhibited a decreased residual hematoma volume assessed by MRI.The biodistribution analysis demonstrated that the organs containing larger quantities of 99mT-c-labeled MSCs after 24 h were the kidneys, lungs, spleen and liver, with no difference among ICH, sham and naive groups. Nevertheless, by analyzing the cerebral distribution of MSCs, we noticed that there was an almost two-fold increase in the quantity of MSCs in the ipsilateral hemisphere in relation to the contralateral hemisphere, only in the ICH group. Finally, in the severe model of ICH, we observed significant differences between the saline and MSCs groups at 20 days in the Rotarod and EBST tests, with the MSCs group exhibiting a better functional outcome. Both the reduction of lesion volume in the model of moderate ICH and the functional improvements in the severe-grade ICH model provide a good perspective for the development of novel therapies using MSCs, consi-dering that, as of yet, there are no specific treatment for ICH.

Keywords: Hemorrhagic stroke; mesenchymal stem cell; collagenase; magnetic resonance Ima-ging.

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ENGINEERING THE PLASMA MEMBRANE OF PANCREATIC BETA CELLS FOR A CELL-BASED TREATMENT IN T1DM.

Zamboni, F.¹; Collins, M. N.¹.

1University Of Limerick, Irlanda.

Type 1 diabetes mellitus (T1DM) results from a specific immune-mediated destruction of pancreatic β cells that leads to the complete loss of insulin production. Since the discovery of insulin in 1921, T1DM has been effectively treated, however, exogenous insulin replacement therapy still does not mimic the physiological pancreatic insulin secretion pattern, which results in the occurrence of life threating hypoglycaemic episodes and in the increase of incidence of macro and microvascular complications. Currently, T1DM treatment is shifting towards pancreatic β cell replacement for the restoration of responsive insulin secretion to blood glucose variations. Although the infusion of isolated islets through the portal vein of the liver is possible, the shortage of donors and the dan-gers of pharmacological immunosuppression makes it an unlikely approach for T1DM. Research is now being carried out to develop novel cell-based therapies for T1DM in order to isolate cells from the immune system and avoid the use of immunosuppression drugs, with the ambition of restoring insulin competence in patients with T1DM. Aims: In this landscape, we propose to immunoprotect pancreatic β cells by bioengineering their plasma membrane using PEG-lipids. Methods: Dipalmi-toyl-glycerol-phosphatidyl ethanolamine (DPPE) 20 mg and maleimide-polyethylene glycol-N-hy-droxysuccinimide (mal-PEG-NHS) 180 mg were dissolved in chloroform and stirred for 24 hours at room temperature to form mal-PEG-lipid. The product was purified by precipitation in diethyl ether followed by centrifugation (600 RPM), where the pellet is obtained after evaporation. Mal-PEG-Li-pids (20 mg) were labelled using 3.1 mg of fluorescein-5-isothiocyanate (FITC). Beta-TC-6 cells derived from pancreatic tumour (insulinoma) had their surface membrane modified by the insertion of FITC-mal-PEG-lipids. A suspension of cells (5x105) were incubated with 1,000 μM of FITC-mal--PEG-lipids for 120 minutes at 37°C. Cells were centrifuged (180 G) and washed with PBS twice. Cell membrane modification was confirmed using confocal microscopy. The integrity of the plasma membrane after the modification was assessed using annexin V labelled with Alexa Fluor 568. Results and Discussion: Palmitoyl was selected to be inserted into the lipid bilayer by hydrophobic adsorption because saturated fatty acids are more stable in the plasma membrane than unsaturated fatty acids; long fatty acids (above 13 carbons) also show to be more stable in the plasma membra-ne than short fatty acids and do not change the membrane fluidity like very long fatty acids (above 21 carbons); and palmitoyl is a native fatty acid of plasma membranes. Mal-PEG was also preferred to modify the plasma membrane over biotin-streptavidin because it is less immunogenic than the former. Surface engineering of pancreatic β cells was possible and showed no harmful effects to the cells. Final considerations: Next steps involve the additional coating of pancreatic β cells using hyaluronic acid derivates that are attached to the plasma membrane by reacting with the maleimide portion of the PEG-lipids. Coating stability and diffusion characteristics will be assessed to validate the coating as a barrier for the immune system while allowing the diffusion and release of glucose and insulin, respectively.

Keywords: Type 1 Diabetes Mellitus; Pancreatic Beta Cells; Immunoprotection; Surface Enginee-ring.


IMUNOMODULAÇÃO COM PLASMA RICO EM PLAQUETAS COMBI-NADO AO BACILLUS CALMETTE-GUÉRIN PARA O TRATAMENTO

DO CÂNCER DE BEXIGA NÃO-MÚSCULO INVASIVO.

Luzo, A. C. M.¹; Dias, L. P.¹; Volpe, B. B. V. B.¹; Durán, M.¹; Galdames, S. E. M.¹; Ferreira, L. A. B.¹; Durán, N.¹; Fávaro, W. J.¹ .

1Universidade Estadual De Campinas, Brasil.

O Plasma Rico em Plaquetas (PRP) é um lisado plaquetário que libera fatores de crescimento capazes de regenerar tecidos e modular a resposta imune através da via de sinalização dos recep-tores Toll-Like (TLR) 2 e 4. O câncer de bexiga urinária é a doença maligna mais comum do trato urinário, sendo que atualmente a terapia padrão ouro do câncer de bexiga urinária não-músculo invasivo (CBNMI) é a imunoterapia intravesical com BCG (Bacillus Calmette-Guerin) após res-secção transuretral do tumor. Contudo, a vacina BCG apresenta reações adversas de intensida-des variadas, desde sintomas irritativos leves até reação sistêmica grave, o que contribui para a interrupção do tratamento e/ou recorrência de tumor de até 30%. Objetivo: Associamos o PRP à imunoterapia com BCG no tratamento do CBNMI quimicamente induzido em ratos e avaliamos a resposta imune mediada pelos TLRs 2 e 4. Materiais e Métodos: O CBNMI foi induzido em ratos Fischer 344 fêmeas por tratamento com N-metil-N-nitrosourea (MNU). Após tratamento com MNU, os animais foram distribuídos em quatro grupos experimentais (n=10/grupo): grupo MNU (câncer), grupo MNU + PRP, Grupo MNU + BCG e grupo MNU + PRP + BCG, além do grupo controle (sem MNU) e controle + PRP. O PRP foi produzido a partir de sangue humano, os quais foram coletados 10ml de sangue total e centrifugado à 100xg, durante 10 minutos. O sobrenadante foi coletado para administração intravesical nos animais dos grupos predeterminados. Após o tratamento, amostras das bexigas urinárias foram submetidas às análises histopatológicas e imunohistoquímicas para os intermediários da via de sinalização dos TLRs (TLR2, TLR4, MyD88, TRIF, IRF-3 e IFN-γ). Resul-tados: O resultado das análises histopatológicas destes grupos experimentais mostrou 40%, 30% e 70% de inibição tumoral para os grupos MNU+BCG, MNU+PRP e MNU+PRP+BCG respectiva-mente. O perfil das imunorreatividades das proteínas TLR2, TLR4, MyD88, TRIF, IRF-3 e IFN-γ revelaram ativação do sistema imune inato nos grupos MNU+BCG e MNU+PRP+BCG. O grupo MNU+BCG o fez através da via dependente de MyD88, ao passo que no grupo MNU+PRP+BCG esta ativação ocorreu tanto pela via depende de MyD88 quanto pela via dependente de TRIF. Dis-cussão: A combinação das imunoterapias entre o PRP e BCG aumentaram os níveis proteicos de todos os antígenos estudados, que atingiram máxima imunorreatividade, mostrando uma modula-ção do sistema imune inato. A produção de citocinas como o IFN exerce ação antitumoral por indu-ção de TRAIL, um potente indutor de morte celular tumoral, sendo que o IFNγ é responsável pela estimulação de vias antiproliferativas e antitumorais em macrófagos e células tumorais. Conclusão: Os resultados obtidos, nas condições estudadas, demonstraram que o PRP xeno, não causou efei-to adverso, imunomodulando os animais. Sua associação ao BCG desencadeou melhor resposta de inibição do crescimento tumoral, quando comparada com a terapêutica de BCG e PRP isolados, podendo ser uma importante estratégia terapêutica para o CBNMI.

Keywords: PRP; Imunomodulação; Câncer.

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RECOMBINANT HUMAN GROWTH FACTORS AND MESENCHYMAL STEM CELLS DERIVED EXOSOMES FOR WOUND HEALING AND

SKIN REGENERATION.

Carreira, A. C. O.¹,²; Aguiar, B. A.¹,²; Fratini, P. C.²; Sogayar, M. C.¹,² .

1Núcleo De Terapia Celular E Molecular, Brasil.2Universidade De São Paulo, Brasil.

The regenerative medicine aims treatments to accelerate the different phases of tissue repair: blood clotting and inflammation, cell proliferation, tissue remodeling. In scarring and deficient tissues, there is an impairment of the ideal levels of growth factors in the wound healing phases. In chronic wounds, protease levels exceed that of their respective inhibitors, leading to destruction of extra-cellular matrix and degradation of growth factors and their receptors. By manipulating the growth factors composition, it is possible to accelerate or modify the process of regeneration and remo-deling of damaged tissues. Currently, Mesenchymal Stem Cells (MSCs) are considered to be pro-mising sources for various types of Cell Therapies in the Regenerative Medicine field. These cells display a regenerative potential and may be used for repair and maintenance of different tissue types, however, the mechanisms by which this repair activity is exerted is not well known yet. In this way, the application of MSCs or their microvesicles/exosomes and growth factors could contribute to the correct repair in a timely manner. First, this study aims to establish a model of dorsal wound healing in nude rats in order to test in vivo biological activity of recombinant human Platelet-derived growth factor (rhPDGF-BB) and Vascular endothelial growth factor (rhVEGF165), abundant fac-tors at Platelet-rich plasm (PRP), but produced in mammalian cells, and, further, use this model to evaluate the role of MSCs and their exosomes, combined with recombinant growth factors, to verify its therapeutic effect on the tissue regeneration. The model of dorsal wound healing was standardi-zed in nude rowett rats using four wounds with surgical punch of 6 mm diameter. rhPDGF-BB and rhVEGF165 were purified using heparin affinity chromatography, quantified by ELISA and 5ug were applied into each injury. The wounds were photodocumented and after seven days the wound skin was collected for histological analysis with Hematoxylin-Eosin, Picro-Sirius and Masson’s Trichrome to evaluate the new tissue formed. The results showed comparative efficacies in both treatments of wounds with rhPDGF-BB or rhVEGF165, with an accelerate cicatrization rate in comparison with the injury without treatment or treated with only the vehicle (saline solution). To improve the cicatrization, for the next step we isolated exosomes from MSCs. We standardized a protocol with three steps: ultrafiltration (10kDa cut), Total Exosome Isolation Reagent kit and ultracentrifugation at 120.000g. The material isolated was evaluated by Western blot assay using CD81 antibody and the presence of exosomes was detected at 26kDa. This material will be applied combined with re-combinant growth factors, described before, at injuries to evaluate its wound potential. We waiting to develop and offer an improved alternative and approach to wound healing treatment for patients and animals with complex chronic ulcers and other damage in the skin, which are difficult to treat and associated with high treatment costs.

Keywords: Wound Healing; PDGF-BB; VEGF; Mesenchymal Stem Cells; Exosome.


RECOMBINANT HUMAN GROWTH FACTORS (PDGF, TGF-Β, VEGFS, BMPS) AS AN EFFECTIVE ALTERNATIVE TO PLATELET-RICH PLAS-

MA (PRP) FOR TISSUE REGENERATION.

Carreira, A. C. O.¹,²; De Paula, G. C. G. M.¹,²; Aguiar , B. A. ¹,²; Campos , R. D. A.²; Levin, G.¹,²; Bel-chior, G. G.¹,²; Sogayar , M. C.¹,² .

1Núcleo de Terapia Celular e Molecular, Brasil.2Universidade de São Paulo, Brasil.

Platelet Rich Plasma (PRP) is a blood cell derived product obtained by centrifugation, which contains a high concentration of growth factors in a small volume of plasma. Its major components include: Platelet-derived Growth Factors (PDGF), Transforming Growth Factors (TGF-β 1 and 3), Fibroblast Growth Factors (FGF) and Vascular Endothelial Growth Factors (VEGF) and Bone Morphogenetic Proteins (BMPs), responsible for the osteoinductive activity of the organic bone matrix identified as being members of the TGF-β family. By applying PRP at the site of injury, a robust healing response is achieved. Since 1950s, PRP application has been widely used in several areas such as Orthope-dics, Dentistry, Cardiology, Ophthalmology, Dermatology for use in complex chronic wound repair, especially in diabetic patients or patients with venous complications, mucosal wounds, connective tissues, pathologies of the musculoskeletal system, fracture surface, bone graft, among others. In scarring and deficient tissues, there is an impairment of the ideal levels of growth factors in the wou-nd healing phases. Therefore, it is important to maintain adequate levels of growth factors in all the different stages of tissue repair. The preparation of PRP is a non-standard procedure and variable results are being observed in the literature, due to the amount of growth factors and/or methods of preparation. Besides that, usually more than one PRP application is required involving a new blood collection for each application in order to accelerate tissue regeneration. The blood collections are invasive for the patient, being expensive in time and labor in the operational procedure, implying in the increase of the cost. The early preparation of PRP with possibility of storage for use in future applications during treatment could be an important solution to avoid the discomfort of patients; however, the problem of the absence of a gold protocol stays. Recombinant growth factors were produced in a safe and reproductive heterologous system using mammalian cells (HEK293, 293T, CHO) to obtain overproducing cell clones, to guarantee their quality and post-translational modifi-cations, which are important for their production or stability. In sequence, the growth factors were purified using heparin affinity chromatography and tested using a specific in vitro biological activity assays for each growth factor. The rhPDGF-BB, rhVEGF, rhTGF-β growth factors application and human PRP were tested in a model of dorsal wound healing in rats or mice (normal and diabetic animals induced with streptozotocin). rhBMPs were applied subcutaneously to induce ectopic bone formation in nude rats. The results showed comparative efficacies in the treatments of wounds with rhPDGF-BB, rhVEGF, rhTGF-β in comparison with PRP application, without significant differences, indicating that the use of recombinant growth factors are so effective as the application of PRP for the tissue regeneration process, as observed in the new tissue with collagen deposition and gra-nulation tissue formation. This work shows the possibility of substitute the PRP application using a safe, effective and reproductive source of growth factors using a mammalian cell platform.

Keywords: Platelet-Rich; Platelet; Tissue Regeneration; Wounds Healing; Growth Factors; Bone Morphogenetic Protein; Platelet-Derived Growth Factor.

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USE OF URINE PROGENITORS CELLS FOR IN VIVO RENAL REPAIR IN A NEW NON-INVASIVE RAT KIDNEY INJURY MODEL USING HIGH

SODIUM OXALATE-DIET.

Crestani, T. A.¹; Steichen, C.¹; Crajoinas, R. O.¹; Jensen, L.¹; Dima, L. L.¹; Girardi, A. C.¹; Krieger, J. E.¹.

1Laboratório De Genética e Cardiologia Molecular, Instituto Do Coração, Universidade De São Paulo, Brasil.

Chronic Kidney Disease (CKD) is a major worldwide public health issue affecting 14% of the general population. Despite the elevated costs in dialysis and extended waiting list for renal transplantation, the research focusing on CKD mechanisms and treatment is limited. Moreover, animal models ac-curately recapitulating the human physiopathology of the disease with systemic complications are limited. We propose a new non-invasive model of CKD in rats and assessed the potential benefits of human Urine Progenitors Cells (UPC) injection to trigger kidney repair. 25 male Wistar rats 7 weeks-old, 250-300g, were obtained from animal care of Heart Institute (São Paulo, Brazil). They received a diet with low calcium concentration (control diet) plus 50 μmol/g of sodium oxalate (OXA diet) for 3 weeks to induce CKD. Control rats received only the control diet. After the induction of the renal dysfunction, we injected UPCs (106 cells / per animal) directly in the renal parenchyma (10 injection points) or under the renal capsule. 7 weeks after injection, to assess renal function, the animals were left for 24 hours in the metabolic cage and had their urine and blood collected. Blood gases, caudal pressure, echocardiography and histological analyzes were evaluated. First, we analyzed the effects of OXA diet and showed that, compared to control rat, rats treated with OXA diet developed a stable CKD with a 60% reduction of the Glomerular Filtration Rate (GFR), elevated blood urea levels and proteinuria. Histological analyzes showed an important tubular atrophy and deposition of collagen, proving a high cortical disorganization and fibrosis, besides the accumulation of OXA crystals in the atrophied tubules. Rats treated with OXA diet also displayed classical com-plications of CKD, such as elevated blood pressure and reduced hematocrit. In addition, functional cardiac analyses revealed that OXA diet triggered diastolic dysfunction, presented with higher E/e’ ratio accompanying significant cardiac hypertrophy. In addition, we showed that both types of UPC injections increased significantly GFR and prevent the GFR decrease after 7 weeks of treatment. The rats treated with injected cells in the renal parenchyma showed an increase in blood urea levels and reduction in blood pressure, reversing completely the effects caused by OXA diet. Altogether, our results showed the feasibility to use a convenient and non-invasive strategy based on a diet rich in sodium oxalate to induce CKD and its classical systemic complications in rats. As this rat model does not involve kidney mass loss or acute toxicity it has a strong potential for research on CKD me-chanisms and treatments. Finally, preliminary results of UPC injection provide evidence that these cells may contribute to improve kidney function and systemic complication, such as blood pressure.

Keywords: Chronic Kidney Disease; Rat Model; Oxalate; Human Urine Progenitors Cell Cardiac Function.

Abstract - Células-Tronco Pluripotentes

ENTENDENDO O MICROAMBIENTE CARDÍACO: CARACTERIZAÇÃO DO SECRETOMA DE CÉLULAS-TRONCO EMBRIONÁRIAS DURAN-

TE A DIFERENCIAÇÃO CARDIOMIOGÊNICA IN VITRO.

Kulig, A. W. R.¹, Pereira, I. T.¹, Dallagiovanna, B.¹, Stimamiglio, M. A.¹.

1Instituto Carlos Chagas, Brasil.

Avanços nas terapias celulares têm demonstrado que muitos dos efeitos observados são conse-quência da secreção de fatores parácrinos pelas células implantadas. Assim, algumas abordagens vêm sendo pesquisadas na busca por uma terapia livre de células. Porém, inicialmente, é preciso aprimorar os conhecimentos a respeito dos nichos teciduais para entender como os fatores in-fluenciam as células locais. Neste sentido, há grande interesse em entender a composição dos diferentes nichos (compostos principalmente de fatores solúveis e matriz extracelular-MEC) e como estes podem modular as respostas de diferentes tipos celulares. Com relação ao nicho cardíaco, ainda existem vários aspectos que precisam ser elucidados, como, por exemplo, a caracterização dos sinais secretados que podem modular o comportamento das células progenitoras do cora-ção. Para entender o microambiente cardíaco, uma das alternativas é caracterizar as moléculas secretadas durante o processo de diferenciação cardiomiogênica, permitindo a identificação de potenciais moléculas que possam contribuir para o reparo de um tecido lesionado. Assim, o obje-tivo deste trabalho é caracterizar o microambiente e as sinalizações celulares presentes durante o comprometimento das células-tronco embrionárias (CTEs) à linhagem cardíaca. As CTEs foram induzidas a diferenciação cardiomiogênica e, em diferentes pontos durante o processo (D1, D4, D8/D9 e D15 – referentes aos dias de diferenciação e surgimento de progenitores), foram coletados os meios condicionados (MCs) e a MEC. Esta última foi obtida através da descelularização dos corpos embrióides formados no processo de diferenciação cardíaca. A composição da MEC e dos MCs foi avaliada por espectrometria de massas e arranjo de anticorpos. Identificamos em torno de 40 proteínas de MEC moduladas nos diferentes tempos analisados e 20 fatores de crescimento e/ou citocinas nos MCs. Muitas destas proteínas estão relacionadas com o desenvolvimento embrioná-rio normal e são necessárias ao processo de diferenciação celular. Estes resultados foram comple-mentados com a análise de dados prévios de sequenciamento de mRNAs também obtidos durante a diferenciação cardiomiogênica. Por esta técnica, identificaram-se mais de 150 genes relaciona-dos a proteínas extracelulares reguladas ao longo do processo de diferenciação. Funcionalmente, avaliamos os MCs em cultivo com células H9c2, células-tronco mesenquimais derivadas de tecido adiposo e células endoteliais, quanto a manutenção da viabilidade e a proliferação celular. Embora nenhum dos meios seja tóxico para os cultivos, observamos diferenças na resposta de um mesmo MC frente aos diferentes tipos celulares. Porém, para entender mais profundamente que tipo de alterações ou influências os MCs podem estar promovendo, novos ensaios são necessários. Este é o primeiro trabalho a caracterizar o secretoma durante a diferenciação cardiomiogênica in vitro. Acreditamos que as abordagens utilizadas neste trabalho fornecem ferramentas para o entendi-mento do microambiente cardíaco, uma vez que as caracterizações realizadas potencialmente contribuem com futuras investigações de proteínas ou vias de sinalização específicas que possam ser moduladas na tentativa de promover a regeneração do coração após lesão.

Keywords: Diferenciação Cardiomiogênica; Secretoma; Células-tronco Embrionárias; Fatores So-lúveis; Matriz Extracelular.

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RESPOSTA IMUNE DURANTE A DIFERENCIAÇÃO DE CÉLULAS-TRON-CO NEURAIS EMBRIONÁRIAS OBTIDAS A PARTIR DE CAMUNDONGOS

APPSWE/PS1DE9, UM MODELO DA DOENÇA DE ALZHEIMER.

Vidal, T. R.1, Pillat, M. M.1.

1Universidade Federal de Santa Maria, Brasil.

A neuroinflamação e a neurogênese anormal são processos inerentes à patogênese da Doença de Alzheimer (DA) e, consequentemente, os sinais que promovem esses processos podem atuar como desencadeadores deletérios iniciais. Além disso, estudos apontaram que as proteínas envol-vidas em doenças neurodegenerativas como a APP (proteína precursora amilóide) e PS (proteína presenilina) diversas vezes desempenham papéis críticos no desenvolvimento precoce do sistema nervoso central (ROGERS e SCHOR, 2010). A familiaridade da DA está associada a um padrão de herança monogênica autossômica dominante (SMITH, 1999). Nesses casos, os quais correspon-dem a até um décimo dos pacientes com DA, em geral, ocorre acometimento precoce, aproximada-mente, na quarta ou quinta década de vida (BERTRAM e TANZI, 2005; SELKOE, 2004). Contudo, não há conhecimento se as alterações na inflamação começam nos estágios embrionários dos portadores de mutação associados à DA familiar. OBJETIVO: Investigar a questão da ocorrência ou não da inflamação na fase embrionária através de camundongos duplos transgênicos, modelo da DA familiar (APPswe/PS1dE9). METODOLOGIA: As células-tronco neurais (também denominadas células progenitoras neurais- NPCs) foram obtidas a partir do telencéfalo de embriões selvagens (WT) no décimo terceiro dia de gestação (E13) e de embriões APPswe/PS1dE9, também no mes-mo período de desenvolvimento embrionário. Essas células proliferam como agregados chama-dos neuroesferas na presença de EGF e FGF-2, mantendo a capacidade de se diferenciarem em neurônios e células gliais mesmo após a retirada desses fatores do meio de cultura. Por meio da tecnologia de microarrays, foram estudados os perfis de expressão gênica do genoma nas células diferenciadas in vitro, obtidas de telencéfalo de embriões WT e APPswe/PS1dE9. RESULTADOS E DISCUSSÃO: Foi observada a ocorrência de uma forte resposta imune com ativação microglial em células diferenciadas obtidas de embriões APPswe/PS1dE9. Conforme a expressão do mRNA de oito genes de inflamação presentes em células APPswe/PS1dE9, verificada através de microarray e RT-qPCR, as células obtidas dos embriões APPswe/PS1dE9 expressaram cerca de 100 vezes mais CCL12, CCL5 (RANTES), CCL3, C3, CX3CR1, TLR2, Iba e TNF do que as células obtidas a partir de embriões WT. Ainda, o secretoma das neuroesferas diferenciadas APPswe/PS1dE9 induziu quimioatração significativa de células mononucleares de sangue periférico (PBMCs) em comparação com o secretoma de células WT, evidenciada pelo ensaio de migração Transwell. Nesse ensaio, realizado em triplicata e com quatro animais em cada grupo (teste t de Student; *** p ˂0.001), os núcleos marcados com DAPI foram utilizados para a contagem de células. CON-CLUSÃO: Dessa forma, os resultados sugeriram que as proteínas APPswe e PS1dE9 mutantes, envolvidas na DA familiar neurodegenerativa, desempenham papéis críticos no desenvolvimento precoce do sistema nervoso central, causando inflamação e ativação microglial.

Keywords: Células-tronco; Alzheimer; Neuroinflamação; Appswe/PS1dE9.


ELECTRICAL STIMULUS AS A KEY COMPONENT FOR MATURATION AND CHARACTERIZATION OF HUMAN INDUCED PLURIPOTENT

STEM CELL-DERIVED CARDIOMYOCYTES.

Crestani, T. A.¹; Steichen, C.¹; Rodrigues, M. V.¹; Vilella-Arias, S. A.¹; Turaça, L. T.¹; Neri, E.¹; Krie-ger, J. E.¹.

1Laboratório de Genética e Cardiologia Molecular, Instituto do Coração, Faculdade de Medicina da Universidade de São Paulo, Brasil.

After an acute myocardial infarction (AMI), cardiomyocytes (CMs) are eliminated and replaced by fibrous tissue with low contractile capacity. The use of human-induced pluripotent stem cells (iPS-Cs) is a promising strategy to restore cardiac function, since they can differentiate into all cell types from the human body, including CMs (iPSC-CMs). However, the complete characterization of cells generated during differentiation as well as the choice of the optimal cell population to use for car-diac repair need to be further assessed. The objectives of this work are: 1) to investigate the effect of electrical stimulus (ES) on the differentiation or maturation process of iPSC-CMs; 2) to evaluate if ES can help to define markers to determine the best iPSC-CMs phenotype to use for therapeutic strategies or disease modeling. iPSCs were reprogrammed from cells isolated from urine and fibro-blasts of healthy individuals from the Heart Institute (São Paulo, Brazil). iPSCs were characterized according to morphology, presence of molecular and protein markers, differentiation capacity in the three germinative lines and chromosome integrity. Then, they were differentiated into cardiomyocy-tes directly in the culture plates (control group) or with ES from day 0 to day 3 of the differentiation (ES group) and were characterized by molecular, cellular and functional parameters. iPSCs clones presented small, juxtaposed, monolayer cells with very well defined margin and expression of pluri-potent genes (SOX2, OCT3/4, NANOG and REX 1). Almost all iPSCs population is positive for pluri-potency markers such as NANOG (98.74%) and SOX2 (97.71%). Immunofluorescence showed the presence and correct localization of pluripotency nuclear (SOX2, NANOG, OCT3/4) and membrane markers (SSEA-4, TRA-1-60, TRA-1-81). 1 iPSC clone formed teratoma when injected in imuno-deficient mice and through histological analysis it was possible to verify the presence of the 3 germ layers. The 3 iPSCs clones that presented normal karyotype were success differentiated into car-diomyocytes and spontaneously beating iPSC-CMs were observed at 8 to 10 days after beginning of differentiation. iPSC-CMs with ES presented contractile cells 71% of the time, while control iPS-C-CMs only 57%. The percentage of positive cells for cardiac progenitor (NKX2-5) was increased for ES group at day 6 of differentiation, while GATA4 and troponin positive cells were higher at day 15. Moreover, ES group showed higher expression of GATA4 and cardiac genes (TNNI3, CALM3 and MYL2) at day 15 as examined by qPCR. Regarding functional parameters, typical calcium (Ca) handling parameters, such as Ca transient amplitude, time to peak and Ca decay rate were signifi-cantly higher in iPSC-CMs with ES compared to the control. In conclusion, these results show that ES can enhance cardiac differentiation efficiency of iPSCs, upregulate cardiac genes and promote functional CMs maturation. This study may contribute to increase the efficiency of the differentiation process of hiPSCs into CMs, and to identify best markers for testing therapeutic iPSC-CMs (after AMI, for example) or disease modeling.

Keywords: Human-Induced Pluripotent Stem Cells; Cardiac Differentiation; Cardiomyocytes; Elec-trical Stimulus.

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OPTIMIZATION OF CONDROCYTES IN 3D CULTURE SYSTEM FOR TISSUE ENGINEERING.

Duailibi, S. E.¹; Santos, J. A.¹; Mironov, V.²; Rezende, R. A.²; Silva, J. V. L.²; Duailibi, M. T.¹.

1Universidade Federal de São Paulo, Laboratório de Engenharia Tecidual e Biofabricação, Brasil.2Divisão de Tecnologias Tridimensionais do Centro de Tecnologia e Informação Renato Archer, Brasil.

Tissue Engineering, is also known as a field of Regenerative Medicine. Condrocytes 3D culture is necessary to obtain a sufficient number of cells for cartilage bioengineered in Bioprinter system. However, the current evidences demonstrate that cellular microenvironment direct cell behavior through influences in signaling pathways. Cells behave more like origin tissue when cultured in three-dimensional environments. The growth of cells in a 3D culture system can optimize uniform tissue spheroids which are chondrospheres or building blocks for fabrication cartilage which can be used in bioprinter in order to provide a natural environment by an additional dimension for exter-nal signals, receptor binding, ECM interactions and ability to design and develop the construction of organs and living microtissues with the deposition layer by layer through spheroids. Objective: Develop a 3D cell culture system for optimizing the cartilage bioengineered. Method: The auricular cartilage cells were cultured until the fourth passage. Agarose mold 2% through were made the mold Microtissue 3D Sigma Aldrich® and placed in 12 well plates , and later added the middle were added to 5x105 cells and maintained in culture until the fourteenth day, where they were centrifuged and posteriorly placed into microtubes 1,5ml marking the spheroids with Mitotracker to assess mi-tochondrial function, hoesth to assess the nucleus and alexxa fluor 488 to analyze the cytoskeleton formation when the cell dispose in sphere form. Results: The analyzes performed in fluorescence microscopy Zeiss® showed that after the formation of the spheroid core remained integrate the active mitochondrial activity and disposition of the cytoskeleton was observed very clearly due to condensation of the extracellular matrix that formed the spheroid. Conclusion: The cells placed in the mold agarose Microtissue 3D Sigma Aldrich® were agreggated up over the days and formed uniform and intact spheroids and demonstrate to be useful for further bioprinter application.

Keywords: 3D Culture System; Condrocytes; Spheroids; Tissue Engineering.


PERFORMANCE OF HUMAN PULP STEM CELLS UNDER 3D CULTU-RE OF HYDROGEL MODIFIED BY PROTEINS.

Duailibi, M. T.¹; Santos, J. A.¹; Duailibi, S. E.¹.

1Universidade Federal de São Paulo, Centro de Terapia Celular e Molecular, Laboratório de Enge-nharia Tecidual e Biofabricação, Brasil.

Human Dental Stem Cell (hDSC) 3D culture is necessary to obtain a sufficient number of cells which allows the tissue engineering of tissues or organs in the Regenerative Medicine. However, current evidences demonstrate that cellular microenvironment direct cell behavior through influen-ces in signaling pathways. Cells behave more like origin tissue when cultured in three-dimensional environments, once in 2D cell culture only a side of the cell’s membrane is in contact with the extra-cellular matrix (ECM) and neighboring cells. The growth of cells in a hydrogel 3D provides a natural environment by an additional dimension for external signals, receptor binding, ECM interactions. In fact, hydrogels are not considered a simple support scaffold but, a complex dynamic biomaterial en-vironment. Objective: Study cell behavior under three combinations of hydrogels and proteins in vi-tro. Methods: Primary mesenchymal cells were obtained from molar tooth buds. Three combinations of hydrogels and proteins were used to cell culture. Namely: Hydrogels with laminin (HdxL), bone marrow (HdxBM), fibronectin (HdxF) and a control group (HdC) with only hydrogel. Proliferation and morphology were evaluated. Results: Morphologically, all groups presented calcified nodules which indicate cell differentiation. The results of cell proliferation demonstrated that HdC group had the hi-ghest cell number loss compared to the other groups, a decrease of 50% between the 7th and 28th day. In contrast, the HdxBM group showed the highest cell proliferation rate, an increase of 17,5% between the 7th and 28th day. In the same period, the HdxL group maintained the cell number and the HdxF group presented a decrease of 18,6%. Conclusion: Proteins presence in 3D cell cultures modifies interactions between culture mean and cells, influencing the cell proliferation rate of the cells under study. Future challenges involve understanding the relationship between cells and pro-teins for the future establishment of 3D culture with adequate properties.

Keywords: 3D Culture; Hydrogel; Human Dental Stem Cells.

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Abstract - Mesenchymal Stem Cells/Adultas

ADSC-DERIVED EXOSOMES IMPROVED SEQUELS OF FOCAL IS-CHEMIA VIA INTRANASAL ADMINISTRATION 24 HOURS AFTER THE

INSULT

Rohden, F¹; Teixeira, L. V.¹; Gionbelli, M. P.¹; Schafhauser, D. D. C.¹; Martins, L. A. M.¹; Guma, F. C. R.¹; De Souza, D. O. G.¹.


Brain ischemia is the main cause of death worldwide. Studies with stem cells have shown great po-tentiation of their paracrine action, which occurs through microvesicles and exosomes released by them, thus doing cell-cell signaling. Researches reveal that the use of exosomes in brain ischemic stroke have shown very promising results. Aims: We investigate the effect of exosomes secreted by mesenchymal stem cells derived from adipose tissue (ADSC) in the recovery of rats induced to focal cerebral ischemia. Methods: We use exosomes originated from ADSC culture (LONZA) and previously characterized (purity, size and concentration). Focal cerebral ischemia was induced by thermo coagulation of pial vessels in adult Wistar rats (CEUA 31888) and the treatment performed after 24 hours of ischemic insult. Animals received intranasal 200 μg / kg of exosomes or PBS. The sensorimotor recovery was evaluated by cylinder test 24 hours before the injury, 72 hours after and weekly until the 42nd day post injury. The anxiety-like activity was evaluated by elevated plus maze test on the 7th day after treatment. Long-term habituation memory was analyzed by open field test on days 7, 21 and 42 after treatment. Results: Treatment with exosomes showed significant reco-very in symmetry (p <0.0001) and anxiolytic-like effect in ischemic animals. However, long-term me-mory was not affected. Final considerations: Currently, stem cell research focuses in its paracrine action. In this work we demonstrate the recovery, mainly in motor function, of animals that suffered cerebral ischemia and were treated with exosomes released by ADSC, evidencing the great impor-tance of this line research in the regeneration of ischemic.

Keywords: Brain Ischemia; Mesenchymal Stem Cells Derived From Adipose Tissue; Exosomes.


ANÁLISE DA CONTRIBUIÇÃO DO LED NOS COMPRIMENTOS DE ONDA DE 630 E 850NM EM DIFERENTES FLUÊNCIAS NA CULTURA

DE CÉLULA-TRONCO MESENQUIMAL.

Bovolato, A. L. D. C.¹; Garcia, H. V.¹; Passian, A. C.¹; Nunes, H. C.¹; Ferreira, R. R.²; Inada, N.³; Bagnato, V. S.³; Golim, M. D A.⁴; Moroz, A⁵; Deffune, E.¹.

1Laboratório de Engenharia Celular, Hemocentro, Faculdade de Medicina de Botucatu, Brasil.2Departamento de Ciências Biologicas, Faculdade de Ciências de Bauru, Brasil.3Centro de Pesquisa em Óptica e Fotônica, Instituto de Física de São Carlos, Brasil.4Laboratório de Citometria de Fluxo, Hemocentro, Hospital das Clinicas de Botucatu, Brasil.5Departamento de Bioprocessos e Biotecnologia, Faculdade de Ciências Farmacêuticas de Araraqua-ra, Brasil.

A Medicina Regenerativa (MR) é uma área em crescente expansão no Brasil e no mundo, a qual procu-ra ampliar a capacidade natural de regeneração dos tecidos através da utilização de células, fatores de proliferação e biomateriais. Um dos ramos da MR é a terapia celular, vertente que utiliza células-tronco (CT), visando a substituição de tecidos funcionalmente ou estruturalmente lesados, apresentando um caráter terapêutico. Na medicina LASERs e LEDs (Light Emitting Diode) vem sendo estudados como ferramenta terapêutica, mostrando possuir capacidade bioestimulatória. Na técnica de fotoestimulação, utiliza-se a luz para ativar moléculas e funções celulares, apresentando o potencial de afetar a proli-feração e diferenciação e o metabolismo da célula, estimulando a fosforilação oxidativa e podendo reduzir a resposta inflamatória local. Entretanto para que essa resposta ocorra, é importante a seleção de um comprimento de onda ideal, uma vez que a utilização de um comprimento inapropriado pode acarretar em resultados contrários aos esperados, como a bioinibição. A aplicabilidade do LED como agente bioestimulador tem sido proposta. O objetivo deste estudo foi avaliar a ação da luz contínua e pulsátil, 630nm e no regime pulsátil a 850nm, para ambos, nas doses de 1, 3, 5 e 10J/cm² avaliando a ação sobre células obtidas pelos métodos de dissociação mecânica (DM). Metodologia: Foram reti-rados blocos de tecido adiposo (TA) de ratos da linhagem Wistar com idade média de 3 meses. Para isolar e caracterizar as CT foi realizado o perfil fenotípico utilizando a técnica de citometria de fluxo com os marcadores CD90/CD44/CD71/CD31/CD11b/CD45. Estabeleceu-se a curva de confluência à 80% entre as passagens além da determinação das citocinas TNF, IFN-ɣ, IL-4 e IL-10. Foram realizados ensaios de micronúcleo e cometa para análise de mutagenicidade e genotoxicidade. Resultados e Dis-cussão: O LED, emitindo luz em 630 nm, nas 4 doses aplicadas (1, 3, 5 e 10 J/cm2), sob luz contínua e pulsátil, determinam uma bioestimulação das CTMs cultivadas in vitro com evidente estímulo à pro-liferação celular. A análise do tempo de confluência a 80% (desdobramento) entre as passagens teve efetiva bioestimulação por influência do LED, em especial nas amostras de DM. Esta ação foi menos evidente nas amostras enzimáticas. Os marcadores imunofenotípico tiveram desempenho esperado. As citocinas identificadas no pós-experimento nas doses de 1 e 3 J/cm2foram IL-4 e IL-10 que possuem atividade anti-inflamatória. A análise de dano do DNA por genotoxicidade, realizada pelo teste cometa, evidencia a ação benéfica da luz no reparo de danos no DNA. No entanto, o regime de irradiação a 3 J/cm2 nos dois regimes de luz induziu ao dano de DNA. O dano de DNA (mutagenicidade) avaliado pelo teste do micronúcleo mostrou que as doses de irradiação de 1 J/cm2 contínuo e/ou pulsátil e 3 J/cm2 luz contínua induziram ao dano irreversível de DNA. Para o regime de 850nm, pulsátil, o desdo-bramento celular mais lento e acarretou na diminuição da confluência celular, especialmente nas doses de 5 e 10J/cm². Na cultura de linfoma Linfoblástico tipo B, em apenas 1 semana de exposição o mesmo comportamento de bioinibição foi encontrado na dose de 10J/cm². As técnicas de fotônicas podem ser usadas para bioestimulação ou bioinibição da linhagem Raji e CTM in vitro, na dependência do regime de luz apropriado.

Keywords: Célula-Tronco Mesenquimal; Terapia Celular; Bioestimulação; LED.

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ANALYSIS OF LIMBAL MESENCHYMAL STEM CELLS EXPANDED ON HUMAN DENUDED AMNIOTIC MEMBRANE PORCINE SMALL IN-

TESTINE SUBMUCOSA SCAFFOLDS.

Naasani, L. I. S.¹; Rodrigues, C.¹; Azevedo, J. G.¹; Damo, A.²; Buchner, S.³; Wink, M. R.¹.

1Univesidade Federal de Ciencias da Saúde de Porto Alegre, Brasil.2Irmandade da Santa Casa de Misericórdia de Porto Alegre, Brasil.3Universidade Federal do Rio Grande do Sul, Brasil.

One of the main limitations in the treatment of corneal injuries is that the number of patients who needs this tissue exceed the availability of donors. These limitations have motivated the develop-ment of strategies using the new technological advances in tissue engineering. Traditional tissue engineering strategies involve the combination of cells, growth factors, and scaffolds that can supply cellular biological components allowing to restore the tissue function. The mesenchymal stem cells found in the limbal stroma (L-MSCs) have a self-renewal potential for multilineage differentiation and immunosuppressive properties that aid them to surviving from host immune rejection, which justifies its use for allograft applications and a suitable biocompatible scaffold that does not alter the cellular properties is necessary to transport these cells to the injured area and help to promote the regeneration. Thus, in this work we isolated the L-MSCs from human sclerocorneal rims and evaluated their potential for multilineage differentiation and the expression of mesenchymal surface markers. These cells were seeded on human amniotic membrane (hAM) and porcine small intesti-ne submucosa (SIS) and we analyzed their viability, actin cytoskeleton, cell density and adhesion, nuclei morphology, and surface markers. Our results showed that cells adhered and integrated on both membranes with a high cell density, an important characteristic for cell therapy. In addition, the analysis of surface markers expression on L-MSCs after two weeks showed a slight increase in the percentages of negative markers for MSCs grown on SIS membrane. Thus, considering a long--term culture, the hAM was considered a better scaffold to maintain the MSCs phenotype. However, regarding the function as scaffolds, SIS was as efficient as the amniotic membrane, considering that these two types of biological matrices maintained the cell viability, actin cytoskeleton, nuclei morphology and mesenchymal phenotype, without causing cell death. Therefore, our data in vitro provide evidences for future pre-clinical studies where these membranes can be used as a support to transport mesenchymal stem cells to the injured area, creating a type of temporary curative, allowing the release of bioactive molecules, such as cytokines and growth factors and then promo-ting the tissue regeneration, both in human and veterinary medicine.

Keywords: Amniotic Membrane; Porcine Small Intestine Submucosa; Limbal Mesenchymal Stem Cells; Scaffolds; Sclerocorneal Rims.


APICAL PAPILLA STEM CELLS: CHANGES IN PROTOCOL ISOLA-TION AND NEW DISCOVERIES.

Macedo, L. M.¹; Silva, L. A. G.¹; Silva, A. C. G. D.²; Valadares, M. C.²; Lima, E. M.²; Kitten, G. T., Castro, C. H. D.¹; Gava, E.¹.

1Departamento de Ciências Fisiologicas, Universidade Federal de Goiás, Brasil.2Faculdade de Farmácia, Universidade Federal de Goiás, Brasil.3Departamento de Morfologia, Universidade Federal de Minas Gerais, Brasil.

Dental apical papilla is an important source of stem cells and the establishment of a protocol to pre-serve the stem cell niche is very important for the development of effective therapies. Therefore, the objective of this study was to establish an innovative propose to stem cell from human apical papilla (SCAPs) isolation aiming to preserve the stem cell niche in in vitro environment. Ten healthy donors aged 13–16 years were invited to donate third molar teeth and the apical papilla region was col-lected. To evaluate the influence of enzymatic digestion in cell migration, explants were submitted to different concentrations of Type I Collagenase (Col), Dispase (Dis) and TrypLE™ Express. The enzymes were neutralized with DMEM/F12 supplemented with fetal bovine serum (FBS). Stem cell markers (CD90, CD44, CD146, p75, STRO-1, CD14, and CD19) were analyzed by flow cytometry. Proliferation and migratory capacity of SCAPs were assessed in a three-dimensional (3D) culture system of Type I Collagen gel matrix. To investigate the influence of the serum supplementation medium, SCAPs were maintained in DMEM/F12 supplemented with (1) 10% fetal bovine serum, (2) 10 % human serum (HS), or (3) 2% HS plus 1% insulin-transferrin-selenite media (ITS). Cell migration from explants incubated in Col 0.5 mg/ml began at 7 days with complete extracellular ma-trix (ECM) preservation. In TrypLE group, the cell migration was observed at 14 days with complete ECM preservation until 21 days. Col 3.0 mg/mL and Col/Dis caused the complete ECM disruption at 21 days and 14 days respectively. Cells expressed positively for CD90, CD44, p75 and CD146, and negatively for STRO-1, CD14 and CD19. Development of 3D culture system was able to support papilla niche, with cells migrating continuously after consecutives passages of the same explant, suggesting the presence of cell niches. HS and HS plus ITS groups presented an innate ability to form spheroid-like clusters with fibroblast- or neuro-like features. Supplementation with HS plus ITS decreased p75, but not CD90 expression. In conclusion, low concentration of digestive enzymes preserved extracellular matrix and may retain stem cell niches in dental papilla tissue. The 3D cul-ture system was able to support papilla stem cell niche and induced striking differences in cell sha-pe, proliferation and migration. HS and HS plus ITS supplementation changed morphological and phenotypic characteristics of SCAPs. Taken together, this study provides that processing methods should be designed to maintain a biologic scaffold composed of apical papilla and that SCAPs dis-play cell-intrinsic differences in response to microenvironmental factors.

Keywords: Dental Apical Papilla; Mesenchymal Stem Cells; Adult Stem Cells; Human Serum; Insu-lin-Transferrin-Selenite Media; ITS; 3D Culture; Niche Stem Cells.

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AVALIAÇÃO CLÍNICA DA APLICAÇÃO DE CÉLULAS TRONCO ME-SENQUIMAIS DE TECIDO ADIPOSO PARA O TRATAMENTO DE

ÚLCERA DE CÓRNEA PERSISTENTE EM CÃES: ESTUDO PILOTO.

Malago, R.¹, Bittencourt, M. K. W.², Pereira, A. L.¹, Barros, M. A.³, Morais, B. P.³, Bittencourt, M. D.⁴, Silva, A. S.⁵, Kerkis, I.⁶,Vasconcellos, J. P. C.¹.

1Universidade Estadual de Campinas, Brasil.2Vetprime Especialidades Veterinárias, Brasil.3Regenera Medicina Veterinaria Avançada, Brasil.4Benvista Oftalmologia, Brasil.5Centro Universitário De Itajuba - Fepi, Brasil.6Instituto Butantan, Brasil.

A úlcera persistente, denominada recentemente na oftalmologia veterinária como Defeito Epitelial Corneano Crônico Espontâneo (SCCED) é uma ceratite ulcerativa superficial, refratária e crônica que se manifesta em cães de meia idade, de raças variadas sendo frequententemente descrita na raça Boxer. Clinicamente a SCCED se apresenta com uma solução de continuidade superficial, não infectada, com borda epitelial não aderida ao estroma adjacente. Apesar da fisiopatologia da doença não estar totalmente esclarecida, são descritas anormalidades morfológicas e funcionais da córnea que retratam dificuldade na cicatrização. Diversos trabalhos sugerem tratamentos clínicos que nem sempre são eficazes, sendo necessária a inter-venção cirúrgica. Apesar dos procedimentos contribuírem para a cicatrização da córnea, são invasivos e ne-cessitam de anestesia geral, logo, o tratamento pode ser bastante desafiador e exigir cuidado e medicação frequente por um tempo prolongado. O presente estudo piloto trata-se de uma série de casos que teve como objetivo avaliar clinicamente a cicatrização das úlceras de córnea persistentes em cães após a aplicação tópica de células tronco mesenquimais (CTM) de tecido adiposo heterólogo, foram avaliados 10 olhos de cães com SCCED e que não apresentavam nenhuma outra comorbidade que contra-indicasse o uso das CTM. Foram feitas 5 aplicações tópicas seriadas de CTM devidamente caracterizadas, na concentração de 106 (Laboratório Regenera Stem Cells®) diluídas em 0,1 mL de solução fisiológica. Foram feitas avaliações posteriores à aplicação até a cicatrização da lesão, de acordo com os critérios baseados na escala adaptada de Hackett–McDonald Ocular Scoring System: blefaroespasmo, congestão e descarga conjuntival, aferição da superfície ocular, severidade da opacidade, vascularização e pigmentação corneana, além do tempo de cicatrização. O tempo médio para a primeira avaliação (T1) foi de 8 dias (±3,5) e 2/10 casos (20%) apresen-taram cicatrização, 4/10 casos (40%) tiveram a cicatrização na segunda avaliação (T2), com tempo médio de 19 dias (±9,1), na terceira avaliação (T3) 2/10 casos (20%) haviam cicatrizado em um tempo médio de 20 dias (±2,2), 1/10 casos (10%) teve a cicatrização com 38 dias e outro, 1/10 casos (10%), foi observada com 46 dias. Todos os animais permaneceram saudáveis ao longo do acompanhamento, apresentaram cicatri-zação das úlceras de córnea após a terapia, ao final da última avaliação foram observados graus variados de vascularização corneana superficial em 8/10 (80%), congestão conjuntival 4/10 (40%) opacidade de córnea em 7/10 casos (70%), descarga conjuntival (secreção seromucosa) em 4/10 casos (40%), pigmen-tação corneana 6/10 (60%) e formação de tecido de granulação em 2/10 casos (20%), sinais clínicos estes que também são observados no processo cicatricial após a técnica de desbridamento cirúrgico descrito na literatura. Apesar de mais estudos comparando a aplicação de terapia celular com outras terapêuticas serem necessários, os resultados deste trabalho sugerem que a aplicação tópica das CTM, tem potencial terapêutico para a cicatrização do SCCED, levando em consideração a sua ação por meio da secreção de fatores tróficos e citocinas, efeitos imunomoduladores e sua capacidade de transdiferenciação, trata-se de uma técnica segura, minimamente invasiva, que não requer anestesia geral e que promoveu cicatrização em 100% dos casos. Keywords: Célula-tronco Mesenquimal Adulta; Úlcera Indolente; Cão; Defeito Epitelial Corneano Crônico Espontâneo.


BPA AND TCDD ACTION ON RAT ADIPOSE-DERIVED STEM CELLS (RASCS) DIFFERENTIATION POTENTIAL.

Nunes, H. C.¹; A. F. A. S.²; H. V. G.²; Cucielo, M. S.²; Scarano, W. R.²; Deffune, E.²; Delella, F. K.².

1Universidade Estadual de Campinas, Brasil.2Universidade Estadual Paulista, Brasil.

Mesenchymal stem cells (MSCs) are a heterogeneous population that proliferates in vitro as plasti-c-adherent cells and have fibroblast-like morphology. They can form colonies in vitro and can diffe-rentiate into bone, cartilage, fat cells, and others. Adipose-derived stem cells (ASCs) originate in the adipose tissue and due to their easy processing and isolation techniques, they are used for regene-rative medicine purposes to prevent and treat diseases occurring in a variety of tissues and organs. Considering practical issues, the use of experimental tissue-engineered constructs demands urgen-cy for approval and implantation, and also an increase in clinical trials activity has been noticed in the last decades. Therefore, an important issue to be considered when working with cell therapy is the possibility for chemical contamination. Endocrine disrupting chemicals (EDCs) are lipophilic compounds closely related to adipose tissue. BPA and TCDD are the best known and relevant EDCs that have been recently studied. They are also called obesogens due to their inductive adipogenic differentiation. Accordingly, this study aimed to access and quantify the differentiation potential of rat adipose-derived stem cells (rASCs) exposed to 1uM, 10uM of BPA and 10nM of TCDD. These con-centrations were previously defined based on the literature by half maximal inhibitory concentration (IC50) and lethal dose (LD) tests. Other cell processes were further investigated. Primary culture of rASCs was harvested and isolated by Wistar Rats. In the fourth passage, cells were characterized for the ASC population by flow Cytometric and tri-lineage differentiation kit analyses. A cytotoxicity assay by MTT was performed to confirm possible deleterious activity of these compounds. Apopto-sis/necrosis by Flow Cytometry and the differentiation potential was also performed. Comet assay was accessed to verify DNA damage. Statistical analysis was based on mean S.E.M. or S.D. The statistical differences among two or more groups were determined by ANOVA, followed by the pos-t-hoc Tukey´s multiple comparison tests vs the respective control group. The statistical differences between two groups were analyzed using student’s t-test. Statistical significance was set at P<0.05. The analysis was performed using Prism software. Data represent mean ± SE from three indepen-dent experiments in duplicate or triplicate repetitions. Results demonstrated that 10uM of BPA and 10nM of TCDD were able to induce proliferation according to MTT analysis. Apoptosis/necrosis assay showed that BPA and TCDD were able to induce cell death. Comet assay analysis presented DNA damage in rASCs from all experimental groups. When cells were quantified in terms of adipo-genic differentiation,1uM of BPA caused significant oil droplets formation, while 10uM of BPA and 10nM of TCDD showed a decrease on adipocyte differentiation. Osteogenic differentiation did not show significant results among groups. The present results evidenced that 1uM BPA concentration had the most expressive outcomes in terms of adipocyte differentiation. Thus, it is reasonable to infer that low doses (1uM) were able to induce this process. Our results showed that rASCs exposed to BPA and TCDD demonstrated alterations on important cellular processes, such as cell prolifera-tion, apoptosis rate, adipogenic differentiation, and DNA damage.

Keywords: Mesenchymal Stem Cells; Adipose-Derived Stem Cells; Endocrine Disruptors; Bisphe-nol A; TCDD; Cell Therapy.

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CÉLULAS-TRONCO MESENQUIMAIS DERIVADAS DA MEDULA ÓS-SEA E OSSO ESPONJOSO: POTENCIAIS CANDIDATAS NA

FORMAÇÃO DE UM NICHO HEMATOPOÉTICO HETEROTÓPICO.

Ferreira, F. U.¹; Costa, P. N. M.¹; Rós, F. A.¹; Rossetti, R.¹; Gomes, R.²; Fogagnolo, F.³; Kashima, S.²; Covas, D. T.².

1Universidade de São Paulo, Brasil.2Fundação Hemocentro de Ribeirão Preto, Brasil.3Hospital das Clínicas de Ribeirão Preto/Faculdade de Medicina de Ribeirão Preto, Brasil.

O nicho hematopoético é um microambiente dotado de diferentes tipos celulares, fatores solúveis e condições específicas de fluxo sanguíneo e tensões de oxigênio que juntos regulam o estado de quies-cência e a capacidade de autorrenovacão e diferenciacão das células-tronco hematopoéticas (CTHs). Acredita-se que dentre as diferentes populações ali presentes, as células-tronco mesenquimais (CTM) exerçam grande contribuição na regulação e sustentação deste microambiente por meio da sua ativi-dade parácrina e o seu potencial de diferenciacão em linhagens mesodérmicas. Diante disto, as CTM são vistas como auxiliares em potencial para a construção de organoides para atuarem como modelo de estudo. O objetivo deste trabalho foi isolar e caracterizar CTM humanas de dois compartimentos medulares (osso esponjoso e medula óssea) visando o uso como candidatas na formação de um nicho hematopoético heterotópico. Para tanto, as CTMs isoladas do osso esponjoso metafisário humano (CTM-OE, n=3) e da medula óssea (CTM-MO, n=3) foram cultivadas sob condições de hipóxia e ca-racterizadas quanto ao perfil imunofenotípico, capacidade de diferenciação mesodérmica, potencial de proliferação, migração e expressão gênica em relação à manutenção e suporte do nicho hematopo-ético. Os OE coletados também foram caracterizados quanto à presença do marcador estromal pro-genitor CD146 por imunohistoquímica. Ambas as populações estudadas apresentaram características condizentes com CTM como aderência ao plástico e morfologia fibroblastoide. O perfil imunofenotípico demonstrou uma população com altos níveis (mais de 90%) dos marcadores mesenquimais CD73, CD90, CD105, HLA-ABC e CD146, baixos níveis ou ausência (menos de 2%) dos marcadores hema-topoéticos CD45, CD14, CD34, HLA-DR e do marcador endotelial CD31. Em relação a capacidade de diferenciação mesodérmica, tanto as CTMs-MO quanto CTMs-OE, foram capazes de se diferenciar em adipócitos, osteócitos e condrócitos, sendo que as CTMs-MO apresentaram maior grau de diferen-ciação adipogênica enquanto que as CTMs-OE um maior potencial de diferenciação osteogênica. Os ensaios de doubling time e migração sugerem que as CTMs apresentam um potencial proliferativo e migratório semelhantes. Ambas populações de CTMs expressaram altos níveis de transcrição de genes relacionados ao nicho hematopoético: PTGES, FGF-2 , VEGF-C , GREM-1 e BMP-4 ,TGF-β1, HGF , VEGF-A , VCAM , RUNX-2, BGLAP e CXCL12. Foi demonstrado marcação positiva para o CD146, in situ, sugerindo que essa região (nos quais foram isoladas as CTMs) pode estar enriquecida por células estromais progenitoras. Portanto, os resultados obtidos até o presente momento demonstram que as células isoladas da MO e do OE podem ser caracterizadas como CTMs, e ambas demonstraram poten-cial proliferativo semelhante e expressão de genes associados a manutenção do nicho hematopoético, tornando-as candidatas em potencial para auxiliar na construção de um nicho hematopoético heterotó-pico. Ambas CTMs isoladas do mesmo microambiente medular porém de compartimentos diferentes, apresentam peculiaridades promissoras para seu uso em estudos de bioengenharia tecidual e terapia celular, podendo futuramente beneficiar a aplicação clínica destas células em medicina regenerativa. Como etapas futuras, tem-se a coleta de novas amostras e a padronização do cultivo desta CTMs em um biomaterial para construção do organoide in vitro e in vivo.

Keywords: Células-tronco Mesenquimais Da Medula Óssea; Células-tronco Mesenquimais Do Osso Esponjoso; Nicho Hematopoético; Nicho Hematopoético Heterotópico.


CHARACTERIZATION OF EXOSOMES RELEASED BY THE ADSCS AND THEIR EFFECTS ON THE MOTOR SYMMETRY OF RATS SUB-

MITTED TO A FOCAL ISCHEMIA MODEL.

Rohden, F.¹; Schafhauser, D. D. C.¹; Gionbelli , M. P.¹; Teixeira, L. V.¹; Colombo, M.¹; De Souza, D. O. G.¹; Guma, F. C. R.¹.

1Universidade Federal do Rio Grande do Sul - UFRGS, Brasil.

Introduction: Hypoxia state from focal cerebral ischemia causes a neurodegenerative condition that can damage a variety of cognitive and motor functions. Using exosomes released by stem cells have shown positive results on tissue regeneration in several situations, including ischemia. Aims: Characterization of exosomes secreted by adipo derivated stem cells - ADSC, and evaluation of its effects on global recovery in rat cerebral ischemia model. Methods: ADSC (LONZA) were cultured for exosomes isolation. Purity, concentration, size, and percentage rate of exosomes were evalua-ted by transmission electron microscopy (TEM), Lowry’s modified method and Zetasizer-zs90 de-vice, respectively. Focal cerebral ischemia was performed by the thermocoagulation of pia blood vessels in male Wistar rats adults. Animals were treated after 24 hours of ischemia with intranasal administration of exosomes (200μg/kg) or PBS (control) in the same volume. Cylinder test evalua-ted symmetry, 24 before the injury, 72h after, and weekly until the 42nd day after treatment. Analy-zes were based in the first 20 touches, with differential score for ipsilateral, contralateral and both. Results: TEM analysis confirmed purity and the efficacy of the isolation method. Exosome has 100 nm of average size, and 6x107 cells secrete 1μg/μl protein (suspension of exosomes) within 48 hours. Cylinder test showed significant improvement in the symmetry of animals treated with exo-somes (p<0.0001) since 7th day until the 42nd. Conclusion: We showed that with a 24-hour window therapy, there was significant improvements in the motor status of rats that underwent to ischemic conditions treated with exosomes released by ADSC.

Keywords: Exosome; Adiposo Derivated Stem Cells; Ischemia.

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DESVENDANDO OS MICROAMBIENTES DO TECIDO ADIPOSO SUB-CUTâNEO E SUA RELEVÂNCIA EM PROTOCOLOS DE ENGENHARIA

DE TECIDOS.

Cunha, B. M. D.¹,²; Côrtes, I.¹,²; Ayala, F. R. R.³; Silva, C. C. D.³; Auxenfans, C.⁴; Sigaudo-Roussel, D.⁵; Baptista, L. S.¹,².

1Núcleo Multidisciplinar de Pesquisa de Biologia em Xerém, Universidade Federal do Rio de Janeiro, Brasil.2Laboratório de Bioengenharia Tecidual, Instituto Nacional de Metrologia, Qualidade e Tecnologia, Brasil.3Serviço de Cirurgia Plástica, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Brasil.4Hospices Civils de Lyon, Hôpital Edouard Herriot, Banque de tissus et cellules, França.5Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, Université de Lyon, França.

O tecido adiposo branco (TAB) se desenvolve em regiões distintas do corpo denominadas depósitos, cujo maior encontra-se na região abdominal compreendendo o TAB subcutâneo, adjacente à pele e o TAB visceral, envolvendo órgãos internos. Além da dicotomia entre TAB subcutâneo e visceral, é pos-sível ainda subdividir o TAB subcutâneo em duas camadas - superficial e profunda - que é separada pela fáscia superficial. A fáscia superficial é formada por fibras de colágeno frouxamente entrelaçadas e apresenta extensões denominada de reticunáculo da cutis superficial que envolve os lóbulos adipo-cíticos, sendo este um nicho de células-tronco ainda não descrito pela literatura. O presente estudo propõe avaliar e comparar os microambientes superficial e profundo do tecido adiposo subcutâneo e do reticunáculo da cutis superficial a fim de investigar a contribuição destas células-tronco/estro-mais de tecido adiposo (ASCs) para a fisiologia e neogênese do tecido adiposo. Primeiramente, as amostras de tecido adiposo foram coletadas de pacientes hígidos submetidos a abdominoplastia no Hospital Universitário Clementino Fraga Filho. Os procedimentos descritos neste estudo foram apro-vados pelo Comitê de Ética em Pesquisa (CEP) deste hospital e estão registrados sob os números de protocolos 145/09 and 076/10. Parte do TAB subcutâneo foi congelado à temperatura de -80ºC em OCT e obtidos cortes de 20 µm de espessura do material. A coloração com Hematoxilina revelou um tecido conjuntivo frouxo presente desde a derme até a fáscia superficial circundando lóbulos lipí-dicos. A imunofluorescência revelou que o CD146 (célula-tronco mesenquimal) está localizado bem internamente aos vasos sanguíneos e o CD34 (pré-adipócito) na adventícia dos vasos sanguíneos. Outra parte dos fragmentos de TAB superficial e profundo e o reticunáluco da cutis superficial foram submetidos à ação da colagenase para obtenção da fração estromal-vascular e posteriormente iso-lamento das ASCs quando mantidas em monocamada. A composição da Fração Estromal-Vascular (SVF, Stromal Vascular Fraction) do tecido adiposo dos diferentes microambientes foi avaliada por citometria de fluxo, onde temos a camada superficial com maior presença de células progenitoras da linhagem adipogênica CD45negCD146negCD31negCD34pos (pré-adipócitos). A indução adipogêni-ca da monocamada de ASCs foi realizada utilizando dulbecco’s modified eagle medium (DMEM) low Glucose; Dexametasona a 10-6M; 0,5 mM de 3- Isobutil-1- Metylxanthine (IBMX); 10μM de Insulina e 200μM de Indometacina suplementado com 10% de soro fetal bovino (SFB); penicilina e estreptomi-cina (PS). O meio controle continha apenas 10% de SFB e PS. O acúmulo lipídico foi avaliado a partir da coloração com Nile Red (20µg/mL) revelando maior acúmulo lipídico a partir das ASCs isoladas do reticunáculo da cutis superficial. Nossos resultados preliminares nos remetem a um possível nicho ainda não descrito pela literatura de ASCs no tecido conjuntivo que conecta a derme à fáscia superfi-cial (reticunáculo da cutis superficial).

Keywords: Tecido Adiposo Subcutâneo; Células-Tronco; Microambiente; Medicina Regenerativa.


DISSOCIATION AND RECONSTITUTION OF MESENCHYMAL STEM CELL MEMBRANES TO GENERATE PARTICLES FOR CELLULAR

THERAPY.

Raymundo, A. C. H.¹, Serafini, M. A.¹, Paz, A. H. D. R.¹, Gonçalves, F. D. C.¹


Mesenchymal stem cells (MSC) are a promising therapy for degenerating and immunological disor-ders. However, MSC therapy is associated with risks and practical difficulties that come with the use of living cells. Cells may transform and even though MSC are believed to have a short survival after intravenous infusion, even a single surviving transformed cell forms a risk for tumor formation. To reduce risks associated with MSC infusion and improve the distribution in the body, we generated MSC membrane particles (MP) from humans (hMP) and C57BL/6 mice (mMP). Methods: MSC were isolated from human and C57BL/6 mouse adipose tissue and the cells were primed with LPS (mice) or IFN-ƴ (human) to enhance their immunomodulatory effects. MP were manually genera-ted by lysing MSC in hypotonic buffer, removal of organelles and soluble proteins by centrifugation and ultrafiltration respectively. Analysis of absolute size distribution and concentration of MP were performed using NanoSight particle tracking and their morphology were evaluated by Confocal microscope. Results and Discussion: NTA indicated that the size of the particles ranged from 64 to 685nm, and the mode size of the samples was 149.2 ± 20.2nm and 125.9 ± 9.1nm for hMP and mMP, respectively. There was no significant difference in size distribution (MP/MSC) between hMP and mMP. Confocal imaging revealed that MP consist of a population of particles heterogeneous in size with most of the particles showing a size of less than 200nm but some showing larger sizes. This result confirms the NTA analysis. This new methodology produces particles with an optimized phy-sical size to avoid trapping in the lung. This new physical conformation of MSC furthermore avoids formation of tumors, decreases the risk for immune responses directed against injected cells and avoids potential changing behavior of the therapeutic such as with living cells. Complications related to the size of MSC during systemic or local injection can be minimized with the MP therapy. Additio-nal studies, both in vitro and in vivo, are needed to improve our understanding the mechanisms of action of this potential immunosuppressive tool.

Keywords: Mesenchymal Stem Cells; Cell Therapy; Membrane Particles.

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EFFECT OF ANGIOTENSIN II AND ANGIOTENSIN-(1-7) IN PROLIFE-RATION OF STEM CELLS FROM HUMAN DENTAL APICAL PAPILLA.

Macedo, L. M.¹; Ávila, R. I.²; Valadares, M. C.²; Lima, E. M.²; Borges, C. L.³; Gava, E.¹; Castro, C. H. D.¹.

1Departamento de Ciências Fisiológicas, Universidade Federal de Goiás, Brasil.2Faculdade de Farmácia, Universidade Federal de Goiás, Brasil.3Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Goiás, Brasil.

Dental papilla has been considered an important source of ectomesenchymal stem cells. Renin--angiotensin System (RAS) is a complex systemic cascade of interacting peptides and enzymes that coordinates a variety of physiological processes. This system has two main biologically active peptides, Ang II and Ang-(1-7). Previous studies have shown that these peptides can influence proliferation of progenitor cells. However, the role of RAS on the stem cells isolated from human dental apical papilla (SCAPs) is completely unknown. Thus, the aim of this study was to identify the presence of RAS components in SCAPs and the effects of the Ang II and Ang-(1-7) in the prolifera-tion these cells. SCAPs were collected from third molar teeth of 12 healthy individuals aged 13-16 years. The primary culture was maintained in DMEM/F12 with 10% fetal bovine serum at 37°C and in 5% CO2. RT-PCR and Western Blotting was used to identify the mRNA and protein expression of the Angiotensin-converting Enzyme (ACE), ACE2, Mas, AT1 and AT2 receptors. Carboxyfluores-cein succinimidyl ester (CFSE) assay was used to evaluate the effect of the Ang II and Ang-(1-7) in the cell proliferation. SCAPs were plated at 5x105 cells/mL for 8 h and incubated with Ang II (10-8 to 10-5 mol/L) or Ang-(1-7) (10-8 to 10-5 mol/L) in presence or absence of Losartan (AT1 receptor antagonist, 10-6 mol/L), PD12319 (AT2 receptor antagonist, 10-6 mol/L) or A-779 (Mas receptor antagonist, 10-6 mol/L). Western Blotting was also used to identify the signaling pathways involved in the proliferative effects of the peptides. mRNA expression and protein levels of ACE, ACE2, and AT1, AT2 and Mas receptors were detected in SCAPs. Both Ang II and Ang-(1-7) increased the stem cell proliferation. These effects were inhibited by PD123319. Ang II decreased total mTOR expression and augmented mTOR phosphorylation. Ang-(1-7) diminished total mTOR, p-AKT and p-GSK-3 beta expression, and increased ERK1/2 phosphorylation. In conclusion, SCAPs present the main RAS components and both Ang II and Ang-(1-7) treatments induced cell proliferation me-diated by AT2 activation. However, while Ang II induced stem cell proliferation through mTOR acti-vation, Ang-(17) stimulated proliferation in an AKT/mTOR and ERK1/2-dependent manner.

Keywords: RAS, Scaps; Ang II; Ang-(1-7); AT2; Stem Cell Proliferation; Carboxyfluorescein Succini-midyl Assay; CFSE; AKT; Mtor; GSK3 Beta; ERK1/2.


EFFECT OF HEPATITIS C DRUGS SOFOSBUVIR AND DACLATAS-VIR TREATMENT ON MESENCHYMAL STEM CELLS VIABILITY, AU-

TOPHAGY AND MIGRATION CAPACITY.

Serafini, M. A.¹; Machado, D. N.¹; Ayres, R.¹; Raymundo, A. C. H.¹; Filippi-Chiela, E. C.¹; Araújo, A. B.²; Silveira, T. R. D.¹; Álvares-Da-Silva, M. R.¹; Gonçalves, F. D. C.¹; Paz, A. H. D. R.¹,².

1Universidade Federal do Rio Grande do Sul, Brasil.2Hospital de Clínicas de Porto Alegre, Brasil.

Mesenchymal stem cells (MSCs) are plastic-aderent, fibroblast-like multipotent adult cells that can differentiate into all cells of mesodermal origin. Recently, several research groups reported MSCs immunoregulatory properties, thus showing potential clinical application in treating immune-based disorders and chronic inflammation. Hepatitis C is characterized by chronic inflammation in the liver, who often leads to fibrosis, cirrhosis, and death. Recent studies in terminal patients of hepatitis B and C have shown MSCs transplantation can improve hepatic function, including cirrhotic patients. To evaluate whether hepatitis C drugs sofosbuvir and daclatasvir treatment affects MSCs normal functions. Methodology: Human chorion-derived MSCs were analyzed for autophagy, cell viability and migration capacity assays. Cells were treated for 24h and 72h with 230ng/mL and 620ng/mL sofosbuvir and 400ng/mL and 1000ng/mL daclatasvir, respectively, in addition to a group with the highest concentrations of both drugs combined. These drugs concentrations were defined using hepatitis C patient’s serum concentrations reported in the literature. Viability and autophagy were assessed by markers annexin V-FITC, propidium iodide and acridin orange, and analyzed by flow citometry. Random migration capacity was assessed by wound healing assay and the distance between cells in the wound was measured in 5 different sites per image using Image J software. Results and Discussion: We found no difference between treatments and control group for neither of the parameters proposed. These preliminary results indicate sofosbuvir and daclatasvir hepatitis C drug treatment does not impair cell viability nor does it alter the autophagy or the migratory capacity of MSCs, thus preserving these MSCs normal functions. These results indicate cell therapy with MSCs could be performed concomitantly with treatment with these drugs. More experiments are ne-cessary to determine whether MSCs immunomodulatory capacity also remain impaired by hepatitis C drug treatment, thus making these cells candidates for concomitant cell therapy for reducing liver inflammation in this disease.

Keywords: MSCs; human mesenchymal stem cells; sofosbuvir; daclatasvir; in vitro; cell therapy; hepatitis.

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ESTUDO COMPARATIVO DE Ɣ-H2AX EM CÉLULAS-TRONCO ME-SENQUIMAIS DERIVADAS DO TECIDO ADIPOSO ABDOMINAL E FA-

CIAL HUMANO FRENTE À RUVA E RUVB.

Silva-Acordi, C.¹; Barros-Delben, P.¹; Gomes, R. S.²; Trentin, A. G.¹.

1Universidade Federal de Santa Catarina, Brasil.2Hospital e Maternidade Dr. Carlos Correa, Brasil.

A descoberta que tecidos adultos têm células tronco (CT) com pluripotencialidade deu início a uma série de pesquisas, principalmente a partir do momento que o tecido adiposo (TA) e a pele tornaram-se fontes de obtenção e diferenciação de CT. Com isso, abriu-se novas perspectivas de tratamento para várias condições fisiopatológicas, com finalidade reparadora ou estética. As célu-las-tronco mesenquimais (CTM) são também encontradas no TA, e sua escolha se deu pelo fácil acesso, minimamente invasivo e pelo alto rendimento celular. O TA, de acordo com sua localização anatômica, apresenta diferentes origens embrionárias e propriedades morfológicas distintas. Estas alterações influenciam na capacidade das CT, como diante do estresse oxidativo. O estresse oxida-tivo, pode comprometer a integridade celular por predispor a danos no DNA. Quebras na dupla-fita do DNA (DBS) são consideradas uma das formas de dano que podem comprometer a capacidade das CTM, e podem ser geradas a partir das radiações solares. Isto desencadeia a fosforilação de H2AX, gerando a isoforma ɣ-H2AX, que é um biomarcador de lesão de DNA. O objetivo deste es-tudo foi analisar, comparativamente, a presença do acúmulo do indicador de lesão de DNA γ-H2AX nas CTM do TA facial e abdominal em estado basal e após exposição às radiações UVA e UVB. Foram avaliados, a morfologia celular (por microscopia óptica de contraste de fase); viabilidade (por ensaio de MTS); e imunofluorescência para acúmulo de ɣ-H2AX em diferentes passagens de cultivo (baixas, médias e altas) no estado basal e após a exposição a radiações UVA e UVB. Os resultados mostraram que as CTM-TA de ambas as origens apresentam aderência ao plástico e morfologia celular fibroblastóide, e que isso é alterado após as radiações. Na RUVA independente da dose não foram constatadas diferenças na viabilidade/proliferação entre as origens celulares, o mesmo não ocorreu com a RUVB. Na imunofluorescência foi demostrada um padrão diferente de focos de ɣ-H2AX entre as RUVA e RUVB, sendo que na RUVA gera focos mais distinguíveis e ambos as células acumulam similarmente nas passagens médias e altas. Sob o estímulo de RUVB as CTM do TA abdominal acumulam mais ɣ-H2AX do que as faciais. Concluímos que as células de ambas as origens apresentam similar capacidade de adesão, morfologia e viabilidade celular, porém, apresentam diferenças na proliferação celular e no acúmulo de dano de DNA entre as di-ferentes passagens. Portanto as células faciais de passagens baixas são sugeridas como mais seguras para aplicações terapêuticas.

Keywords: ɣ-H2AX; células-tronco; RUVA; RUVB.


EVALUATION OF ZOLEDRONATE AND BIOACTIVE COMPOUNDS IN MESENCHYMAL STEM CELLS EXTRACTED FROM DECIDUOUS

TEETH.

Machado, G. M.¹,²; Kasper, R. H.¹; Maurmann, N.²,³; Brew, M. C.¹; Do Couto, M.¹; Wiltgen, A.¹; Mahl, C. R. W.¹; Bavaresco, C. S.¹; Pranke, P.²,³,⁴.

1Universidade Luterana do Brasil, Brasil.2Laboratório de Hematologia e Células-tronco, Brasil.3Programa de Pós Graduação em Fisiologia, Universidade Federal do Rio Grande do Sul, Brasil.4Instituto de Pesquisa com Células-tronco, Brasil.

The osteonecrosis of the jaw is often a reported clinical complication in patients who are medicated with bisphosphonates (such as zoledronate – ZOL) for bone pathologies. However, the use of bio-active compositions is being a promising strategy used in the regeneration of lost tissue. Therefore, the main aim of the study has been to evaluate in mesenchymal stem cells (MSCs), the cellular viability after treatment with ZOL and bioactive compositions. The MSCs were treated with ZOL in different concentrations (2.5, 5 or 10 μM for 1 day and 1, 3, 5 or 10 μM for 4 days) and with bioac-tive compositios such as ascorbic acid-2 phosphate (ASAP) (172 μM), sodium fluoride (NaF) (50 μM) and glycine (10 μM) for 4 days. Furthermore, the bioactive compositions that showed the best cellular viability was tested for cytoprotective potential after ZOL treatment. The viability evaluation was performed through the test (bromide of 3- (4,5- dimethylthiazol-2-yl) –2,5- diphenyltetrazolium) (MTT) after the treatments. The results of the cells treated with ZOL showed no statistically signi-ficant difference in every concentration in a 1 day period (F (3.49)= 0.587; p=0.63). The results of the average absorbance ± standard deviation (SD), related to cell viability after 1 day of ZOL tre-atment were: 0.279 ± 0.046 for the control group, 0.297 ± 0.040 for the 2.5 μM, 0.294 ± 0.038 for the 5 μM and 0.292 ± 0.028 for the 10 μM. After 4 days, there was a significant statistical difference in every concentrations relative to the control; the results of the average absorbance ± SD were: 0.336 ± 0.045 for the control group, 0.224 ± 0.029 for the 1 μM (p<0.01), 0.212 ± 0.012 for the 3 μM (p<0.01), 0.188 ± 0.057 for the 5 μM (p<0.01) and 0.084 ± 0.026 for the 10 μM (p<0.01). Among the bioactive compositions, ASAP was the only one that significantly increased the MSCs viability. The glycine demonstrated a decrease in the cell viability. After 4 days, the average absorbance ± SD were: 0.396 ± 0.050 for the control, 0.461 ± 0.039 for the ASAP (p<0.05), 0.360 ± 0.039 for the NaF (p=ns) and 0.332 ± 0.025 for the glycine (p<0.01). The cells treated with ASAP were statistically different from the glycine and NaF (ps<0.01). However, although only ASAP increased the cellular viability when associated with ZOL, it demonstrated an absorbance increase, but did not demons-trate the cytoprotective feature in the concentration used, with 0.188 ± 0.057 being the the average absorbance ± SD for cells treated with 5 μM of ZOL and 0.233 ± 0.053 for 5 μM of ZOL+ASAP (p>0.05). Furthermore, it is suggested that ascorbic acid is a promising bioactive compositions for the tissue regeneration, although it does not protect against the cytotoxicity of the studied drug.

Keywords: Stem Cells; Bifosfonates; Osteonecrosis; Bioactive Compositions.

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GRX, A LIVER STEM CELL LINE, IS NOT AFFECTED BY HEPATITIS C SOFOSBUVIR AND DACLATASVIR DRUG TREATMENT IN VITRO.

Serafini, M. A.¹; Ayres, R.¹; Raymundo, A. C. H.¹; Filippi-Chiela, E. C.¹; Silveira, T. R. D.¹; Álvares--Da-Silva, M. R.¹; Gonçalves, F. D. C.¹; Paz, A. H. D. R.¹,².

1Universidade Federal do Rio Grande do Sul, Brasil.2Hospital de Clínicas de Porto Alegre, Brasil.

Hepatitis C is characterized by chronic inflammation in the liver, who often leads to fibrosis, cirrho-sis, and death. Upon inflammatory conditions, hepatic stellate cells (HSCs) are known to promote liver fibrosis by changing its profile from quiescent to fibrogenic, where these cells produce great quantities of extracellular matrix which replaces hepatic functional tissue, impairing liver function. Neverthless, recent studies reported HSCs to act as stem cells in the liver tissue, being able to di-fferentiate into hepatocytes and thereby promoting tissue regeneration. In this report, we aimed to evaluate sofosbuvir and daclatasvir treatment on hepatic stellate cells seeking to see if drugs com-monly used for the treatment of hepatitis would affect the endogenous liver stem cell. To evaluate whether hepatitis C drugs sofosbuvir and daclatasvir treatment affects hepatic stellate cells normal functions. Methodology: GRX cells (hepatic stellate cells line) were analyzed for autophagy, cell cycle, cell proliferation, cell viability and oil red staining assays. Cells were treated for 24h and 72h with 230ng/mL and 620ng/mL sofosbuvir and 400ng/mL and 1000ng/mL daclatasvir, respectively, in addition to a group with the highest concentrations of both drugs combined. These drugs con-centrations were defined using hepatitis C patient’s serum concentration found in the literature. Cell viability, cell cycle and autophagy were assessed by markers annexin V-FITC, propidium iodide and acridin orange, respectively, and analyzed by flow cytometry. Fibrogenic or quiescent differentiation was evaluated by intracellular lipid droplets staining with Oil Red O after 9 days of treatment, and percentage of stained area was assessed using Image J software. Cell proliferation was evaluated by cumulative population doubling assay for 7 days, and the cells were counted every 72h. Results and Discussion: We found no difference between treatments in neither of the parameters tested, indicating Hepatitis C sofosbuvir and daclatasvir drug treatment does not affect hepatic stellate cells autophagy, cell viability, cell proliferation or cell cycle, nor does it alter cell differentiation into quies-cent or fibrogenic profile. However, we observed a tendency to a slower proliferation rate (1.316 population double score) in the group treated with 620ng/mL sofosbuvir and 1000ng/mL daclatasvir combined while control group population double score was 2.565. Our preliminary results show the hepatic stellate cell line GRX is not affected by sofosbuvir and daclatasvir treatment, thus indicating these drugs do not alter HSCs natural cell profile.

Keywords: Hepatic Stellate Cells; Hepatitis C; Adult Stem Cells; In Vitro; Sofosbuvir; Daclatasvir.


IMUNOSSENSOR PARA IDENTIFICAÇÃO DE CÉLULAS-TRONCO MESENQUIMAIS DE COELHO.

Bovolato, A. L. D. C.¹; Carandina, R. F.¹; Bertanha, M.²; Alves, A. L. G.³; Golim, M. D A.⁴; Zambuzzi, W. F.⁵; Moroz, A.⁶; Simões, R. P.⁶; Moraes, M. L.⁷; Deffune, E.¹.

1Laboratório de Engenharia Celular, Hemocentro, Faculdade de Medicina de Botucatu, Brasil .2Departamento de Cirurgia e Ortopedia, Faculdade de Medicina de Botucatu, Brasil.3Departamento de Cirurgia de Grandes Animais, Faculdade de Medicina Veterinária e Zootecnia de Botucatu, Brasil.4Laboratório de Citometria de Fluxo, Hemocentro, Hospital das Clinicas de Botucatu , Brasil 5Departamento de Bioquímica, Instituto de Biociências de Botucatu, Brasil.6Departamento de Bioprocessos e Biotecnologia, Faculdade de Ciências Farmacêuticas de Arara-quara, Brasil.7Laboratório do Centro Nacional de Pesquisa em Energia e Materiais, Instituto de Ciências e Tec-nologia de São José dos Campos, Brasil.

Diante do crescente uso de células-tronco em medicina regenerativa aliada a importância de diag-nóstico rápido, os imunossensores surgem como ferramentas que propiciam rapidez, seletividade e sensibilidade. Foi investigada a construção de um imunossensor para identificação de células--tronco mesenquimais de coelho frente à inexistência no mercado, de marcadores específicos para a espécie. Com o objetivo de identificar a célula-tronco mesenquimal (CTM) em amostras biológi-cas, foi proposto a construção de filmes pela técnica layer-by-layer (LbL) com solução de fibroina de seda, PEI e quitosana cada qual associado com anticorpo monoclonal de especificidade contra CTM produzido home made (HM) para aplicação em imunossensores eletroquímicos. Metodolo-gia: Inicialmente foi caracterizado o anticorpo monoclonal produzido por técnicas imunoquímicas. A classe do anticorpo produzido é IgG1κ com especificidade anti-CD 90. O crescimento dos filmes foi confirmado por voltametria cíclica (VC) pela área dos voltamogramas. Resultados e Discussão: A distorção dos voltamogramas foi observada com maior adsorção, considerado como cobertura completa do eletrodo. Foi padronizado e produzido o biofilme de bicamadas para fixação dos an-ticorpos monoclonais murinos anti-CD90 HM com solução de quitosana. A análise dos resultados obtidos com imunossensor comparando-se com a citometria de fluxo identificam a elevada sensi-bilidade do mesmo para a confirmação da presença de CTM em amostras biológicas, havendo ne-cessidade de mais desenvolvimento para a quantificação das células. A partir de uma concentração de 103 CTM e leitura em 20 minutos já se obtém resposta evidenciando o reconhecimento espe-cífico de CTM de coelho, rato, cavalo e humano, de menor concentração celular do que a técnica gold standard por citometria de fluxo. Análises estatísticas definem que para obter o melhor rendi-mento do imunossensor o tempo de incubação ideal é de 30 minutos. A detecção eletroquímica da solução de anticorpos anti-CD90 HM foi feita por VC, e foi observada uma mudança nas correntes obtidas em potenciais positivos, principalmente em 0,28V e também da área dos voltamogramas.

Keywords: Imunossensores; Célula-tronco Mesenquimal; Anticorpos Monoclonais; Biomarcadores; Filme Automontado.

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INFLUENCE OF AMNIOTIC MEMBRANE SCAFFOLD ON THE EX-PRESSION OF ADHESION MOLECULES IN STROMAL STEM CELLS

Vedovatto, S.¹; Bertoni, A. P. S.¹; Souza, A. F. D.²; Wink, M. R.¹.

1Universidade Federal do Rio Grande do Sul, Brasil.2Universidade Federal de Ciências da Saúde de Porto Alegre, Brasil.

Among the various biological materials used as scaffold, the amniotic membrane (AM) is one of greater availability, being a promising matrix for the delivery of cells. The association of the amniotic membrane with stromal stem cells has demonstrated a potential for tissue regeneration, requiring a better understanding of the interaction of this biological matrix with stromal stem cells and its pos-sible influence on cell adhesion and migration. For this purpose, the expression of genes involved in these cellular processes was evaluated by quantitative PCR to clarify probable mechanisms of influence of the membrane on the cells. To characterize the human amniotic membrane in vitro as a scaffold for human adipose derived stromal stem cells (ADSCs) evaluating the expression of genes involved with cell adhesion and migration. Cell culture of ADSCs was performed and then immunophenotyped by flow cytometry. The cells were divided in two groups: one cultured with the decellularized amniotic membrane and one of conventional cell culture. Total RNA was extrac-ted from the two groups for the production of complementary DNA and polymerase chain reaction (PCR) was performed by qualitative and quantitative methods for beta-actin, TATA box, glyceral-dehyde-3-phosphate dehydrogenase, fibronectin, N-cadherin and vimentin. The PCR results were analyzed by the 2-ΔΔCt method. Results: The cells demonstrated mesenchymal characteristics and through quantitative PCR they suggested TBP and ACTB as normalizing genes suitable for genetic analysis. The results obtained from real time RT-qPCR for adhesion and proliferation genes presen-ted an important variability and for this reason we didn’t perform statistical analysis. We believe that the biological diversity of patients and other technical issues could affect the expression of these genes. More independent cell cultures are needed to provide more robust results. However, it can be observed that there was a slight increase in fibronectin and N-cadherin expressions, suggesting ADSCs in contact with AM can improve cellular adhesion. Discussion: The possibility of using the amniotic membrane as a dermal substitute associating it with stem cells aims at faster recovery of damaged tissues and opens space for questions about the interaction of this scaffold with the cells and the relation of both biological materials with the bed of the wound. The expression of bioactive molecules by the stromal cells is influenced by the environment and can generate responses or suppress signals according to external stimuli which, in the case of the amniotic membrane, may be involved in its structure rich in collagen, elastin, laminin and fibronectin. The cell adhesion properties in scaffolds for tissue engineering are among the most critical characteristics that may affect its effi-ciency for application in regenerative medicine, hence a superior expression of N-cadherin, vimentin and fibronectin in the group of ADSCs grown with the AM can indicate it as a scaffold that makes stem cell therapy more suitable for clinical application as a dermal substitute. The study allowed us to consider the amniotic membrane as a suitable scaffold for stromal stem cells derived from adipo-se tissue and it may be suggested that it is involved in up-regulation of cell adhesion genes when co-cultured.

Keywords: Human Adipose-Derived Stem Cells; Amniotic Membrane; Cell Adhesion.


INFLUENCE OF THE SOURCE OF MESENCHYMAL STEM CELLS FOR TISSUE ENGINEERING: DERMAL VERSUS ADIPOSE TISSUE IN

SKIN WOUND HEALING.

Zomer, H. D.¹; Jeremias, T. D. S.¹; Trentin, A. G.¹,².

1 Universidade Federal de Santa Catarina – UFSC, Brasil.2 Instituto Nacional de Ciência e Tecnologia em Medicina Regenerativa, Brasil.

The skin is constantly challenged by external factors and highly susceptible to traumas. When inju-red, cutaneous repair occurs by wound healing, a complex process comprising inflammation, proli-feration and remodeling phases, which restores homeostasis but not tissue functionality. Large skin wounds such as burns lead to secondary disorders and death if not quickly stabilized. Thus, new strategies are necessary to accelerate wound closure and improve the healing quality. In this con-text, tissue engineering and cell therapies arise as promising alternatives. Mesenchymal stem cells (MSCs), found in virtually all adult tissues, are easy to obtain and have high proliferation capacity in vitro. Despite sharing stem cell characteristics such as self-renew, differentiation to mesodermal tissues and immunophenotype, MSCs from distinct niches can present individual features. There-fore, studying diverse sources of MSCs is important to develop future therapeutic approaches. This study aimed to compare MSCs from human abdominal dermis (DSC) and adipose tissue (ASC) in association with Integra™ matrix during the process of skin wound healing. DSC and ASC used in this study were previously isolated from human abdominoplasty skin samples and presented similar immunophenotypic expression of CD90+, CD73+, CD105+, CD34- and CD45- and mesodermal differentiation potential to adipocytes, osteocytes and chondrocytes. DSC and ASC were cultivated in Integra™ matrix and used as therapy to excisional skin wounds in mice C57bl6. Control groups received only the Integra™ without cells. Wounds were evaluated clinically and histologically after 3, 7, 21 and 60 days. Results showed a higher number of nucleated cells per field analyzed inside the matrix in DSC (81.17 ± 35.3 SD) and ASC (102.5 ± 31.75 SD) groups in comparison with the control (39.33 ± 15.9 SD) in the inflammatory phase of wound healing (day 3). Grafts were more pinkish, a clinical sign of integration, in DSC and ASC groups in comparison with the control during the proliferation phase (day 7). Accordingly, histological analysis showed a thicker granulation tissue and more blood vessels in cell-treated groups than the control group (angiogenesis: 0.58 ± 0.1 SD in DSC, 0.6 ± 0.1 SD in ASC and 0.33 ± 0.06 SD in control, per field). After 21 days, the wounds were significantly more closed in DSC (90.27% ± 18.78 SD) and ASC (86.88% ± 24.5 SD) groups than control (55.78% ± 29.06 SD). At both early (day 21) and late (day 60) remodeling phase, the neoepidermis was more similar to the normal epidermis and more skin appendices (as hair follicles and sebaceous glands) were present in cell-treated groups than the control. Besides, Integra™ de-gradation and collagen density was significantly higher and more similar to normal skin in DSC trea-ted group than the control group. In late remodeling phase, DSC treated group presented a smaller scar and a higher score of elastic fibers than the control group. Together, these results suggest that the source of MSC influence in the clinical outcome, however, both DSC and ASC presented faster wound closure and better healing features than the control. In conclusion, association of both DSC and ASC to Integra™ represents promising therapeutic strategy to improve human cutaneous re-pair. Furthermore, the use of DSC results in better overall quality of healing than ASC. This study was supported by CNPq, CAPES / PDSE, MCTIC and INCT-REGENERA.

Keywords: Cutaneous Repair; Mesenchymal Stromal Cells; MSC; Translational Study; Histology.

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INFLUÊNCIA DO DIÂMETRO NAS PROPRIEDADES MORFO-FUNCIO-NAIS DE ESFEROIDES DE CÉLULAS-TRONCO DE TECIDO ADIPOSO

HUMANO BIOFABRICADOS DE MODO AUTOMATIZADO.

Miranda, T.B.¹,²; Faria, M.R³; Charelli, L. E.⁴; Miranda, G.A.S¹,²; Beatrici, A⁵; Granjeiro, J. M.³; Silva, K. R.⁴; Baptista, L. S.¹,².

1Núcleo de Pesquisa Multidisciplinar em Biologia - NUMPEX-Bio, Brasil.2Universidade Federal do Rio de Janeiro, Brasil.3Laboratório de Bioengenharia Tecidual, Diretoria de Metrologia Aplicado às Ciências da Vida, Institu-to Nacional de Metrologia, Qualidade e Tecnologia, Brasil.4Programa de Pós-Graduação em Biotecnologia, Instituto Nacional de Metrologia, Qualidade e Tec-nologia, Brasil.5Divisão de Metrologia Mecânica, Instituto Nacional de Metrologia, Qualidade e Tecnologia, Brasil.

O cultivo 3D de células, em especial os esferoides, vem sendo cada vez mais utilizado em aplicações nas áreas de terapia celular e engenharia tecidual, uma vez que a interação das células com a matriz extracelular neste sistema mimetiza de forma mais fiel o microambiente tecidual quando comparado com sistemas de cultivos bidimensionais. Um aspecto a ser considerado é o tamanho destes esfe-roides e o quanto o seu diâmetro pode influenciar suas propriedades físico-químicas e funcionais. O presente trabalho tem como objetivo avaliar a influência do diâmetro de esferoides de células-tronco/estromais de tecido adiposo humano (ASC, Adipose-derived Stromal/Stem cells) em suas proprieda-des morfo-funcionais. Esferoides de ASC foram produzidos após semeadura automatizada (EpMotion 5070, Eppendorf) 1,8x10e5 , 4,3x10e5 e 1,3x10e6 de células em hidrogéis de agarose previamente moldados com 81 microrressecções a partir de um molde de silicone, conforme recomendações do fabricante (Microtissues, Inc.). Os esferoides foram mantidos em estufa úmida a 37ºC e atmosfera com 5% de CO2 , em meio de cultivo composto por DMEM suplementado com albumina 1,25µg/ml, ácido ascórbico 50µg/ml, insulina 10µg/ml, transferrina 10µg/ml, selênio 0,01µg/ml e penincilina 100UI/ml e estreptomicina 100µg/ml. O diâmetro dos 81 esferoides obtidos a partir de cada quanti-dade de células utilizadas foi obtido a partir de fotomicrografias obtidas sete dias após a semeadura, com auxílio de um microscópio invertido acoplado a uma câmera e do software Axiovision LES4. Após a obtenção das imagens, os esferoides foram coletados, fixados e processados rotineiramente para avaliação histológica por HE (Hematoxilina e Eosina) e por MEV (microscopia eletrônica de varredu-ra). O sobrenadante do cultivo dos esferoides foi coletado e armazenado a -80o.C para avaliação da secreção de IL-6, VEGF e RANTES por citometria de fluxo, através da metodologia CBA (Cytometric Bead Array, BD Biosciences). A partir da semeadura automatizada de 1,8x10e5 , 4,3x10e5 e 1,3x10e6 células para formar 81 esferoides cada, foram obtidos esferoides com diâmetros de 196,5µm ± 34,3; 343,8 ± 53,1 ; 418,9µm ± 16,2, respectivamente. A avaliação histológica mostrou que os esferoides não apresentaram regiões com morfologia típica de morte celular. Já a avaliação por MEV revelou um micro tecido com material extracelular fibrilar semelhante a componentes de matriz extracelu-lar. A quantidade de IL-6 e VEGF produzida por célula foi maior nos esferoides de maior tamanho: 3,43x10e-5 , 14,99x10e-5 e 24,37x10e-5 pg/mL/célula de VEGF nos esferoides pequeno, médio e grande, respectivamente, e 0, 5,21x10-5 e 9,46x10-5 pg/mL/célula de IL-6 nos esferoides peque-no, médio e grande, respectivamente. Portanto, o tamanho dos esferoides influencia a capacidade das células de sintetizarem as moléculas avaliadas. Sabendo-se que o tamanho dos esferoides tem influência sobre sua funcionalidade, este deve ser um parâmetro a ser considerado em aplicações terapêuticas e biotecnológicas. Avaliações mecânicas e da viabilidade dos esferoides de diferentes tamanhos estão em curso.

Keywords: Esferoides; Automatização; Células-tronco Estromais; Diâmetro; Biofabricação.


IN VITRO EVALUATION OF MESENCHYMAL STEM CELL BEHA-VIOUR IN SCAFFOLDS CONTAINING DIFFERENT CONCENTRA-

TIONS OF ALGINATE AND SODIUM CHLORIDE AS A STRATEGY IN REGENERATIVE MEDICINE.

Albrecht, C. L.¹,²; Maurmann, N.¹,²; Pranke, P.¹,²,³.

1Laboratório de Hematologia e Células-tronco, Universidade Federal do Rio Grande do Sul, Brasil.2Programa de Pós Graduação em Fisiologia, Universidade Federal do Rio Grande do Sul, Brasil.3Instituto de Pesquisa com Células-tronco, Brasil.

Alginate is a biomaterial widely used in the areas of odontology, pharmacy and food industry, and has been studied also due to its potential applications in regenerative medicine. It is a naturally occurring polymer obtained from different species of brown algae. In a certain pH range, with the addition of a bivalent ion such as calcium, it is capable of forming a hydrogel with properties similar to the extracellular matrix, becoming a potential tool in tissue engineering. The aim of this study was to evaluate which concentrations of calcium, alginate and mesenchymal stem cells (MSC) are ideal to the construction of scaffolds as a strategy in regenerative medicine. First, stem cells were treated only with calcium chloride, in order to analyse the toxicity of the excess of the ion to MSC. Thereafter, MSC, in a density of 400.000/well, were mixed to alginate 1% (w/v) and treated with different concentrations of calcium chloride (0; 25; 50; 75 and 100mM) to reticulate the biomaterial. As control, cells were cultivated as usual in the tissue culture plate. In another experiment, 400.000 cells/well were mixed to 0.5% and 1% alginate, and 50mM of calcium chloride were added. Ultima-tely, MSC, in the densities of 100.000, 200.000 and 400.000 per well were added to 1% alginate and treated with 50mM of calcium chloride. Experiments were performed with MSC obtained from human exfoliated deciduous teeth; cell viability was assessed via MTT assay and visualized with fluorescence microscopy after fluorescein diacetate and propidium iodide staining. As a measure of cytotoxicity, the lactate dehydrogenase enzyme (LDH) was dosed and free calcium was quantified. Results showed that after a few hours, even the smallest excess of calcium was toxic to the cells in the absence of alginate. Moreover, it was demonstrated that, after seven days, no significant statis-tical difference between scaffolds with 1% alginate, crosslinked with 50mM of calcium and 100.000 cells and the control wells (with the same concentration of MSC). However, in higher concentrations (especially 400.000/well) it was observed that cell viability was superior in the scaffolds. Further-more, free calcium concentrations remained constant and LDH dosages corroborated with the data above. After seven days, the average absorbances of 400.000 cells in 1% alginate scaffolds with 25 and 50mM of calcium were superior to control; however, experiments have demonstrated that 25mM were not sufficient for completely reticulating alginate, giving rise to soft gels. In the same way, 1% alginate scaffolds showed better results in terms of viability when compared to control, and 0.5% alginate generated very malleable scaffolds, leading to the loss of a number of cells during manipulations. These results suggest that alginate provides a three-dimensional microenvironment which seems to favour survival of big quantities of cells, while the same amount of cells cultivated in the tissue culture plate may not have the same conditions. Besides that, 1% alginate and 50mM calcium chloride demonstrated the best results in mimicking, as far as possible, the characteristics of a natural extracellular matrix.

Keywords: Tissue Engineering; Regenerative Medicine; Mesenchymal Stem Cells; Biomaterials; Alginate; Calcium.

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MEMBRANE PARTICLES OF MESENCHYMAL STEM CELLS AND CONDITIONED MEDIUM POLARIZES MACROPHAGES IN VITRO TO

AN ANTI-INFLAMMATORY PHENOTYPE.

Raymundo, A. C. H.¹; Serafini, M. A.¹; Azambuja, J H.²; Braganhol, E.²; Gonçalves, F. D. C.¹; Paz, A. H. D. R.¹.1Universidade Federal do Rio Grande do Sul, Brasil.2Universidade Federal de Ciências da Saúde de Porto Alegre, Brasil.

The immunomodulatory properties of mesenchymal stem cells (MSC) have been extensively stu-died over recent years. It is believed that these cells can induce immune cells, such as macropha-ges, from a pro-inflammatory profile (M1) to an anti-inflammatory profile (M2). In this way basic and clinical studies have demonstrated the effect of MCS cell therapy to treat inflammatory conditions. However, there are controversial studies about the location, the persistence and the risks of MSC transplantation. Tracking studies have shown that most MSCs are trapped in the lungs due to their size and have a short-term survival in the body after intravenous infusion. In addition, after 24 hours of infusion, MSCs tend to disappear from the lungs suggesting that these cells pass their effects to resident cells. In order to increase the biodistribution of the therapeutic effect of MSC, avoiding cellular entrapment in pulmonary capillaries, as well as providing safer therapy based on MSC po-tential our research group is studying alternative cell therapies such as the use of bioactive factors secreted by MSC (MSC-BF) and the generation of membrane particles from MSC (MSC-MP). To evaluate the immunomodulatory effects of MSC-BF and MSC-MP on the polarization of murine macrophages. Methods: C57BL/6 male mice were used to collect mesenchymal stem cells from epididymal adipose tissue and peritoneal macrophages to perform cell cultures. After culture and expansion of the MSC, these cells were stimulated with LPS, and their conditioned medium (MSC--BF) was collected. Particles from MSC plasmatic membrane were generated by cell lysis and ultra-centrifugation, and their size were analyzed by Nanoparticle Tracking Analysis (NanoSight NS300). The peritoneal macrophages were stimulated with LPS (pro-inflammatory environment control) and IL-4 (anti-inflammatory environment control) and co-cultured with MSC, MSC-MP and MSC-BF to evaluate the macrophage polarization. The cultures were evaluated for CD206 marker by flow cy-tometry (anti-inflammatory macrophage marker). Results and Discussion: Nanoparticle Tracking Analysis shown that the majority of MSC-MPs demonstrated a size of 114nm. Macrophages CD206 expression revealed different results based on the co-culture condition: MSC (MFI= 6.195), MSC--MP (MFI= 4.582) or MSC-BF (MFI= 4.066) indicating that cell-cell contact potentiate the effects of cell culture. With regards to these preliminary results we can conclude that membrane interaction promoted better macrophage polarization into an anti-inflammatory profile in vitro instead of the use of bioactive factors. We can also suggest the feasibility of the membrane particles application to promote the interaction with macrophages.

Keywords: Mesenchymal Stem Cells; Macrophage Polarization; Membrane Particles; Immunomo-dulation.


MESENCHYMAL STEM CELL-DERIVED FACTORS PROTECT THE INTESTINES FROM EXPERIMENTAL COLITIS IN ORGAN CULTURE.

Gonçalves, F.D.C.¹; Aramburu Serafini, M.¹; Henzel Raymundo, A. C.¹; Pfaffenseller, B.¹; Bergmann Araújo, A.¹; Visioli, F.²; Paz, A.H.¹.

1Hospital de Clínicas de Porto Alegre, Brasil.2Universidade Federal do Rio Grande do Sul, Brasil.

Although the mesenchymal stromal cells (MSC) have shown therapeutic potential in intestinal tissue repair, controversy remains concerning to the short survival and their poor biodistribution in recipient tissues suggesting that the therapeutic effects afforded by MSC transplantation are related to the paracrine interactions between MSC and resident cells. Therefore, this paracrine mechanism may have therapeutic potential for cell-free treatment strategies using MSC-secreted bioactive factors. To investigate the paracrine role of MSC in three-dimensional culture of mouse colon with DSS--induced colitis. Methods: Acute colitis was induced in mice by oral administration of 2% dextran sulfate sodium (DSS) for 7 days. Inflammatory responses were assessed on the basis of clinical signs, morphological, and histopathological parameters. On days 2 and 5, colonic explants were re-moved and a three-dimensional culture model was performed for cell biological investigations in an in vivo-like environment. The structural integrity of the intestinal mucosa was tested by treating the cultures with MSC or conditioned medium (CM) for 24 h. The tissue was then prepared for histology/immunohistochemistry, and the culture supernatants were assayed for Il-6 production. Results and Discussion: Histological analysis demonstrated MSC and CM treatment reduced colon damage in organ culture. An increased in cell proliferation (Ki-67 staining) was observed after CM treatment in colonic explants. Additionally, the MSC treatment was able to reduce CD3+ cells. The therapeutic effect of MSC and CM was most likely mediated by the downregulation of pro-inflammatory cytokine IL-6. Accordingly, the intestinal in vitro model has demonstrated to be potentially useful for studying cellular interactions in a three-dimensional cell arrangement. Moreover, both MSC and CM treat-ments provide strong evidence that they can alleviated the colonic damage in organ culture. These results suggest MSC-secreted factors are capable of providing support without cell transplantation to protect from colon inflammation caused by DSS-induced colitis.

Keywords: Organ Culture; Three-Dimensional Culture; DSS-Induced Colitis; Conditioned Medium; Mesenchymal Stromal Cell; Cell Therapy.

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MODULAÇÃO DA MIGRAÇÃO E MORFOLOGIA CELULAR DE QUE-RATINÓCITOS A PARTIR DE MEIO CONDICIONADO DE CÉLULAS ESTROMAIS MESENQUIMAIS DERIVADAS DE TECIDO ADIPOSO

HUMANO.

Zanatelli, C.¹; Rodrigues, C.¹; Wink, M. R.¹; Naasani, L. I. S.¹; Paim, T. C.¹; Azevedo, J. G.¹.

1Universidade Federal de Ciências da Saúde de Porto Alegre, Brasil. A pele, por se tratar de um tecido externo está propensa a uma série de agressões, tais como trau-ma mecânico, queimaduras ou feridas crônicas. A cicatrização de feridas é o processo pelo qual o tecido do corpo se conserta após um trauma, abrangendo uma série de sinalizações químicas, deposição de matriz extracelular, proteólise e proliferação celular. A re-epitelização da pele no local da injúria também requer a migração rápida e coordenada de queratinócitos para o leito da ferida, possibilitando o remodelamento e funcionalidade do tecido regenerado.Terapias de regeneração epitelial vêm sendo desenvolvidas com células e matrizes através de diferentes técnicas de cultivo e tem demonstrado um grande potencial terapêutico na cicatrização de lesões de pele. As células estromais mesenquimais (MSCs) são células multipotentes, com capacidade de diferenciação em células especializadas, tais como osteoblastos, condrócitos e adipócitos. Possuem capacidade de auto-renovação e viabilidade em longo prazo. Além da função de reposição celular, secretam elementos tróficos e imunomoduladores que podem ser úteis no tratamento de uma série de con-dições clínicas. A capacidade regenerativa da pele é orquestrada por diferentes tipos de células, incluindo células estromais mesenquimais (MSCs). Seu papel na cicatrização de feridas vem sendo amplamente estudado, devido à sua capacidade de produzir um secretoma capaz de modular seu microambiente. Neste trabalho, investigamos a influência do meio condicionado (MC) proveniente de MSCs de tecido adiposo na resposta mecânica de culturas de queratinócitos HaCaT. O estudo de parâmetros celulares, como a morfologia e migração, são de grande interesse devido ao seu papel importante nos processos biológicos tais como embriogênese, morfogênese tecidual e rege-neração. As MSCs foram isoladas de tecido adiposo abdominal humano proveniente lipoaspirados humanos doados (REC-ISCMPA nº882968), o tecido foi processado e as células foram isoladas e cultivadas em atmosfera controlada. A identidade mesenquimal das células foi confirmada através de diferenciação celular. Ao confirmar as características das células, estas foram expandidas em laboratório para a confecção do meio condicionado (MC). Os queratinócitos humanos imortalizados (HaCat) tiveram sua viabilidade analisada através de contagem com azul de tripan, a migração celular foi avaliada através do ensaio “scratch wound assay”, e a morfologia celular foi analisada através de coloração do citoesqueleto por actina e o núcleo foi avaliado através de Análise Mor-fométrica Nuclear (NMA) realizada por software. As MSCs derivadas do tecido adiposo exibiram morfologia fusiforme alongada (semelhante a fibroblastos), potencial de autorrenovação e aderên-cia plástica. Sua multipotência foi demonstrada pela incubação das células em meios capazes de promover a diferenciação nas linhagens condrogênicas, adipogênicas e osteogênicas. Culturas de queratinócitos tratadas com MC revelaram aumento nas taxas de proliferação, quando comparadas ao controle negativo em 24 horas de cultivo e migraram mais rapidamente, fechando maior parte da lesão no ensaio de migração. Este estudo permitiu evidenciar que os fatores secretados por MSCs derivadas de tecido adiposo humano tem o potencial de regular a forma e causar a disseminação de queratinócitos humanos, esse conhecimento é essencial para melhor estabelecer a segurança e a eficácia das terapias baseadas em células.

Keywords: Regeneração; Pele; Reparo.


NEUROPROTECTIVE EFFECTS OF INTRAVITREAL INJECTION OF TRANSGENIC MESENCHYMAL STEM CELLS EXPRESSING HIGF-1

IN MOUSE VISUAL SYSTEM AFTER OPTIC NERVE CRUSH.

Vasques, J. F.1; Abreu, C. A.1; Souza, B. S. F.2; Soares, M. B. P.2; Mendez-Otero, R.1.

1Universidade Federal do Rio de Janeiro, Brasil.2Fundação Oswaldo Cruz, Brasil.

Retinal ganglion cells (RGC), like other central nervous system neurons, have reduced regenerative capacity and, once damaged, the axons of adult RGC do not regenerate at long distances through the optic nerve. In addition, RGCs are extremely sensitive to axonal damage and most of them die after injury to the optic nerve. There are, currently, no clinically applicable therapies capable of protecting RGC and increasing the regeneration of their axons in an efficient and prolonged way. Recently, bone marrow-derived cells have been used to increase regeneration and/or functional re-covey in several animal models of neurological diseases. In visual system, mesenchymal stem cells (MSC) were able to increase the survival of RGC in glaucoma models and after axonal transection or optic nerve crush, as demonstrated by several previous studies, including from our group. Howe-ver, even inducing a significant increase in survival and regeneration of RGC, MSC therapy was not able to promote functional recovery. In this work our main objective is to potentiate the regenerative response of cell therapy through the use of a mouse transgenic MSC line expressing the human tro-phic factor IGF-1 (mMSC-hIGF1), which has neuroprotective and neuroregenerative action. Metho-dology: SvEv129 adult mice were submitted to optic nerve crush and intravitreal injection of vehicle or 90,000 or 200,000 mMSC-hIGF1/control-mMSC. 14 days after surgery retinas were dissected and immunohistochemistry for TUJ1, marker of RGC, was performed. Results and discussion: Intra-vitreal injection of 90,000 mMSC-IGF1 cells after optic nerve crush was enough to induce the same significant neuroprotection evoked by 200,000 control-mMSC, leading to an increase of about 45% in the number of RGC compared to animals treated with PBS. Injection of 200,000 mMSC-hIGF1 promoted an even more robust and significant neuroprotective response, with an increase of about 90% in the number of RGC detected in relation to the PBS group. These data suggest that IGF-1 expression is capable of significantly potentiate the neuroprotective action of MSCs. It remains to be determined whether this effect is sustained after long periods and if it also results in an increase in axonal regeneration through the optic nerve and visual function recovery.

Keywords: Mesenchymal Stem Cells; Optic Nerve Crush; Neuroprotection IGF-1.

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NON-HYPERTROPHIC CARTILAGE BY SCAFFOLD-FREE TISSUE ENGINEERING VALIDATED BY COMPARATIVE SECRETOME

ANALYSIS.

Côrtes, I.¹; Matsui, R. A.¹; Beatrici, A.²; Azevedo, M. S.¹; Souza, K. L.¹; Delolme, F.³; Moali, C.³; Gran-jeiro, J. M.²; Baptista, L. S.¹,⁴.

1Núcleo de Pesquisa Multidisciplinar em Biologia - NUMPEX-Bio, Brasil.2Instituto Nacional de Metrologia, Qualidade e Tecnologia, Brasil.3Centre National de la Recherche Scientifique, Universidade de Lyon, França.4Universidade Federal do Rio de Janeiro, Brasil.

Articular cartilage has a poor capacity of regeneration and promising adult stem cell sources for car-tilage regeneration include adipose stem/stromal cells (ASCs) from subcutaneous adipose tissue. In this study, we attempted to engineer a non-hypertrophic cartilage scaffold-free tissue engineering, represented by spheroids. Human lipoaspirate samples and cartilage fragments from nasal septum were obtained from healthy donors that underwent aesthetic surgery procedures (Protocols 145-09 and 146-09). Cell suspension was seeded in a micromolded agarose hydrogel for spheroids forma-tion for three experimental groups: Induced ASC spheroids; Non-induced ASC spheroids and Carti-lage progenitor cells (CPC) spheroids. For chondrogenic induction, ASC spheroids were maintained under hypoxia (10% O2 and 5% CO2) in culture medium supplemented with 10 ng/ml TGF-β3 and 10-8 M dexamethasone. ASC and CPC spheroids were kept at 37 °C with 5% CO2 and atmospheric O2, in absence of chondrogenic inducers. All experimental groups were maintained for until 21 days. Induced and non-induced ASC spheroids showed most viable cells identified by 7AAD exclusion strategy. The level of TGF-β1 in supernatant evaluated by multiplex assay of induced ASC spheroids significantly decreases comparing day 21 to 7 (p = 0.0020) and low level of TGF-β3 was detected in supernatant of non-induced ASC spheroids at days 7 (p = 0.0022), 14 (p = 0.0022) and 21 (p = 0.0022) compared to induced ASC spheroids. Related to interleukins levels, the supernatant of non--induced and induced ASC spheroids showed similar high levels for IL-6 (p = 0.3282) and IL-8 (p = 0.7137) at day 7. The supernatant of induced ASC spheroids showed a decrease at day 21 for IL-6 (p < 0.0001) and for IL-8 (p < 0.0001) compared to day 7. The master gene for chondrogenesis – SOX9 – was significantly upregulated at day 14 (p = 0.0436) and day 21 (p = 0.0004) compared to day 7 in induced ASC spheroids. SOX5 (p = 0.0029) and SOX6 (p < 0.0001) were significantly upregulated at day 21 compared to day 7. Induced ASC spheroids were completely positive for collagen type I, type II, type VI and aggrecan. The intensity of staining for collagen type I was low compared to collagen type II, the typical collagen from hyaline cartilage. Interestingly, collagen type X, a typical collagen from hypertrophic cartilage, decreased its intensity along induced ASC spheroids culture. Induced ASC spheroids showed a significantly high force module at day 7 (p = 0.0003), 14 (p < 0.0001) and 21 (p = 0.0007) compared to non-induced ASC spheroids evaluated by Microsquiser equipment. Se-cretome (proteomic of cell culture supernatant at day 21) was analyzed by LC-MS/MS. Experiments were performed on a QExactive HF mass spectrometer operating with Xcalibur software (version 4.0). We identified up to 644 proteins, including proteins related to skeletal development, extracellular matrix remodeling and collagen synthesis. ASC induced spheroids showed high viability, a high level of TGF- β3 and SOX-9 gene expression besides a high force module. These spheroids showed the typical extracellular matrix components of cartilage, most of them confirmed in secretome. More im-portantly the collagen type X was not identified in secretome and its intensity of staining decreases along induced ASC spheroid culture. We can conclude that a non-hypertrophic phenotype was rea-ched by induced ASC spheroids.

Keywords: Secretome; Spheroids; Adipose Derived Stem/Stromal Cells (Ascs); Chondrogenesis.


OSTEOGENIC POTENTIAL OF MULTICELLULAR SPHEROIDS CONS-TITUTED BY HUMAN BURSA SUBACROMIALIS-DERIVED MESEN-

CHYMAL STEM CELLS.

Amaral, A. C.¹; Erlo, G.¹; Moreira, S. B.¹; De Carvalho, R. A.¹; Dernowsek, J. A.²; Rezende, R. A.²; Da Silva, J. V. L.².

1Universidade de Araraquara, Brasil.2Centro de Tecnologia da Informação Renato Archer, Brasil.

Regenerative Medicine is a branch of medicine that has markedly advanced in recent years. Achie-vements are mainly related to use of mesenchymal stem cells (MSCs), associated or not with biolo-gical supports and/or bioactive molecules, with intention of improving the efficiency of tissue regene-ration process. Although several studies have shown the potential of MSC therapy, especially on the bone healing process, there is a need to expand the knowledge in order to enable its clinical utilizaty in an effective and safe way. Parameters as obtaining the source of MSC and culture procedures in cell expansion phase are known to be able to influence the cellular niche and, consequently, the therapeutic potential of this technique. OBJECTIVE: To analyze the osteogenic potential of multicellular spheroids from human bursa subacromialis-derived mesenchymal stem cells (hBSD-MSC). METHODOLOGY: Fragments of the bursa subacromialis were extracted from a 48 years old volunteer by arthroscopic procedure and submitted to a process to isolate the mesenchymal cell contingent. Upon reaching third passage, the mesenchymal profile of cells was determined by adipogenic and osteogenic differentiation and flow cytometry. Multicellular spheroids were consti-tuted by non-adherent surface culture technique. After 5 days of culture, spheroids started to be cultivated in osteogenic medium for an additional period of 14 days. The osteogenic differentiation potential of hBSDMSC in spheroids was determined by alizarin red staining technique. RESULTS: Characterization procedures confirmed the mesenchymal profile of cells isolated from human bursa subacromialis. After culturing on a non-adherent surface, cells constituted multicellular spheroids, which reached an average diameter of 200 μm after x time. Alizarin red staining demonstrated the osteogenic differentiation potential of hBSDMSC cultured as multicellular spheroids. hBSDMSC, cultured as multicellular spheroids in an “in vitro” expansion phase, constitute a promising alternati-ve for research and clinical applications in bone regenerative medicine strategies.

Keywords: Mesenchymal Stem Cells; Regenerative Medicine; Cell Therapy; Multicellular Spheroids.

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PLACENTA-DERIVED MESENCHYMAL STEM CELLS CULTURE ON PMMA SCAFFOLDS AS A 3D PUTATIVE MODEL OF HEMATOPOIETIC

STEM CELLS NICHE IN VITRO.

Lucas, R. P.¹; Belchior, L. F.¹; Alvarez-Silva, M.¹.

1 Universidade Federal de Santa Catarina – UFSC, Brasil.

The umbilical cord blood (UCB) have practical and ethical advantage over classical sources of hematopoietic stem/progenitor cells (HSPCs) for transplantation. Nevertheless, the small volume of blood available in the vascular networks of umbilical cord blood and placenta compromises its clinical use for transplantation into adults due to lower counts of HSPCs. One challenge of tissue en-gineering is reproducing the biological bone marrow microenvironment where the HSPCs naturally expand. The use of mesenchymal stem cells (MSCs) associated with three-dimensional (3D) scaf-folds is a good approach because they represent keystone players in the marrow niche that modula-tes the behavior of HSPCs through biochemical signals that positively favors it symmetric divisions. The objective of this study was to evaluate the interaction and morphology of human placenta-deri-ved mesenchymal stem cells (P-MSCs) in a 3D culture system based on poly(methyl methacrylate) (PMMA) scaffolds. For this purpose, full-term health human placentas were collected in the Depart-ment of Obstetrics from the University Hospital of the Federal University of Santa Catarina. All pro-cedures were approved by the Human Being Research Ethics Committee of the University (Protocol Number 198/03). Cells were isolated based on their culture plastic adhesion from the chorionic villi of the human placenta which compose one of the HSPCs niche during embryonic development. As described in previous work, P-MSCs were morphologically and functionally characterized in vitro, having fibroblast-like morphology, and exhibiting ability to differentiate to adipogenic and osteogenic mesodermal linages. We also checked the cell ability to form colonies when cultured at low density. In addition, porous poly(methyl methacrylate) (PMMA) scaffolds were fabricated using a modifica-tion of the commonly used salt-fusion particulate leeching method with the polymerization of methyl methacrylate monomer mixed with 2,2′-Azobis(2-methylpropionitrile (AIBN). Stereomicroscope and scanning electron microscopy (SEM) analysis revealed test groups with typical spongy 3D struc-ture with well-interconnected macropores and porosities ranging from 71,71% to 75,14%. These biomaterials correlate with bone trabeculae. We also observed micro-topographies pattern on the wall surfaces of all produced biomaterials. Finally, it was possible to culture the P-MSCs in the PMMA scaffolds. SEM analysis revealed that P-MSCs were firmly attached to the PMMA scaffolds and they were individually distributed or forming interconnections and clusters. The cells showed multiple morphologies with elongated or strained shape. Confocal microscopy analysis showed the presence of long and parallel actin bundles. Based on our results we concluded that this 3D culture system of P-MSCs in PMMA scaffolds may be an attractive model for ex-vivo expansion of HSPCs in association with P-MSCs as stromal elements of the niche.

Keywords: Mesenchymal Stem Cells; Human Placenta; PMMA Scaffolds; Hematopoietic Stem Cells Niche.


PROLIFERAÇÃO DE CÉLULAS-TRONCO MESENQUIMAIS DERIVA-DAS DE POLPA DE DENTE PERMANENTE HUMANO EM POLÍME-

ROS TEMOPLÁSTICOS ABS E PLA IMPRESSOS EM 3D.

Barchiki, F.¹; Senegaglia, A. C.¹; Boldrini Leite, L. M.¹; Hochuli, A. H. D.¹; Micosky, A.¹; Kuligovski, C.²; Correa Dominguez, A.²; Moura, S. A. B.³; Brofman, P. R. S.¹

1Pontifícia Universidade Católica do Paraná, Brasil.2Instituto Carlos Chagas, Brasil.3Universidade Federal do Rio Grande do Norte, Brasil.

Introdução: A necessidade de alternativas para o reparo e substituição de tecidos vivos acometidos por traumas ou alterações patológicas impulsionou o desenvolvimento de materiais sintéticos bio-compatíveis. O poli (ácido lático) (PLA) é um biomaterial muito utilizado e promissor, principalmente por ser de origem orgânica. O acrilonitrila-butadieno-estireno (ABS) é um polímero barato e suas propriedades físico-químicas sugerem sua utilização como biomaterial. PLA e ABS podem ser utiliza-dos em impressoras 3D e sua associação com células-tronco mesenquimais derivadas de polpa de dente humano (DPSC) poderá proporcionar uma aplicação clínica personalizada. Objetivo: Analisar a proliferação e viabilidade das DPSC cultivadas sobre protótipos em ABS e PLA impressos em 3D. Metodologia: Aprovado pelo Comitê de Ética e Pesquisa da PUCPR, parecer 1.838.022. Discos de ABS e PLA (1,8cm de diâmetro X 2mm de espessura) foram impressos em 3D, lavados e esterilizados por óxido de etileno. As DPSC foram isoladas por digestão enzimática de dentes permanentes (n=5) de pacientes em tratamento ortodôntico. As células foram cultivadas com IMDM, antibiótico, soro fetal bovino em incubadora a 37°C, 5% de CO2. Após a primeira passagem (P1) as células foram transdu-zidas, passando a expressar luciferase. Esta enzima degrada D-Luciferina (150ug/mL) ofertada nas análises gerando energia luminosa que pode ser captada e mensurada pelo equipamento IVIS Lumi-na II. Foram plaqueadas 30.000 células (P3) na região central poço controle e dos discos de ABS e PLA, após 24 horas as células foram lavadas com tampão fosfato salina para remoção de células não aderidas. Os polímeros foram transferidos para outra placa, de modo que somente células aderidas foram analisadas. A proliferação e migração celular sobre a superfície dos polímeros foram mensu-radas pela emissão de sinal luminoso das células por 7 dias. A viabilidade celular foi verificada pela técnica de citometria de fluxo no FACS Verse utilizando o corante vital 7-AAD e anexina previamente e posteriormente a 14 dias de cultivo sobre os polímeros. Resultados e Discussão: Das amostras de DPSC estudadas, três apresentaram semelhança no tempo de proliferação até a confluência (~13 dias) e número de células (~4,8XE6) na P1. A análise da proliferação celular na P3 demonstrou que as células estavam aderidas aos polímeros e que houve intensificação do sinal bioluminescente captado no decorrer das análises (intervalo de 7 dias). A proliferação e a migração celular foram mais eviden-tes no controle, seguidos pelas amostras de PLA e ABS. Estes dados corroboram outros da literatura que analisaram proliferação de tipos celulares distintos nas superfícies de PLA e ABS. A viabilidade das DPSC previamente ao cultivo sobre os polímeros testados foi 98,4% enquanto que após cultivo na superfície do ABS foi 96,6% e do PLA 96,2%. A análise com anexina mostrou reatividade (0,18%) nas DPSC antes do cultivo sobre os polímeros e valores aproximados foram observados nas células cultivadas sobre ABS (1,22%) e PLA (1,62%). A redução da viabilidade após o cultivo direto sobre os polímeros ABS e PLA também foi demostrado em outro estudo. Conclusão: A proliferação e viabili-dade das DPSC cultivadas sobre polímeros comerciais e de baixo custo ABS e PLA demonstram um potencial uso na bioengenharia tecidual. Os resultados são promissores no âmbito das pesquisas biomédicas e encorajam a realização de estudos adicionais.

Keywords: Células-tronco Mesenquimais; Polpa De Dente Permanente; Poli (Ácido Lático) (PLA); Acrilonitrila-Butadieno-Estireno (ABS); Impressão 3D.

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RELATO DE CASO DE TRATAMENTO DE FERIDA, POR COMPLI-CAÇÃO PÓS-OPERATÓRIA, COM PLASMA RICO EM PLAQUETAS

(PRP).

Borim, H. H.¹; Kobayashi, E. M. ¹; Daleffe, D.²; Lemos, A. C. L.¹; Villas-Bôas; G. L.¹; De Souza, I. M.¹; Gongora, J. S.¹; Morikawa, K. A.¹; Bastos, R. L.¹; Jorge, V. M. H.¹.

1Universidade Estadual de Maringá, Brasil.2Hospital Universitário Regional de Maringá, Brasil.

As feridas podem ser classificadas quanto a etiologia, complexidade e tempo de existência. As fe-ridas simples, evoluem para a resolução de forma espontânea, seguindo as fases da cicatrização. Já as complexas, podem necessitar de intervenção para a sua resolução. Feridas crônicas são aquelas que não cicatrizam espontaneamente em 3 meses e que, frequentemente, apresentam como complicação processos infecciosos, podendo ser complexas, sobretudo quando associadas com patologias sistêmicas que prejudicam o processo de cicatrização. Assim, se faz necessário o uso de curativos, que consistem em meio terapêutico utilizados com finalidades diversas em feri-das, tanto para limpeza, quanto para absorção, drenagem ou auxílio na sua resolução. O presente trabalho relata o caso clínico de uma paciente, 73 anos, portadora de ferida cirúrgica pós-fratura em tornozelo direito há 5 anos e tratada com plasma rico em plaquetas (PRP), uma preparação de plasma autólogo com elevação na concentração de trombócitos. Os dados coletados foram obtidos através da anamnese, exame físico e registro fotográfico da ferida. A correção cirúrgica da fratura não foi feita de imediato, devido a edema e bolhas na pele, sendo adiada para tratar a infecção superficial. Foi operada com fixação interna com placa e parafusos e fio de Kirschner. Após 1 se-mana de alta (40 dias pós fratura), a ferida cirúrgica abriu e expôs os materiais de fixação interna e os tendões dos extensores dos dedos do pé. A infecção aumentou a ferida e a exposição óssea e tendínea, impossibilitando qualquer outra técnica de cobertura cutânea. Foi proposto curativos seriados com infiltração perilesional de PRP (6 mL), para aceleração do processo de cicatrização cutânea, uma vez que já havia infecção óssea e articular. Com 4 meses de tratamento foi realizada cirurgia de aproximação dos bordos, pois, a infecção estava debelada. No mês seguinte foram reti-rados os parafusos, após constatação de calo ósseo. Foi liberada para caminhar com carga parcial no início, para ganho de movimento da articulação tibiotalar. A dor era nível 6 (escala de 0 a 10) no início da fisioterapia (10 meses pós fratura) e persistiu por 4 anos, quando começou a diminuir e, atualmente, só ocorre após caminhar por mais de 12 quadras. Tem boa mobilidade do tornozelo, consegue caminhar com sapatos de salto, sem instabilidade e com bom posicionamento e apoio do pé. Marcha normal. Métodos tradicionais utilizados na terapia de lesões nem sempre alcançam o resultado desejado, o que se deve, por vezes, à não presença de Fatores de Crescimento (FCs). O PRP auxilia nesse aspecto, visto sua capacidade de liberação de FCs, como Fator de Crescimento Derivado de Plaquetas, Fator de Transformação do Crescimento, Fator de Crescimento Endotelial Vascular, Fator de Crescimento Semelhante à Insulina, e propriedades miogênicas e quimiotáti-cas. A partir da degranulação de grânulos alfa plaquetários, os FCs liberados pelo PRP auxiliam a cicatrização ao atrair células não diferenciadas para a matriz recém-formada e estimulam sua divisão celular. Além disso, modula a liberação de citocinas, limitando o processo inflamatório, en-quanto mantém atividade antimicrobiana contra agentes como Escherichia coli e Staphylococcus aureus, incluindo meticilina resistente. Com o relato deste caso, tem-se a intenção de demonstrar uma alternativa de tratamento para casos de feridas crônicas que não apresentam melhora com os tratamentos tradicionais.

Keywords: Plasma Rico Em Plaquetas; Cicatrização De Feridas.


THE SECRETOME OF ADIPOSE STEM CELLS HUMAN PROTECTS AGAINST GENOTOXICITY GENERATED BY OXIDATIVE STRESS.

Silva-Acordi, C.¹; Delben, P. B.¹; Fagundes, A. C.¹; Gomes, R. S.²; Trentin, A. G.¹.

1Universidade Federal de Santa Catarina, Brasil.2Hospital e Maternidade Dr. Carlos Correa, Brasil.

Human life expectancy is increasing due to the quality of life improvement. Likewise, pathologies associated with aging are also growing and aggravating social and economic sectors. The aging process is related to the increase of damage in DNA whit loss of genetic integrity. The accumule of the stem cells damaged contributes to the progress of human aging. Adipose stem cells (hASC) secretome has cytoprotective effect and reduce damage associated with early cellular senescence. The objective of this research is to evaluate the effect of hASC-conditioned medium in the upkeep of human mesenchymal stem cell integrity genetic. Methods: To achieve our objective, in this rese-arch we analyzed nuclear anomaly by blockade of cytokinesis by cytochalasin B assay (CBMN) and immunodetection genotoxicity 8-Oxig.Our results showed a reduction of nucleocytoplasmic bridge and micronucleus, indicators of chromosomal instability and reduction of the presence of 8-Oxig, which suggests reduction of oxidative damages in the DNA in the groups treated with secretome of hASC. In conclusion, the secretome from hASC protects mesenchymal stem cells against genetic instability. Consequently, this secretome has the potential to preserve the stem cell status that is lost during human aging.

Keywords: Secretome; Hasc.

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USING HUMAN ADIPOSE STEM CELLS SPHEROIDS AS NOVEL APPROACH FOR ASSESSMENT OF SILVER CHLORIDE NANOPARTI-

CLES CHRONIC EXPOSURE.

Charelli, L. E.¹; Muller, N. V.¹; Silva, K. R.¹; Sant’anna, C.¹; Baptista, L. S.². 1Instituto Nacional de Metrologia, Qualidade e Tecnologia, Brasil.2Universidade Federal do Rio de Janeiro, Brasil.

Human mesenchymal stem cells (hMSC), such as adipose stem cells (ASCs), play a crucial role in tissue regeneration, mainly through direct differentiation or secreting anti-inflammatory/repair mole-cules at the injury site. Hence, they are widely used in tissue bioengineering. Due its antimicrobial properties, silver-based nanoparticles (AgNPs) have been extensively used as coating for biome-dical implants, including scaffolds seeded with hMSC. Chronic ASCs exposure to nanoparticles -at sub-lethal levels- may occur due to NPS coating in skin implants or even from sunscreens and cosmetics that contains it. Compared with monolayer culture, 3D culture system is able to enhance stemness, anti-inflammatory and proangiogenic properties of ASCs. Indeed, the use of 3D culture in toxicology assays has shown an increase of accuracy of in vitro drug assessment, mainly due to cell-matrix architecture seen in 3D cultures, that promotes biological barriers, resulting in diffusion and transport conditions more similar with those found in vivo. To standardize 3D spheroids culture system as new methodology for nanotoxicological in vitro assays. For this purpose, we evaluated the effect of sub-lethal (5, 10 and 25µg/mL) concentrations of silver chloride nanoparticles (AgCl--NPs), on ASCs spheroid cultures, throughout 21 days of treatment. Human lipoaspirate samples were obtained from healthy donors. This study was approved by the Research Ethics Committee of the Clementino Fraga Filho University Hospital (UFRJ-Protocol 145-09). After the ASCs isolation, the monolayer was maintained in culture flasks (37ºC and 5%CO2) until to reach the confluence of 2x106 cells. Then, ASCs were harvested and seeded into a micro-molded non-adhesive hydrogel with 81 circular recesses (2% agarose -UltrapureAgarose, Invitrogen- in 0.9% NaCl), according to the manufacturer’s recommendations (Microtissue Inc). ASCs spheroids were treated with sub-le-thal concentrations (5,10and 25µg/mL) of AgCl-NPs for 21days. Cell morphology was accessed by phase contrast microscopy. Oxidative stress(ROS) and cells viability were accessed by flow cytometry. The ultrastructural morphology was examined by scanning and transmission electron microscopy. The quantification of cytokines in ASCs supernatants was performed by Cytometric Bead Array (BD) and a multiplex immunoassay BioPlex MAGPIX (BioRad), respectively. Also, the qualitative analysis of viability was examined by fluorescence microscopy, using the kit LIVE/DEAD (Thermo Fisher). ASCs spheroids morphology and diameter remained widely unaltered after light microscopy evaluation. No statistical significance was found for cell viability. ROS levels increased significantly for the group treated with 5 and 10µg/mL at day 7 (p=0.0395, p=0.0266, respectively). Ultrastructural analysis showed cell damage only on the surface of ASCs spheroids treated with 10µg/mL. Nevertheless, it was observed that -all ASCs spheroids treated with AgCl-NPs- had signi-ficant (p<0.05) alterations on their secretory profile, mainly IL-6, IL-8, and IL-1β, at specific concen-trations and kinetics. Transforming growth factor - β1 and -β2 was also altered significantly (p<0.05) throughout the treatment period. Our results indicate that the sub-lethal concentrations of AgCl-NPs tested altered the secretory profile of ASC spheroids, which may lead to long-term disorder in tissue homeostasis (e.g. subcutaneous adipose tissue exposed by products containing these nanoparti-cles).

Keywords: Spheroids; Adipose Stem Cells; 3D culture; Interleukin secretion; Silver Chloride Nano-particles; alternative method.

Abstract - Scaffold

CORRELATION BETWEEN THE SOFTNESS OF HYALURONIC ACID MICROPARTICULATE SCAFFOLDS AND THE PROLIFERATION OF

HUMAN ADIPOSE MESENCHYMAL CELLS.

Santana, M. H. A¹; Luzo, A. C. ¹; Shimojo, A. M.¹.

1Universidade Estadual De Campinas, Brasil.

Hyaluronic acid (HA) is a naturally occurring biomacromolecule found in most connective tissues and is particularly concentrated in synovial fluid. HA chemical structure is composed by disacchari-de units of D-glucuronic acid and D-N-acetylglucosamine, linked together through alternating beta-1,4 and beta-1,3 glycosidic bonds. HA polymer structure is characterized by large and linear chains in which the number of repeat disaccharide units leads to a molecular mass of 400 Da. Nowadays, HA has been widely used as a viscosupplement in orthopedics, in plastic, intraocular and abdominal surgeries and wound healing. High molecular weight hyaluronic acid (HA) associated to platelet--rich plasma (PRP) has been used for better effects in wound healing and regenerative medicine. However, the fluid or plain HA is very soluble in aqueous medium, and modifications are necessary to provide structural stability. HA molecular structure presents sites for covalent modification, such as carboxylic acid and hydroxyl, which have been explored for production of useful materials for bio-medical applications, as well as to the production of scaffolds for cell cultivation. This study aimed to prepare and characterize various crosslinked HA based scaffolds, structured in microparticles, and to evaluate their performance on “in vitro” proliferation of human adipose mesenchymal cells (AdMSCs) stimulated by the growth factors from PRP. The rational for using crosslinked HA is to in-crease stability against degradation and increase the residence time “in vivo” compared to non-cros-slinked fHA, as well as the structuration in microparticles provides a larger surface area for cell pro-liferation. HA was crosslinked with 14-butanediol diglycidyl ether (BDDE), forming HA-BDDE gels, and autocrosslinked in its ester bonds by means of intermediate activation, giving HA- ACP gels. These gels also were treated with chitosan for attenuation of HA negative charge. The mean diame-ter of the microparticles was 200µm approximately. PRP was prepared by centrifugation of whole blood and activated by autologous thrombin and calcium chloride (aPRP). The gels were evaluated in a proportion of 200mg of crosslinked HA to 200µL aPRP. The rheological characterization shown all gels were predominantly viscoelastic. The viscoelasticity increased from HA-BDDE:PRP > HA--ACP-CHI:PRP> HA-ACP:PRP> aPRP. Non-crosslinked HA did not present a gel behavior. The complex module, G*, which is related with rigidity of the material, decreased from HA-BDDE:PRP > HA-ACP-CHI:PRP> HA-ACP:PRP> aPRP The range of G* was from 1500 (HA-BDDE) to 47 (HA-ACP:PRP) and 21 (aPRP). The “in vitro” culture of AdMSCs shown higher proliferation in HA--ACP-CHI:PRP scaffolds and aPRP. The lower cell proliferation was obtained in HA-BDDE:PRP. Therefore, despite the much larger surface area provided by the fibrin fibers in aPRP, and also the instability of te HA-ACP:PRP microparticles, there was a clear correlation between the softness (lower rigidity) and AdMSCs proliferation. The softness surface of the HA-ACP-CHI :PRP micropar-ticles added to its viscoelasticity provided a favorable environment to adhesion and proliferation of AdMSCs. These results are relevant to the development of HA based scaffolds for applications in healing and regenerative trerapies.

Keywords: Hyaluronic Acid; Microparticles; Scaffolds; Human Adipose Mesenchymal Cells.

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Abstract - Scaffold

DYNAMIC FUNCTIONALIZATION OF VASCULAR SCAFFOLDS.

Braghirolli, D. I.¹,²; Teixeira, G. B.³; Pranke, P¹,²,⁴.

1Laboratório de Hematologia e Células-tronco, Universidade Federal do Rio Grande do Sul, Brasil.2Programa de pós-graduação em Fisiologia, Universidade Federal do Rio Grande do Sul, Brasil.3Universidade Federal do Rio Grande do Sul, Brasil.4Instituto de Pesquisa com Células-tronco, Brasil.

Currently, there is a great lack of vascular grafts suitable for use in vessel replacement surgeries, es-pecially for application in small vessels. The commercially available synthetic supports have a high failure rate due to thrombosis and intimal hyperplasia.Tissue engineering (TE), therefore, has been evaluated as an alternative for the development of vascular substitutes. In TE, tubular scaffolds can be produced and associated with drugs in order to prevent the formation of thrombi in their interiors guaranteeing the sucess of its clinical application. However, the geometry of these scaffolds can mi-nimize their binding to biomolecules, especially in their lumen. This study has aimed to evaluate the use of bioreactors to functionalize dynamically the internal surface of vascular scaffolds with the he-parin anticoagulant. Tubular scaffolds of poly(caprolactone) with 6 mm diameter were produced by electrospinning. The scaffolds were inserted into rotating bioreactor chambers and their lumen were filled with 2M NaOH for 30 minutes. Subsequently, the supports were rinsed with water and then left in contact with 2 mL of a fresh solution of 1% (w/v) heparin, 5 mg/mL N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) and 5 mg/mL N-hydroxysuccinimide (NDS). Bioreactors were rotated at 1 rpm, overnight. After functionalization, the scaffolds were removed from the chambers and cut into 1 cm2 pieces. Following this, these samples were submitted to the blue toluidine test for determi-nation of heparin content. As a control, flat scaffolds, with 1 cm2, were statically functionalized. For this, the heparin solution was dispensed on the surface of the scaffolds and maintained overnight. The blue toluidine test demonstrated that the heparin content was significantly higher in statistically functionalized biomaterials. The tubular scaffolds exhibited 131.9 /cm2 of heparin. Meanwhile, the scaffolds functionalized by the static method showed 143.5 μg/cm2 of heparin on their surface. This result was already expected. In static functionalization, there is no fluid movement and, thus, the chemical bonds between biomolecules and 3D structures can be maximized. Although the heparin binding was lower when dynamic functionalization was performed, this method is ideal for the as-sociation of drugs to tubular structures. The rotating bioreactor allows for a constant contact of the biomolecules with the entire internal wall of the material structure, ensuring homogenous functio-nalization. Moreover, the concentration of an anticoagulant, obtained with the dynamic process, is enough to confer antithrombogenic properties to the scaffolds. In accordance with previous studies of this research group, the obtained concentration prevents the formation of thrombi on the bioma-terial surface. Another important feature of the homogeneous heparin functionalization of scaffolds is that they can reduce the proliferation of smooth muscle cells and thus, prevent the occurrence of neointimal hyperplasia.The present work has demonstrated that the use of rotating bioreactors allows for the attachment of heparin to the internal surface of tubular scaffolds in an adequate con-centration for their thrombogenic activity. The homogenous functionalization of tubular biomaterials with heparin can prevent thrombosis and intimal hyperplasia in their lumen, the two major causes of failure of the vascular grafts.

Keywords: Tubular Scaffolds; Vascular Scaffolds; Vascular Tissue Engineering; Bioreactor.

Abstract - Scaffold

ESTUDO DAS PROPRIEDADES ADESIVAS E CITOTÓXICAS DE MEMBRANAS POLIMÉRICAS BIOSMART NANOTECHNOLOGY EM

CULTURAS DE CÉLULAS TRONCO MESENQUIMAIS.

Kohori, N. A.¹; Bovolato, A. L. C.¹; Silva, R.R.²; Barud, H.S²; Meneguin, A. B.²; Deffune, E.¹.

1Laboratório De Engenharia Celular, Hemocentro, Faculdade De Medicina, Brasil; 2Biosmart Nanotechnology, Incubadora Municipal De Empresas De Araraquara, Brasil.

Considerando todas as lesões provocadas pelo contato direto de fontes prejuduciais físicas, quí-micas ou biológicas, cerca de um milhão de pessoas sofrem por queimaduras todo ano no Brasil (Ministério da Saúde). Devido a esse número alarmante, há esforços em conjunto para a pesquisa e desenvolvimento de produtos ou propostas terapêuticas que aliviem a dor e restaurem a integri-dade da pele. Nesse contexto, a empresa BioSmart Nanotechnology, startup que tem como uma de suas linhas de P&D a produção de membranas para a área de feridas crônicas ou queimaduras por métodos inovadores utilizando-se da biotecnologia. Dessa forma, os novos produtos na saúde necessitam de testes de citotoxicidade e padronizações em células in vitro, para que posteriormen-te sejam desenvolvidos ensaios em animais dentro da nova ótica de reduzir os experimentos in vivo. O presente estudo fundamenta-se na pesquisa e estabelecimento de técnicas ex vivo a fim de avaliar as propriedades adesivas e citotóxicas de membranas poliméricas em um estudo cego, qualificando sua utilização como scaffolds para terapêutica. Objetivo: Analisar a interação célula--membrana polimérica da BioSmart denominadas SF e GG a fim de investigar um potencial produto para reparação de tecidos lesados. Metodologia: Os métodos de esterilização Sterrad (St) e Óxido de Etileno (ETO) foram comparados através da aplicação de 10^5 células tronco mesenquimais advindas de tecido adiposo murino (CTM-TAm), com avaliação da adesão em 24 e 48 horas a fim de quantificar as citocinas do sobrenadante e as células aderidas à membrana. Já a citotoxicidade foi verificada de forma direta e indireta através dos índices de apoptose e necrose celular por con-tagem celular e citometria de fluxo utilizando Kit BD® de anexina V e Iodeto de propídeo. Resulta-dos e Discussão: Os índices de apoptose e necrose evidenciaram que o St foi superior ao ETO no estudo comparativo das duas metodologias de esterilização, pois as maiores taxas de mortalidade foram demostradas nas membranas SF.ETO e GG.ETO, o que pode inviabilizar o uso do ETO por deixar resíduos tóxicos nas membranas. A viabilidade evidenciou maior quantidade de células no controle (CTRL) seguido das diferentes apresentações das membranas: CTRL (96,39%) > GG.St (77,82%) > SF.St (65,54%) > GG.ETO (50,71%). Já na contagem celular foi obtido um resultado semelhante desta ordem, trocando apenas SF.St e GG.ETO de posição, no entanto, é importante ressaltar que as membranas SF.St tiveram maior desempenho visual na cultura e expansão de cé-lulas aderentes, apesar disso a análise de citotoxicidade direta ficou comprometida pois as células não se desprenderam completamente dos scaffolds. Os ensaios de adesão e citotoxicidade direta mostraram superioridade da membrana GG indicando seu potencial como um veículo de liberação celular. Enquanto a SF apresentou características peculiares: a matriz extra-celular secretada pe-las CTM-TAm são de intensa adesividade à membrana, este fato ocasionou a formação de corpos organóides, aliados ao crescimento acelerado das células aderidas. Pode-se concluir que os resul-tados in vitro são positivos revelando, portanto, indicadores para continuar sua avaliação, como por exemplo estudos por microscopia eletrônica de varredura a fim de uma análise exata da interação célula-membrana, em especial, da membrana SF para futura comercialização como substituto da metilcelulose em cultura de medula óssea.

Key Words: Citotoxicidade; Scaffolds; Adesividade.

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Abstract - Scaffold

LIVER BIOSCAFFOLD AS A NEW STRATEGY FOR LIVER REGENE-RATION: A SYSTEMATIC REVIEW.

Rossi, E. A¹; Mesquita, L. F. Q. D. ¹; Souza, B. S².

1Universidade de Salvador, Brasil.2Fundação Oswaldo Cruz, Brasil.

Diseases that lead to hepatic failure continue to grow in the world and liver transplantation is the only curative option for patients with end stage liver disease. Unfortunately, the treatment is severely limited by some issues, including the low availability of donor organs. Recently, liver decellularized scaffolds have been being proposed as a novel strategy to overcome these issues. Many advances have been achieved in the last years, making this technique much more feasible. This systematic review describes chronologically the progress of this promising technique and highlight future chal-lenges in the field. We adapted from the rules proposed in the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guide. We search in Scielo (http://www.scielo.org), Pubmed (http://www.ncbi.nlm.nih.gov/pubmed) and Lilacs (http://lilacs.bvsalud.org) databases were searched between December 2017 and April of 2018. An additional manual search using references contained in main selected articles was also performed. A window of ten years without language limitations was used. Searches were performed using the descriptive terms “liver”, “recellulariza-tion” and “decellularization” based on the medical terms of the National Library of Medicine (https://meshb.nlm.nih.gov/search). The Boolean expressions “AND” was used in order to find the registries in which “liver” was associated with at least one of the other descriptive terms listed. Adding all da-tabases, 1238 articles were found. After analyzing article titles, it was observed that some articles were found in more than one database, while others did not fit with the criterion established for this study. In the end, 22 articles were elected for the review. As the result of international efforts, the evolution of this technique in the last few years becomes clear, but the last step (recellularization) needs improvement to be viable in any clinical trial. Such advances made hepatic bioartificial organ much more realistic, with great hopes for future clinical applications, and an immense contribution for medicine. We hope that this technique will continue to develop rapidly, so that we could have it implemented clinically in the near future, in order to generate improvements especially to those patients in line for liver transplant.

Keywords: Bioengineering; Biomaterials; Liver; Organ transplantation; Regenerative medicine.

Abstract - Scaffold

PROPERTIES OF CHITOSAN CHLORIDE MODIFIED WITH CALCIUM NANOPHOSPHATE COMPLEX.

Paz, L. R.¹; Pighinelli, L. ¹; Guimarães, F. M. ²; Kolling, L. F. P. ¹; Broquá, J¹; Kmiec, M. ¹; Persson, P.¹; Lisboa, C¹; Dias, P. S. R. ¹; Piva, G.A. ².

1Universidade Luterana do Brasil, Brasil. 2Universidade Federal do Rio Grande do Sul, Brasil.

The compositions of chitosan and calcium phosphates are formed from the joining of two or more organic and / or inorganic materials, which give the structure favorable characteristic to develop biomaterials. Among many characteristics, we highlight the bioactivity, biodegradability and biocom-patibility with human tissues. The chemical characteristics of chitosan, calcium β-triphosphate and hydroxyapatite indicate the possibility of obtaining chitosan hydrochloride complex modified with bi-component nano ceramic. Biocomposite development of bi-component chitosan as biomaterial alternative applied to regenerative medicine and tissue engineering. METHODOLOGY: Initially, the solution of chitosan hydrochloride with a polymer content of 2% (m/ m) was prepared in a solution of 2L of 0.9% HCl solution (v/v). The salt-making process occurs by constant stirring performed by a mixer at room temperature (25 °C). Three solutions (A, B and C) were prepared. In solution A was added 2% (m/m) of β-TCP, in solution B 0.5% (m/m) of HAp and solution C was obtained from the mixture of solutions A and B, having the following composition: 2% (m/m) of β-TCP and 0.5% (m/m) of HAp. It is noted that the polymer concentration and HCl content of all solutions were maintained unchanged, that is, 2% (m/m) polymer and 0.9% (v/v) acid. After obtaining the solutions, they unde-rwent the following quantitative and qualitative analyzes: - Fourier-transform infrared spectroscopy (FTIR); - Particle size determination of commercial β-TCP and HAp; - Scanning electron micros-copy (SEM) study of commercial calcium phosphate particulates; - Determination of particle size and Zeta potential of calcium phosphates in chitosan solution. RESULTS AND DISCUSSIONS: The FTIR and SEM analyzes indicate that the morphologies of the powders differ in shape. It also showed that they have great agglomeration capacity. Particle size analysis of commercial calcium phosphates indicated the presence of about 10% of phosphate particles at the nanoscale. The chitosan dissolution process using a solution of β - TCP microparticles dissolved with 0.9% HCl re-duced the number of β - TCP particles to the nano size, resulting in a solution of chitosan / β - TCP hydrochloride salt in nanoparticles, suggesting a strong ionic interaction between the calcium phos-phate and the chitosan amine groups, generating a bi-component nano ceramic. The analyzes also indicate that hydroxyapatite is less soluble than β - TCP. The solution with the smallest particle size and stability was solution C, which had a range of 12.8 - 58 nm and a zeta potential of 52.9 ± 4.0 mV.It was observed that, by adding β-TCP to the chitosan solution, the phosphate passed from mi-cro-scale to nano; During mixing of the HAp and β-TCP particles contained in the chitosan solution the HAp particles immediately dissolved, resulting in a clear solution showing a great stability and a beneficial effect by the size of the nanoparticles within a range of 12.8-58.0 nm; This work showed a new method of obtaining solutions of chitosan hydrochloride with nano ceramic using hydroxyapa-tite and micro - size β - TCP.

Keywords: Chitosan; Chitosan Salt; Calcium Phosphate; Functional Biopolymer; Medical Applica-tions.

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Abstract - Scaffold

REGENERATED CELLULOSE SPONGE AS SACRIFICIAL TEMPLATE FOR THE SYNTHESIS OF THREE-DIMENSIONAL POROUS ALUMI-

NA-SILICA SCAFFOLD FOR TISSUE ENGINEERING.

Claro, A. M. ¹; Alves, C. C. ²; Sábio, R. M. ²; Caiut , J. M. A. ²; Ribeiro, S. J. L. ³; Barud, H. S¹.

1Universidade de Araraquara, Brasil. 2Universidade de São Paulo, Brasil. 3Universidade Estadual Paulista, Brasil.

Tissue engineering has emerged as a multidisciplinary field that aims to improve health and quality of life by restoring, maintaining and improving the functions of tissues and organs. Cells and scaffol-ds are the two major components of tissue engineering. Different cell lines can be used depending on the tissue from which they will be isolated from and the damaged site where the scaffold will be applied on. Scaffolds act as a support for cells, thus facilitating cell adhesion, proliferation, mor-phogenesis, differentiation, and extracellular matrix (ECM) production. Once three-dimension (3D) porous scaffolds can better simulate the native three-dimensional architecture of in vivo systems than conventional 2D cultures, they are able to support tissue regeneration. A desirable biomate-rial to be used as scaffold must be biocompatible and provide a variety of shapes and sizes, and also be tough enough to be used in locations where there is impact load.This study aimed to use regenerated cellulose sponge as sacrificial template for the synthesis of three-dimensional porous alumina (Al2O3) - silica (SiO2) scaffold from boehmite-GPTS (glycidoxypropyltrimethoxysilane) sols for issue engineering application. Boehmite sol was prepared by Gieselmann (1989) methodology, by hydrolyzing aluminum-tri-sec-butoxide in distilled water at 83°C. In order to obtain the hybrid sol, GPTS was added to the boehmite sol under vigorous stirring. Regenerated cellulose sponge was coated with the boehmite-GPTS sol. Drying was performed at 60°C for 12h. Finally, regenerated cellulose sponge coated with the boehmite-GPTS sol was converted into porous alumina-silica sca-ffold via thermal treatment at 500°C for 4h in air. Regenerated cellulose and the resulting products have been characterized by Fourier transform‐infrared (FT‐IR), optical microscopy, X‐ray diffrac-tion (XRD), X-ray micro computed tomography (micro-CT), nitrogen adsorption-desorption analysis, and aluminium-27 and silicon-29 nuclear magnetic resonance (NMR). Taken together, the analysis results indicate that regenerated cellulose was properly covered by boehmite-GPTS and that after thermal treatment, cellulose was eliminated and there was conversion of the hybrid into an alumi-na-silica porous matrix. Regenerated cellulose sponge was used as sacrificial template in order to obtain porous alumina-silica scaffold. This approach appears as a viable alternative to produce a wide range of three-dimensional scaffolds in a variety of shapes for tissue engineering application.

Keywords: Tissue Engineering; Scaffold; Regenerated Cellulose Sponge.

Abstract - Scaffold

SCAFFOLD PRODUCTION OF NANOFIBROUS POLYCAPROLACTO-NE AND ASCORBIC ACID 2-PHOSPHATE MAGNESIUM.

Kasper, R. H. ¹,²; Machado, G. M.¹; Maurmann, N.²,³; Brew, M¹; Do Couto, M. ¹; Wiltgen, A. ¹; Mahl, C. ¹; Bavaresco, C. S. ¹; Pranke, P.²,³,⁴.

1Universidade Luterana Do Brasil, Brasil. 2Laboratório de Hematologia e Células-tronco, Universidade Federal do Rio Grande do Sul, Brasil.3Programa de Pós-graduação em Fisiologia, Universidade Federal do Rio Grande do Sul, Brasil.4Instituto de Pesquisa com Células-tronco, Brasil.

Tissue engineering involves the search for biological substitute materials favorable for the develo-pment and regeneration of new tissue. Polycaprolactone (PCL) is one of the synthetic polymers of the first choice for this purpose. Ascorbic acid is a bioactive compound with very promising antioxi-dant functionality for use in medicine.The aim of this study has been to cultivate stem cells on PCL scaffolds with ascorbic acid 2-phosphate (ASAP). The stem cells were isolated from human teeth and characterized in the laboratory. To produce the scaffolds, PCL (MW=80,000) was dissolved in tetrahydrofuran:N, N-dimethyl formamide (7:3) to create a 10% (w/v) solution. The set-up to con-duct the electrospinning included a high voltage of 19 kV, a collector to needle distance of 15 cm, and a constant flow rate of 1.14 mL/h. Scaffolds of PCL and PCL/ASAP were produced. The ASAP concentration was 25 µg/ml per scaffold. A total of 30,000 cells/well were seeded in tissue culture plates (TCP) used as a control group or on the scaffolds in a 24-well plate and the analysis was performed after 7 days. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) test. Statistical analyses were performed by ANOVA (P<0.05). A statistical difference was not found between the TCP, PCL and PCL/ASAP groups (p = 0.096). The average ± standard deviation (SD) values of absorbance obtained related to cell viability were: 0.16 ± 0.02 for the TCP, 0.20 ± 0.02 for the PCL) and 0.21 ± 0.03 for the PCL/ASAP). Therefore, both groups are non-cytotoxic for stem cells and are safe for tissue regeneration studies.

Keywords: Ascorbic Acid; Polycaprolactone; Regenerative Medicine; Scaffold.

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Abstract - Scaffold

STERILIZATION PROCESSES OF NATURAL SCAFFOLDS FOR TIS-SUE ENGINEERING.

Iwamoto, L. A. D. S.¹; Duailibi, M. T. ¹; Lourenço, F. R. ²; Iwamoto, G. Y. ³; Oliveira, D. C. ⁴; Duailibi, S. E. ¹.

1Universidade Federal de São Paulo, Centro de Terapia Celular e Molecular, Laboratório de Enge-nharia Tecidual e Biofabricação, Brasil. 2Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Brasil. 3Universidade Federal de São Paulo, Laboratório 3M, Brasil. 4Universidade Federal de São Paulo, Laboratório de Pesquisas em Saúde, Brasil.

Tissue Engineering (TE) is a multidisciplinary science dedicated to producing organs and human parts replaced by traumatic lesions, degenerative diseases or agenesis. One of its stages is the production of biocompatible frameworks for application in Regenerative Medicine. These structures are known as scaffolds, they are similar to the original tissue in texture and porosity. Moreover, it drives the behavior of the cells that will be sown, leading to a construct. The treatment of extensive losses is very difficult and challenging, as the complete recovery of the anatomical and functional integrity of damaged tissues not only provides physical and psychological improvement but also can mean the survival of the patients. The use of natural scaffolds involves the removal of living cells from the organ/tissue and let it read to be implanted in humans, and its biosafety implies being sterile, and with its macro and micro-geometry preserved. Objective: Evaluate the efficiency of ste-rilization after demineralization and decellularization of the teeth, enabling them as natural scaffol-ds. Methods: The samples were submitted to demineralizing/decellularising solutions in sequence sterilizing them. All of them were processed with 28% EDTA, 9% hydrogen peroxide, enzymatic detergent; they were divided into 5 groups: G1 - control, G2 - sterilization with dry heat, G3 - sterili-zation with autoclave, G4 - sterilization with ethylene oxide; G5 - sterilization with Gamma radiation. The evolution of demineralization and decellularization was accompanied by weighing, analytical techniques SEM (Scanning Electron Microscopy), optical images and radiography. Samples were weighed before and after the sterilization process. The sterility test evaluation were performed to evaluate the microbial growth. The results were statistically analyzed by Friedman, Kruskal-Wallis, Qui-square test and Fisher’s exact test. The level of rejection of the null hypothesis was set at 0.05 or 5%. The presented results indicates all the 4 sterillization methods presented equivalent good performance, radiographically there were no evident advantage for any of them. At groups G2 and G4 the color of the samples did not change, while in group G3 the root portion became yellowish and in G5 the coronary portion became greyed. In G5, the microhardness test presented lower levels. Conclusion: Although the demineralization/decellularization processes are not sufficient to eliminate all microorganisms present in the evaluated samples, the sterilization processes performed have been shown to be effective in eliminating the bioburden.

Keywords: Sterilization Processes; Tissue Engineering; Demineralization/ Decellularization.

Abstract - Tissue engineering

AVALIAÇÃO DA CINÉTICA E DAS ALTERAÇÕES MORFOLÓGICAS DA FUSÃO DE ESFEROIDES DE CÉLULAS-TRONCO/ESTROMAIS

DE TECIDO ADIPOSO HUMANO.

Miranda, G. A. S. D C.¹, Kronemberger, G. S.², Granjeiro, J. M.², Silva, K. R.¹, Baptista, L. S.¹.

1Universidade Federal do Rio de Janeiro, Brasil.2Instituto Nacional De Metrologia, Qualidade E Tecnologia, Brasil.

A fusão de esferoides é um processo fundamental para a biofabricação de tecidos e fornece um modelo simplificado de análise de formação de tecidos, uma vez que pode ser quantificado com maior facilidade e com maior rendimento do que em tecidos complexos. A compreensão e oti-mização do processo de fusão tecidual é essencial para construir tecidos e órgãos a partir de esferoides individuais. O objetivo deste trabalho é avaliar o processo de fusão de esferoides de células-tronco/estromais de tecido adiposo humano no seu estado indiferenciado e diferenciado para a via osteogênica, através de um análise cinética e morfológica. Para isso, as células foram semeadas em hidrogéis de agarose (2x106 células/hidrogel) previamente moldados com 81 mi-crorressecções a partir de um molde de silicone, conforme recomendações do fabricante (Micro-tissues, Inc.). Os esferoides formados foram mantidos em meio de cultivo composto por albumina 1,25µg/ml, ácido ascórbico 50µg/mL, insulina 0,01 mg/ml, transferrina 0,01mg/ml selênio 0,01µg/ml e penincilina 100UI/ml e estreptomicina 100µg/ml (esferoides indiferenciados) ou em meio de indução de diferenciação (meio condrogênico por 2 semanas composto de Dexametasona 10-8mM e TGFβ-3 10ng/mL seguido de meio osteogênico por 3 semanas composto de β-glicerofosfato 10mM e Dexametasona 10-7mM - esferoides diferenciados) por até 5 semanas. Após este período, os esferoides foram coletados e delicadamente dispostos em poços de placa de 96 poços previa-mente recobertos com agarose. Quatro esferoides por poço foram dispostos em proximidade uns com os outros e mantidos em estufa úmida a 37o.C e atmosfera com 5% de CO2. A partir daí, o processo de fusão de esferoides indiferenciados e diferenciados foi acompanhado por microscopia óptica de contraste de fase, obtendo-se fotos nos tempos de 0 e 3 horas, bem como de 1, 2, 3, 6 e 7 dias após o plaqueamento para a fusão, com auxílio de um microscópio invertido, acoplado a uma câmera e do software Axiovision LES4. O processo de fusão foi avaliado após 3 e 7 dias do plaqueamento para a fusão através de análises histológicas realizadas em cortes corados por he-matoxilina e eosina. Sete dias após o plaqueamento, o processo de fusão também foi avaliado por microscopia eletrônica de varredura. Em todos os poços avaliados, observou-se que os esferoides indiferenciados fusionaram-se mais rapidamente do que os esferoides diferenciados. No 3o. dia após o plaqueamento da fusão, os esferoides indiferenciados encontravam-se parcialmente fusio-nados, enquanto que os esferoides diferenciados encontravam-se apenas com contato superficial. Os esferoides indiferenciados apresentaram ainda uma subpopulação de células morfologicamen-te distinta orientada para a região de fusão dos esferoides. Ao final de 7 dias do plaqueamento da fusão, foi observado que os esferoides induzidos apresentaram-se pouco fusionados, porém com projeções interligando-os em pontos onde os esferoides não se encontravam interligados ante-riormente. Portanto, a capacidade de fusão de esferoides induzidos à diferenciação osteogênica é inferior à capacidade dos esferoides indiferenciados. A presença de projeções entre os esferoides induzidos dispostos em aproximação para a fusão sugere um mecanismo de fusão diferente do que ocorre com os esferoides indiferenciados. Avaliações de viabilidade dos esferoides após a fusão estão em curso, bem como análises quantitativas da cinética de fusão.

Keywords: Engenharia De Tecidos; Fusão; Esferoides; Biofabricação.

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APLICAÇÃO DA ENGENHARIA DE TECIDOS NO REPARO DE CARTI-LAGEM ARTICULAR EM CÃES.

Tobin, G. C.¹, Nardi, N. B.¹, Witz, M. I.¹, Baja, K. G.¹, Silveira, M. D.¹.

1Universidade Luterana Do Brasil, Brasil.

A terapia celular e principalmente a engenharia de tecidos têm sido intensamente exploradas nos últimos anos para o reparo da cartilagem articular, com emprego de várias combinações de bio-materiais, células e moléculas sinalizadoras. As células-tronco mesenquimais do tecido adiposo (CTTAs) são uma população de células-tronco mesenquimais que recebe especial atenção devido a sua facilidade de coleta, abundância e potencial para regeneração. Objetivos: Desenvolver uma combinação envolvendo CTTAs e hidrogel para uso em estudo clínico de reparo de cartilagem articular em cães. A displasia coxofemoral canina é caracterizada por instabilidade e luxação ou subluxação de quadris. Estes resultados trarão uma contribuição para esta patologia específica, o estabelecimento de competência na engenharia de tecidos e a possível expansão para outras patologias humanas. Metodologia: CTTAs foram coletadas de cães sadios, colocadas em cultivo e caracterizadas quanto a morfologia, potencial de proliferação e diferenciação. As células foram combinadas a vários tipos de biomateriais, com avaliação da viabilidade, proliferação e condro-gênese das células nestas combinações, sendo definido para o estudo clinico o hidrogel de acido hialurônico (Synvisc, Novartis, SP). No estudo clinico em andamento, quatorze cães com displasia coxofemoral já foram incluídos - 6 receberam CTTAs, 4 CTTAs associadas a Synvisc, e 4 controles com tratamento placebo. Análises clínicas e radiológicas são realizadas aos 30, 60 e 90 dias. O projeto foi aprovado pelo CEUA da ULBRA (nº 2016.82). Resultados: CTTAs caninas foram isola-das e cultivadas, mostrando morfologia, proliferação e capacidade de diferenciação características deste tipo celular, mantendo sua morfologia e boa proliferação. A capacidade de diferenciação das CTTAs foi confirmada por reação com os corantes específicos para cada linhagem. As células combinadas a Synvisc foram capazes de diferenciar em tecido cartilaginoso, conforme avaliado por reação com Alcian Blue e análise histológica. O estudo clínico com os cães acompanhados até o presente momento mostrou que os cães tratados com terapia intra-articular de CTTAs associadas ao hidrogel Synvisc apresentam melhores resultados e em menor tempo (60 dias), com melhoras significativas em vários parâmetros ao fim do tratamento. Um dos parâmetros observados em que houve significativa melhora foi atrofia muscular, com alguns pacientes zerando a atrofia muscular pela possibilidade de retorno às suas atividades fisicas com diminuição significativa da dor. O grupo tratado só com células apresentou melhora evidente aos 90 dias, e o grupo placebo teve agrava-mento dos sinais clínicos. Discussão: com estes resultados evidencia-se que as células mantêm as características de células-tronco mesenquimais do tecido adiposo quando isoladas e cultivas, incluindo a capacidade de condrogênese, na presença ou ausência do biomaterial. Ainda, com a presença do synvisc o efeito regenerador destas células é potencializado, comprovado tanto pelos estudos in vitro como no estudo clínico com os cães. A combinação de CTTAs caninas e o synvisc tem ótimo potencial condrogênico. O menor tempo para observação da melhora nos pacientes do estudo clínico se deve ao Synvisc que incrementa a ação das CTTAs potencializando a sua ação de reparo tecidual e antecipando a melhora do paciente com significativo aumento na qualidade de vida. Este estudo está em andamento.

Keywords: Biomaterial; Synvisc; Mesenquimal; Displasia; Condrogênese.


APPLICATION OF FGF-2/PLGA MICROFIBERS IN SPINAL CORD TIS-SUE ENGINEERING.

Reis, K. P.¹,²; Sperling, L. E.¹,²; Teixeira, C.³; Paim, Á.¹,²; Alcântara, B.¹,²; Pranke, P.¹,²,⁴.

1Laboratório De Hematologia E Células-Tronco, Universidade Federal Do Rio Grande Do Sul, Bra-sil.2Programa De Pós-Graduação Em Fisiologia, Universidade Federal Do Rio Grande Do Sul, Brasil.3Universidade Federal De Ciências Da Saúde De Porto Alegre, Brasil.4Instituto De Pesquisa Com Células-Tronco, Brasil.

Spinal cord injury (SCI) is a major public health issue that affects approximately 2.5 million patients worldwide. A promising approach for treatment is represented by the use of scaffolds, which can provide a structural support able to bridge the gap between the lesion and the injury site. Basic fibroblast growth factor (FGF-2) promotes SCI tissue repair but is easily degradable and presents collateral effects in systemic administration. In order to address the stability issue and avoid the sys-temic effects, FGF-2 was encapsulated into core-shell microfibers by coaxial electrospinning and its in vivo potential was studied in a hemisection rat model of SCI. Methodology The morphology and diameter of the fibers were characterized by scanning electron microscopy. The microfibers were implanted in a hemisection SCI rat model. The study was approved by the local ethics committee. The animals were divided into three groups (1) SCI control, (2) PLGA scaffold and (3) FGF-2/PLGA scaffold. Locomotor test was performed weekly for 6 weeks. After this time, histological analyses were performed and expression of nestin and GFAP was quantified by flow cytometry. Results and discussion The results demonstrated that coaxial electrospinning resulted in a uniform microfiber morphology. The groups that received implanted scaffolds showed the tendency to have higher sco-res in the locomotor evaluation than the SCI control group. A significant difference was observed 28 days after SCI in the PLGA scaffold and FGF-2/PLGA scaffold groups when compared to the SCI control. The addition of FGF-2 into the microfibers seemed to have no better effect than the PLGA scaffold alone on the locomotor recovery after SCI. This result suggests a lower amount of FGF-2 was delivered from the microfibers. Therefore, only the presence of the scaffolds was sufficient to promote functional recovery. The histological analysis showed incomplete degradation of the sca-ffold and the presence of tissue in the hemisection cavity. The existence of a scaffold at the injury site is important to support tissue regeneration, that is, the scaffold degradation should occur in a ti-mely fashion allowing the regenerated nerve tissue to become mature enough to be self-supporting. There was a decrease in GFAP expression in the scaffold groups compared to the SCI control. This result indicates a reduction in glial scaring at the local lesion. There was no significant difference of the number of nestin expressing cells among the groups. These results indicate the potential of the microfiber scaffolds to promote locomotor recovery after spinal cord injury and a reduction in glial scaring at the injury site. Therefore, the studied scaffolds have an excellent potential in SCI tissue engineering. However, it is necessary to increase the concentration of the FGF-2 within the microfi-bers to observe the synergetic effects of the scaffolds and the growth factor in this model.

Keywords: Spinal Cord Injury; FGF-2; Coaxial Electrospinning.

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BONE BIOFABRICATION FROM HUMAN ADIPOSE STEM/STROMAL CELL SPHEROIDS SEEDED INTO PLA/CHA 3D PRINTED SCAFFOLD.

Kronemberger, G. S.¹; Palhares, T. N.²; Rossi, A. M.²; Granjeiro, J. M.³ Baptista, L. S.⁴.

1Instituto Nacional De Metrologia, Qualidade e Tecnologia, Brasil.2Centro Brasileiro de Pesquisas Físicas, Brasil.3Universidade Federal Fluminense, Brasil.4Universidade Federal Do Rio De Janeiro, Brasil.

New bone formation is desirable in a variety of pathological states and also in critical-size defects. Adipose-derived stromal/stem cells (ASCs) can be considered a promising cell source for tissue engineering because of its easy accessibility and abundance. We proposed a novel tissue engine-ering approach based on adult stem cells spheroids seeded into scaffolds. The aim of the study is to interact ASCs spheroids with a 3D printed composite scaffold of polylactic acid (PLA) and nanos-tructured carbonated hidroxiapatite (CHA). ASCs were harvested by a mechanical dissociation of human lipoaspirates obtained according to local research ethics committee. ASCs were seeded in a non-adhesive agarose hydrogel micromolded with 81 ressections and maintained in a medium containing ascorbic acid 50 µM, insulin 5 mg/mL, transferrin 5 mg/mL and selenium 5ug/mL (ITS) and human albumin 1.25 μg/mL. After 24 hours, one spheroid was formed in each resection. Then, spheroids were collected from the hydrogel, manually seeded on the surface of a PLA/CHA compo-site scaffold and maintained for one week in culture. The interaction of spheroids with the scaffold was evaluated by SEM. It was observed that migrating cells from spheroids spread over the scaffold surface. Furthermore, some spheroids have changed their morphology, interacting with a larger area of biomaterial. We also observed that cells from spheroids proliferated and produced extra-cellular matrix components over the biomaterial, improving cell adhesion with the scaffold surface. Therefore, it was possible to produce an engineered constructed from human ASCs spheroids see-ded into PLA/CHA scaffolds for bone tissue engineering applications. Our perspective is to implant the engineered constructed into a calvaria defect model to evaluate bone regeneration in vivo.

Keywords: Bone; Spheroids; 3D Scaffold; Tissue Engineering.


CARACTERIZAÇÃO DE ENXERTOS DE VÁLVULA CARDÍACA PORCINA OBTIDOS POR DOIS PROCESSOS DE

DESCELULARIZAÇÃO.

Leitolis, A.¹; Mendonça, J. G. R.²; Sus, P. H.²; Da Costa, F. D. A.²; Stimamiglio, M. A.³ Dominguez, A. C.³.

1Universidade Federal do Paraná, Brasil.2Pontifícia Universidade Católica do Paraná, Brasil.3Instituto Carlos Chagas, Brasil.

Válvulas cardíacas são estruturas responsáveis por manter o fluxo sanguíneo unidirecional no in-terior do coração, alterações no seu funcionamento podem ocorrer devido a doenças ou infecções prévias, de modo que, em casos graves, poderá ser necessária a substituição da válvula nativa por uma prótese. Nesse tipo de cirurgia, diferentes modelos biológicos podem ser utilizados, entre-tanto, tais modelos podem induzir respostas imunes decorrentes da sua viabilidade celular. Nesse sentido, a descelularização, procedimento para eliminação das células alogênicas do tecido, vem sendo testado como alternativa para melhorar a biocompatibilidade das biopróteses. Além disso, recentes descobertas indicam que as vesículas extracelulares (VEs), partículas delimitadas por uma bicamada lipídica, tais como, exossomos e microvesículas, podem ter um importante papel no transplante de órgãos. Estudos anteriores em modelos animais já reportaram a participação dessas estruturas no transplante renal, de medula óssea e cardíaco. O objetivo do presente trabalho foi avaliar a eficiência de dois processos de descelularização de válvulas cardíacas porcinas e avaliar in vitro a biocompatibilidade desses enxertos. Inicialmente, válvulas cardíacas pulmonares porci-nas obtidas de abatedouro credenciado, foram descelularizadas com solução de SDS 0,1% (Grupo 1) ou solução de SDS 0,1% seguida de incubação com tampão hipertônico (10 Mm Tris-HCl e 2,5 M NaCl) e 20 UI/ml de Benzonase (Grupo 2). Posteriormente, os tecidos obtidos foram submetidos a protocolos de confirmação da remoção do conteúdo celular através da marcação com DAPI e extração de DNA, além da caracterização da composição e aspecto da matriz extracelular (MEC) e ensaio de citotoxicidade. Para a verificação da biocompatibilidade desses enxertos, as cúspides obtidas dos dois grupos foram cocultivadas com células-tronco mesenquimais e células endoteliais (HUVEC). Nossos resultados mostraram ausência de núcleos nas cúspides e conduto das válvu-las de ambos os grupos, porém a quantificação do DNA residual extraído revelou uma diminuição significativa na quantidade desse material somente nas válvulas do grupo 2. Para verificar se os protocolos utilizados causaram prejuízo nos tecidos, foram realizadas marcações para elastina, colágeno e GAG. Enquanto o conteúdo e a disposição desses componentes não foram alterados no grupo 1, uma acentuada redução de marcação para colágeno foi observada no grupo 2. Adicio-nalmente, os tecidos de ambos os grupos apresentaram-se não citotóxicos. Quanto aos ensaios de biocompatibilidade, as cúspides obtidas dos dois grupos funcionaram como um arcabouço tanto para células-tronco quanto para células endoteliais. Experimentos para avaliar a capacidade das vesículas extracelulares em favorecerem a biocompatibilidade dos enxertos descelularizados ainda estão sendo conduzidos. A descelularização com baixas concentrações de SDS produziu tecidos de matriz extracelular conservada (colágeno, Elastina e GAG), entretanto, a remoção mais eficiente do DNA só foi alcançada por meio da incubação dos tecidos com tampão hipertônico e uma enzima nuclease (Benzonase). Além disso, cúspides de válvulas descelularizadas foram recelularizadas com células humanas com potencial de serem utilizadas para recelularização ex vivo.

Keywords: Válvula Cardíaca; Descelularização; SDS; Benzonase.

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DEVELOPMENT OF A CONDUIT OF PLGA-GELATIN ALIGNED NANO-FIBERS PRODUCED BY ELECTROSPINNING SEEDED WITH STEM

CELLS FOR NERVE REGENERATION.

Pozzobon, L G ¹, Sperling, L. E.¹,², Pranke, P.¹,²,³.

1Laboratório De Hematologia E Células-Tronco, Universidade Federal Do Rio Grande Do Sul, Bra-sil.2Programa De Pós-Graduação Em Fisiologia, Universidade Federal Do Rio Grande Do Sul, Brasil.3Instituto De Pesquisa Com Células-Tronco, Brasil.

A promising alternative to conventional nerve grafting is the use of artificial grafts made from biode-gradable and biocompatible materials and supportive cells. In this study, a poly (lactic-co-glycolic acid) (PLGA) conduit of aligned nanofibers was produced by the electrospinning method and se-eded with mouse embryonic stem cells (mESCs) that were subsequently differentiated into neural precursor cells. Scaffolds of aligned fibers with an average diameter of 0.90±0.36 µm, an alignment coefficient of 0.817±0.07 and an 112.5±0.12º contact angle were produced. A treatment with gelatin increased the fiber diameter to 1.05±0.32 µm, reduced the alignment coefficient to 0.655±0.045 and made the scaffold very hydrophilic with a contact angle of 0°. The conduits seeded with murine embryonic stem cells (mESCs) were submitted to a neural differentiation protocol and the cell pro-liferation, differentiation and viability were analyzed. The real time PCR analysis showed that the cells were upregulating the expression of neural markers nestin and beta3 tubulin. The MTT assay showed that the cells maintained viable and proliferated after 7 days in culture, showing better via-bility on gelatin treated conduits. Moreover, the Live/Dead assay indicated high cell viability, with almost no dead cells. Confocal images of phalloidin/DAPI staining showed that the cells adhered and proliferated widely, in fully adaptation with the biomaterial. In conclusion, a method is presented here for producing conduits made of aligned biocompatible and biodegradable PLGA nanofibers with a very good cell-conduit interaction as promising regenerative product for nerve grafting.

Keywords: Biomaterial; Stem Cells; Nerve Graft; Conduit; Aligned Nanofibers; Tissue Engeneering; Regenerative Medicine.


DEVELOPMENT OF A RED PROPOLIS-ENCAPSULATED NANOCAP-SULE HYDROGEL TO BE USE IN REGENERATIVE MEDICINE.

Steffens, D.¹; Salvador, S. F.²; Dos Santos, P. R.¹; Pranke, P.¹; Moura E Silva, S.¹; Khademhosseini, A.³; Ely, M. R.¹ Henriques, J. A. P.¹.

1Universidade De Caxias Do Sul, Brasil.2Universidade Federal Do Sul, Brasil.3University Of California, Estados Unidos.

Skin substitutes are in high demand for the treatment of burns and wounds. GelMA hydrogels are 3D polymeric structures with high aqueous content, used with the aim of mimicking ECM. The an-ti-inflamatory, antimicrobial and regenerative properties of red propolis (RP) are interesting for skin healing and therefore, hydrogels with RP lipid core nanocapsules were constructed. Two groups of nanocapsule suspensions were developed: PCL, a Poly-ε-Caprolactone nanocapsule without RP and Prop, a PCL nanocapsule with 5 mg/mL of RP. They were evaluated for size, polidispersivity (PDI) and zeta potencial (ZP). The quantification of the propolis content in the nanocapsules was also analyzed. Morphology, pore area, wettability and degradability were evaluated for 1) pure hy-drogel, 2) hydrogels with PCL nanocapsules and 3) hydrogels with Prop nanocapsules. As prelimi-nary results, on day 0, the PCL nanocapsules were shown to have a size of 293±36 nm, 1.8 of PDI and -17mV of ZP. When propolis was added to the system, the size was 271±40 nm, the PDI was 1.3 and the ZP was -23.5mV. After 30 days, both groups remained stable, with the ZP for PCL of -25.7mV and for the Prop -30 mV. The size and PDI for the PCL was 257.7±19 and 1.3, and 267±35 nm and 1.15 for the Prop. The quantification showed that 94.9% of the RP was within the nanocap-sules. All the hydrogels showed a heterogeneous porous structure. The surface porous size varied greatly in all the groups, as follows: 35.56±56.1 µm, 13.52±16.58 µm and 24.4±29.48 µm for groups 1, 2 and 3. The same pattern occurred with the internal pores, in which were found 35.56±56.1 µm, 161.85±276.44 µm and 56.9±135 µm for groups 1, 2 and 3, respectively. The wettability was higher than 490% for all the groups tested. The degradability test showed a slight degradation in the first 6 hours for all the groups. At 24 hours, the hydrogels with propolis nanocapsules had the highest level of degradation, with 34.5% of weight loss. The same pattern was observed after 7 days with 61.5% of degradation in this group. In conclusion, although several evaluations are still required, promising biomaterial are undergoing investigation for use in skin tissue engineering.

Keywords: Gelma Hydrogel; Red Propolis; Nanocapsules; Biomaterials; Skin Susbtitute.

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DEVELOPMENT OF BIPHASIC SCAFFOLDS OF COLLAGEN TYPE I FOR PERIPHERAL NERVE REGENERATION.

Millan, D.¹; Jimenez, R. A.¹; Silva, S.²; Mano, J.²; Fontanilla, M. R.¹.

1Universidad Nacional De Colombia, Colômbia.2University Of Aveiro, Portugal.

Biomaterials have been used to produce conduits that protect and connect sectioned peripheral nerve stumps preventing the formation of neuromas and fibrous tissue between the stumps of the nerves (Faroni A, et al. 2015). Major drawbacks of current commercial devices are the size of the injury and their low effectiveness rates and low functional recovery rates (Sarker M, et al, 2018). Often, the standard diameters of these devices cannot be adapted to the diameter of the nerve injured at the time of surgery (Kehoe S, et al. 2012). Hence, the need to develop products that provide microstructural and cellular microenvironments that accelerates axonal regeneration. This work developed biphasic scaffolds of collagen type I that can form conduits with inner unidirectional and outer multidirectional pores, which could be used for regeneration of injured peripheral nerves. The above mentioned scaffold organization was designed to mimic the orientation of collagen fibers on the peripheral nerve and to provide a mechanical barrier for migration of unwanted cells. The scaffolds prepared can be folded to form a tubular conduit adapted to the diameter of the sectioned nerve stump. The unidirectional and multidirectional sections of the scaffolds were prepared using different concentrations of collagen (5 mg/g and 8 mg/g, respectively). Scaffolds were cross-linked using different concentrations of glutaraldehyde (0.02%; 0.04%; 0.06%; 0.08% and 0.1%). Physico-chemical and mechanical properties, and cytocompatibility of the obtained scaffolds were evalua-ted. Scanning electron microscopy analysis showed scaffolds with two different zones: one with uni-directional pores and other with multidirectional pores. The pore size in the scaffold-unidirectional zone was larger than the one in the multidirectional zone. Infrared spectra of the scaffolds showed the characteristic bands of collagen type I indicating that its native structure was preserved (León et al 2016; Suesca et al 2017). No changes were seen in the spectra of samples made with different pore orientation, different collagen concentrations, and different cross-linking percentages. All the scaffolds had water contact angles smaller than 90° indicating they are moderately hydrophilic (Yuan and Lee 2013). The hydrophilicity of the scaffolds did not depend on the concentration of collagen and the concentration of glutaraldehyde. It was found that cross-linking percentage of scaffolds de-creased when collagen concentration increased and was directly proportional to the concentration of glutaraldehyde used. Data from Z potential evaluations showed that scaffold-surface negative charge increased when glutaraldehyde concentration increased. On the other hand, tensile stress data demonstrated that the elastic modulus increased proportionally when the concentration of glutaraldehyde increased. Finally, cell viability assays demonstrated the cytocompatible of the sca-ffolds. Overall, data suggest that the biphasic scaffolds can form conduits with inner unidirectional and outer multidirectional pores. The type I collagen scaffolds developed are biocompatible and have physicochemical and mechanical properties that can promote their bioactivity when used in regeneration of injured peripheral nerves.

Keywords: Biphasic Scaffolds Of Collagen Type I; Unidirectional Pores; Multidirectional Pores; Ner-ve Conduits Regeneration.


HISTOLOGICAL AND BIOCOMPABILITY EVALUATION OF THE BOVINE PERICARDIUM AFTER SOFT DESCELLULARIZARION

PROCESS FOR THE USE IN VALVE BIOPROTHESIS.

Stimamiglio, M. A.¹, Heuschkel, M. A.¹, Roderjan, J. G.², Suss, P. H.², Luzia, C. A. O.², Affonso Da Costa, F. D.², Correa, A.¹.

1Instituto Carlos Chagas, Fundação Oswaldo Cruz, Brasil.2Pontifícia Universidade Católica Do Paraná, Brasil.

Bioprosthetic heart valves are extensively applied in patients with valvulopathies requiring valve replacement. However, conventional bioprosthesis present limitations related to its rupture and cal-cification, mainly by the use of chemical crosslinkers. In this context, aiming the development of a valvular bioprosthesis that overcome these limitations, the purpose of this study is to evaluate the structural integrity and biocompatibility of the bovine pericardium (BP) after a soft decellularization process with a 0.1% sodium dodecyl sulfate (SDS) solution. The efficacy of BP decellularization was accessed by extracting genomic DNA followed by agarose gel electrophoresis, Hematoxylin/Eosin staining (H&E) and nuclear labeling with 4’,6-diamino-2-phenylindole (DAPI). Immunofluorescence was performed in order to visualize the proteins of the extracellular matrix (ECM) and the immuno-genic xenoantigen galactose-alpha-1,3-galactose (alpha-gal), found on the membrane of mamma-lian cells except humans and higher primates. The biocompatibility of the tissue was evaluated by its culture with mesenchymal stem cells derived from human adipose tissue (hASCs). Thus, decellularization process promoted a mean reduction of 77% of the amount of DNA in the samples. Cell nuclei were not visible in H&E and DAPI staining, despite the presence of DNA bands larger than 200 bp in the agarose gel. The content and arrangement of type I, III, IV collagens, decorin and elastin were not altered with the decellularization. However, laminin and lumicam exhibited reduced tissue labeling, as well as the cytoskeleton protein vimentin. Scanning electron microscopy showed a loosening of the fibers on the fibrous face of the decellularized tissue compared to the fresh one. In addition, the decellularization protocol has proven to be efficient in removing the xenoantigen al-pha-gal. The decellularized BP was non-cytotoxic in vitro and allowed the adhesion of hASCs after 24 h in culture. Finally, after 7 days in culture the tissue scaffold became repopulated by hASCs and after 30 days the ECM protein pro-collagen I was seen in the repopulated scaffold. Taken together, these results strengthen the idea that a balance between elimination of imunnogenic components, resorting to soft chemical treatment, and preservation of the tissue structure is desirable to ultima-tely achieve an active pericardium prone to recellularization. These characteristics indicate that soft BP decellularization with 0,1% SDS solution allows the acquirement of a suitable scaffold for cell repopulation and for the development of valvular bioprostheses.

Keywords: Valvular Bioprothesis; Bovine Pericardium; Extracellular Matrix; Tissue Decellularization.

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HUMAN ADIPOSE STEM/STROMAL CELL SPHEROIDS AS A MODEL OF OSTEOGENESIS.

Kronemberger, G. S.¹; Beatrici, A.¹; Silva, K. R.¹; Granjeiro, J. M.²; Baptista, L. S.³.

1Instituto Nacional De Metrologia, Qualidade e Tecnologia, Brasil.2Universidade Federal Fluminense, Brasil.3Universidade Federal Do Rio De Janeiro, Brasil.

New bone formation may be desirable in a variety of clinical settings, such as osteoporosis and non--union fractures. Alternatively, stem cells derived from adipose tissue represent a promising cellular source for the formation of this tissue through tissue engineering, because of its easy accessibility and abundance. In this context, a new approach in the field of tissue engineering corresponds to the use of spheroids as modular units for engineering biological tissues. In this way, the aim of this study is to induce and padronize the osteogenesis process using human adipose tissue derived stem cells (ASC) in a three-dimensional micromolded agarose hydrogel culture. Human lipoaspirate samples were obtained according to the local research ethics committee and human adipose tissue stem cells were isolated by mechanical dissociation. One group of spheroids were maintained for three weeks in an osteogenic induction medium composed of 10 mM β-glycerophosphate, 10-7 mM Dexamethasone and 200 mM hrBMP-7 (human recombinant bone morphogenic protein 7). Another group of spheroids were first maintained for two weeks in a chondrogenic medium composed of 10-8 mM Dexamethasone and 10 ng/mL TGFβ-3. Then, the medium was exchanged for an osteo-genic one composed of 10 mM β-glycerophosphate and 10-7 mM Dexamethasone for three weeks. Initially, spheroids diameter measurements were performed. In control spheroids, the range of dia-meter was 300-350 µM, in hrBMP-7 induced spheroids the range of diameter was between 350-400 µM and in TGFβ-3 induced spheroids the range of diameter was 400-450 µM. Regarding cell viability, this was shown to be high for the control and induced spheroids, once 85% of cells were negative for 7-AAD DNA intercalant. Hematoxylin and Eosin histological analyzes were performed to evaluate cell morphology and Alizarin Red O staining to identify extracellular mineralization, whe-re we observed the presence of calcium deposits only in both induced conditions. The molecular analyses by reversal transcriptional polymerase chain reaction (rtPCR) showed a high relative ex-pression of alkaline phosphatase (ALP) in hrBMP-7 induced spheroids when compared with control spheroids and a low relative expression of chondrogenesis master gene Sox9 in hrBMP-7 induced spheroids. Simultaneously, immunohistochemistry was performed using antibodies to components of bone extracellular matrix, as type I collagen, osteocalcin, osteopontin, biglycan and tenascin C, where the TGFβ-3 induced spheroids showed a better distribution of those molecules in the last week of culture when compared to hrBMP-7 induced and control spheroids. The surface of sphe-roids was visualized by scanning electron microscopy, in addition to an elemental EDS analysis for the calcium and phosphate elements, present only in induced spheroids of both conditions. At the end, mechanical resistance to compression were evaluated utilizing Microsquisher (Cell Scale), in which TGFβ-3 induced spheroids presented a better performance in the third week of culture, re-aching values of 400 µN of force, while control and hrBMP-7 induced spheroids presented values of 20-30 µN of force. We were able to compare two different protocols for osteogenic induction in ASC spheroids and realize that TGFβ-3 induced spheroids showed a better response in analyses of osteogenic differentiation efficiency.

Keywords: Spheroids; Osteogenesis; Tissue Engineering.


MACROPHAGE POLARIZATION IN SKIN WOUNDS TREATED WITH A DERMAL SUBSTITUTE ASSOCIATED WITH MESENCHYMAL STEM

CELLS.

Zomer, H. D.¹,², Jeremias, T. D. S.¹, Ratner, B.², Trentin, A. G.¹,³.

1Universidade Federal de Santa Catarina, Brasil.2University of Washington, Brasil.3Instituto Nacional de Ciência e Tecnologia em Medicina Regenerativa, Brasil.

Large skin wounds such as burns lead to secondary disorders and death if not quickly stabilized. Therefore, novel strategies combining cell therapy, tissue engineering and regenerative medicine have been developed to treat these challenging wounds. Currently, the gold standard treatment involves the use of dermal templates, such as Integra™ matrix, a dermal substitute composed of collagen and chondroitin sulfate. In contrast, mesenchymal stem cells (MSCs) shows wide range of therapeutic applications due their differentiation potential and paracrine effects. Recent studies suggest that MSCs induces the shift of macrophages from pro-inflamatory phenotype (M1) to pro--healing phenotype (M2). The aim of this study was to verify the macrophage polarization in skin wounds treated with Integra™ matrix associated with human MSCs. Dermal and adipose derived mesenchymal stem cells (DSC and ASC, respectively), were isolated from human abdominoplasties discards. The MSCs were characterized with regard to their immunophenotype and mesodermal differentiation potential and associated with the Integra matrix. Wounds were induced in the back of C57bl/6 mice and treated with Integra™ (control group), Integra™ with DSC or Integra™ with ASC. Wounds were measured over time and samples were harvested after 3 and 21 days for immunohis-tochemistry evaluation of F4/80 (macrophage marker), iNOS (M1 marker) and MMR (M2 marker). DSC and ASC expressed mesenchymal markers (CD90, CD73 and CD105 and do not express hematopoietic markers (CD45 and CD34) and both MSCs were able to generate adipocytes, osteo-cytes and chondrocytes. DSC and ASC were adequately associated with Integra™ in vitro and pro-moted higher closure (90.27% ± 18.78 SD and 86.88% ± 24.5 SD, respectively) of skin wounds after 21 days than control (55.78% ± 29.06 SD). At day 3, there were more macrophages in the control group than MSCs groups (60.81% ± 7.1 SD versus 45.32% ±9.0 in DSC and 40.29% ± 5.63 SD in ASC), but the percentage of macrophages was similar between control and cell groups after 21 days (43.33% ±4.27 SD in control group, 44.91% ±3.87 SD in DSC and 46.93% ±7.9 SD in ASC). The percentage of macrophages decreased from day 3 to 21 in control group, while it remained stable in the cell groups, suggesting an anti-inflammatory effect of MSCs in the inflammatory phase of healing. Accordingly, at both 3 and 21 days, there were significantly more iNOS (M1) macrophages and less MMR (M2) in the control group than DSC and ASC groups (3 days: 83.27% ±5.34 SD iNOS in control versus 65.95% ±6.36 SD in DSC and 64.53% ±12.79 SD in ASC and 45% ±6.63 SD MMR in control versus 68.59% ±9.49 SD in DSC and 62.02% ±10.35 SD in ASC; 21 days: 68.83% ±5.15 SD iNOS in control versus 42.30% ±11.25 SD in DSC and 46.44% ±12.68 SD in ASC and 51.76% ±7.93 SD MMR in control versus 66.69% ± 9.85 SD in DSC and 63.91% ± 8.52 SD in ASC). The percentage of iNOS in each group decreased over time, but the percentage of MMR remained similar. Togheter, these results demonstrate the immunomodulatory and anti-inflammatory effects of DSC and ASC in skin wound healing and highlights both types of MSCs as promissing sources of cells for future therapies. The association of Integra™ matrix with mesenchymal stem cells promote faster wound closure and an immunomodulation by the shift of macrophages polarization from a pro-inflammatory phenotype to a pro-healing phenotype.

Keywords: Cutaneous Repair; Mesenchymal Stromal Cells; MSC; Translational Research; Inflamma-tion; Immunomodulation.

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RASTREABILIDADE NOS RESULTADOS DE MEDIÇÃO DO MÓDULO DE YOUNG PARA ESFROIDES CELULARES.

Beatrici, A.¹; Boldrini, L.¹; Granjeiro, J. M.¹ Baptista, L. S.².

1Instituto Nacional De Metrologia, Qualidade E Tecnologia, Brasil.2Universidade Federal Do Rio De Janeiro, Brasil.

O desenvolvimento das técnicas de medição e produção de tecidos in vitro têm permitido a melhoria das caracterizações das propriedades dos constructos engenheirados. Uma das necessidades da biofabricação e bioimpressão é a homogeneidade, morfológica e biomecânica, dos tecidos cons-truídos a partir de esferoides celulares. Apesar da utilização de métodos estatísticos na análise dos resultados pouco tem sido desenvolvido para a rastreabilidade dos resultados e modelagem da estimativa de incertezas desses métodos de caracterização. O desvio padrão de um resultado de medição é apenas uma parte da estimativa de incerteza, frequentemente nem é a mais importante. Na tentativa de melhorar as análises de resultados na Engenharia Tecidual, e assim determinação mais correta das característica biomecânicas dos constructos, foi desenvolvida a modelagem da estimativa de incerteza para testes compressivos em um dispositivo de placas paralelas. Os resul-tados de medição do Módulo de Young (módulo de compressibilidade) para esferoides celulares são independentes da sua origem (células tronco, cartilagem, osso, etc.). Objetivos: i) Desenvolver a modelagem de estimativa de incerteza dos resultados de medição do Módulo de Young para es-feroides celulares. ii) Prover rastreabilidade em resultados de medição do módulo de Young para esferoides celulares em testes compressivos com uso de dispositivo de placas paralelas. Metodo-logia: As principais influências na incerteza de medição são identificadas com o uso de um diagra-ma de Ishikawa. A partir da equação do Módulo de Young para esferas comprimidas entre placas paralelas foi aplicado o método de Kragten para a estimativa da incerteza combinada. A calibração na força foi realizada com o uso de um comparador de massa analítico utilizando uma coleção de pesos padrão rastreada ao kilograma. A calibração dimensional foi realizada com uma grade de ca-libração rastreada ao metro. Essa abordagem pode ser aplicada apenas a constructos que exibiam simetria esférica e diâmetros entre 300 a 1000 micrometros e com compressão máxima de 25% do diâmetro inicial. Os resultados de medição foram obtidos da análise de esferoides de células tron-co derivadas de tecido adiposo, diferenciadas em cartilagem ou diferenciadas em células ósseas. Resultados e Discussão: Foram realizadas medições para estimativa da repetibilidade e reprodu-tibilidade dos resultados bem como de todas as grandezas de entrada. As incertezas expandidas relativas típicas obtidas para o Módulo de Young (Y) ficaram por volta de 5%. Essa avaliação das incertezas envolvidas nos resultados de medição estão subordinadas a duas premissas assumidas na modelagem. Primeiro, apesar de tecidos nativos como o ósseo e a cartilagem possuírem proprie-dades anisotrópicas, especialmente relativas a cargas compressivas ou extensivas, na formação dos esferoides celulares é assumido, por simetria da formação dos agregados celulares, que essas propriedades são isotrópicas. Conclusões: A abordagem desenvolvida permitiu a determinação dos resultados de medição com incertezas de medição expandidas menores que a variabilidade apre-sentada pelos esferoides celulares, sendo possível identificar diferenças entre a origem das células (tecido adiposo), tipos de indução a diferenciação bem como o tempo de indução. Esse resultado permitirá comparações com resultados de outros centros de pesquisa de forma mais confiável.

Keywords: Biometrologia; Rastreabilidade; Módulo de Young.


“SCAFFOLDS” DE POLIÁCIDO LÁCTICO (PLA) OBTIDOS POR MA-NUFATURA ADITIVA FUNCIONALIZADAS PELO PLASMA DE

OXIGÊNIO PARA APLICAÇÃO NA ENGENHARIA DE TECIDOS.

Machado, L. G.¹; Rangel, E. C.², Cruz, S.³; Iemma, M. R. D. C.¹, Barud, H. S.¹

1Universidade de Araraquara, Araraquara, SP, Brasil.2Universidade Estadual Paulista, Brasil.3Universidade Federal de São Carlos, Brasil.

A engenharia de tecidos visa criar possibilidades de regeneração, reposição de órgãos e tecidos. O poliácido láctico (PLA) é um polímero que vem recebendo atenção da comunidade científica com inúmeros estudos, devido a suas propriedades mecânicas e térmicas. Isto se torna possível rea-lizando a intersecção de matrizes e moléculas terapêuticas. É um poliéster alifático termoplástico que é derivado de recursos renováveis como o milho, beterraba e outros amidos. A manufatura aditiva é uma forma de prototipagem rápida que trabalha com extrusão controlada de um filamento termoplástico. Ela deposita sobre uma mesa camada a camada, dessa forma constrói o objeto ma-nufaturado por camadas (scaffold) para a utilização na engenharia de tecidos. A fim de melhorar as propriedades de biocompatibilidade do PLA (ângulo de contato, rugosidade, adesão e proliferação) optamos por realizar a funcionalização do polímero. Aplicamos a funcionalização por plasma. O plasma apresenta a vantagem de modificar somente a superfície. A modificação superficial exerci-da por elétrons, íons e radicais altera a molhabilidade e a ativação da superfície se dá com novos grupos polares funcionais como: carbonila, carboxila, éter, amina e hidroxila. A substituição de gru-pos etil com oxigênio na superfície do polímero, aumenta a energia da superfície do polímero livre (nanômetros) sem alterar as propriedades do material. Objetivos – Obter scaffolds de PLA através da manufatura aditiva (prototipagem rápida) e funcionalizar sua superfície com plasma de oxigênio. Metodologia – A manufatura aditiva constrói camada a camada com alto nível de detalhamento e precisão do defeito a ser reconstruído. Impressora 3D de manufatura aditiva, “Boa Impressão”, modelo Stella, Curitiba-PR. Utilizamos uma extrusora com bico de 0.2 mm e temperatura inicial na primeira camada de 200ºC e camadas subsequentes 195ºC. Os scaffolds foram modelados no software Autodesk Inventor CAD (desenho assistido por computador) com diâmetro e altura res-pectivamente de 10 mm e 1 mm e exportado no formato STL. Utilizamos filamento Movitech com diâmetro de 1,75mm para a extrusão do PLA. O plasma de oxigênio é concebido através de um sistema que é composto por um reator de aço inoxidável (~ 5,2 x 10-3 m³) contendo em seu interior dois eletrodos circulares paralelos com 11,9 cm de diâmetro, separados por 5 cm. Após o scaffold sofrer a ação da ablação pelo plasma de oxigênio realizamos a caracterização físico - química. Resultados e Discussão - Avaliamos o ângulo de contato (água) sobre o scaffold que foi modificado por plasma de oxigênio em diferentes períodos de ablação e este apresentou maior hidrofilicidade superficial. Utilizamos a microscopia de força atômica AFM para averiguar a rugosidade superficial e obtivemos aumento de até 450% na rugosidade do scaffold em comparação ao scaffold sem tratamento. Submetemos o scaffold funcionalizado ao teste de proliferação e viabilidade celular com 100.000 células Osteo - 1 semeadas em meio DMEM por 72 horas, conseguiu -se melhoria significativa deste comportamento celular (adesão e proliferação). Até o presente momento en-tende-se que PLA ao ser funcionalizado a temperatura ambiente por plasma de oxigênio agrega propriedades mecânicas, físicas e biológicas que viabilizarão a criação de scaffolds de PLA para serem utilizados na engenharia de tecidos.

Keywords: Scaffold; Manufatura Aditiva; Poliácido Láctico; Plasma De Oxigênio.

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THROMBOSPONDIN-1 PROTEIN SILENCING IN ADIPOSE STEM CELL SPHEROIDS.

Charelli, L. E.¹, Matsui, R. A.¹, Baptista, L. S.².

1Instituto Nacional De Metrologia, Qualidade E Tecnologia, Brasil.2Universidade Federal Do Rio De Janeiro, Brasil.

Osteoarthritis (OA) is caused by mechanical and biological events that destabilize the joint tissues homeostasis. This pathology is commonly associated with loss of joint cartilage, synovial inflamma-tion, and the formation of osteophytes. Furthermore, angiogenesis has been described as main event that leads to these effects. Aiming to bridge the gap in joint cartilage repair, strategies that target events of angiogenesis has emerged as OA therapeutics approach. Our research group has performed the secretome of adipose stem cells spheroids induced to the chondrogenesis and identi-fied several molecules related to skeletal development, osteogenesis, chondrogenesis and anti-an-giogenesis, such as the protein Thrombospondin-1. This protein has been described as having an-ti-angiogenic and anti-hypertrophic properties. Therefore, our objective is to support the importance of TSP-1 in chondrogenesis from ASC spheroids. Human ASCs were obtained from subcutaneous lipoaspirate samples, from healthy donors (18 to 55 years old), according to the Research Ethics Committee of the Clementino Fraga Filho University Hospital (UFRJ-protocol 146/09). After ASCs isolation, the monolayer culture was maintained on 24 well plates, at 37ºC with 5% CO2 and 21% of O2. Immunohistochemistry (IHC) for anti-TSP-1 and Western blot analysis in the culture super-natant was performed in ASC spheroids (i.e. 3D culture system). The silencing standardization was made in the kinetics of 18,48,36 and 72 h, using the kit HiPerfect Transfection reagent (Qiagen). The quantification of gene expression was performed with Real-Time PCR System (7500-Applied Biosystems). The ASCs viability and morphology - after mRNA silencing- was examined using pha-se contrast microscopy and the kit LIVE/DEAD (Thermo Fisher). Western Blot analysis from the culture supernatant of induced ASC spheroids revealed a high intensity of bands correlated with TSP-1, corroborating the secretome analysis. The IHC attested the presence of TSP-1 in induced ASC spheroids. The folding change for the kinetics of 48 h showed a significant (p=0,00394704) decrease for ASC monolayer that underwent silencing, compared with not silenced ASC . ASC morphology and viability was not altered by the TSP-1 silencing. Preliminary results showed an ef-ficient silencing of the TSP-1 protein in ASC monolayer. TSP-1 silencing is being performed in ASC spheroids.

Keywords: Protein Silencing; Thrombospondin-1 (TSP-1); Adipose Derived Stem Cells (ASC); Chondrogenesis; Spheroids.


TRANSPLANTE ORTOTÓPICO DE TRAQUÉIAS PREPARADAS POR TÉCNICAS DE ENGENHARIA CELULAR: MODELO EM COELHOS.

Nunes, H. C.¹; Evaristo, T. C.²; Souza, A. V. G.²; W. M.², Bagnato, V. S.³; Ferreira, R. R.²; Cardoso, P. F. G.⁴ Fabro, A. T.³; Deffune, E.²; Cataneo, D. C.².

1Universidade Estadual De Campinas, Brasil.2Universidade Estadual Paulista, Brasil.3Universidade De São Paulo, Brasil.4Instituto Do Coração, Universidade De São Paulo, Brasil.

Tendo em vista o desafio do tratamento de injúrias traqueais e o progresso na produção desse órgão por técnicas de engenharia de tecidos, faz com que o interesse nessa área cresça de forma conside-rável, já que ainda não há um tratamento padrão descrito na literatura acerca das lesões traqueais extensas. Assim, este estudo teve como objetivo a construção de modelo de traqueia para enxerto uti-lizando técnicas de engenharia de tecidos. Para tanto, foram realizados 5 e 10 ciclos de descelulariza-ção nas traqueias de coelhos doadores seguindo etapas: congelamento/descongelamento sem adição de nenhum crioprotetor, banho ultrassônico no aparelho Ultrasoniccleaner (Uniquemodel USC 1400®) na frequência de 40 Khz durante 10 minutos, irradiação utilizando LED com comprimento de onda de 450nm ± 20nm na dose de 90 J/cm2 totalizando 60 minutos de exposição, adição do detergente deoxi-colato de sódio a 4% sob agitação a 180 rpm no C24 Incubatorshaker durante 48 horas. Paralelamente, foram realizadas expansão e caracterização das células-tronco mesenquimais dos coelhos receptores com posterior diferenciação dessas células em condrócitos e células de músculo liso. Finalmente, foi realizada a rescelularização das traqueias com aplicação das células-tronco mesenquimais, dos con-drócitos e das células de músculo liso dos coelhos receptores nas superfícies externas dos arcabouços produzidos para os transplantes traqueais. Após 5 ciclos repetidos do protocolo de descelularização utilizado, ainda é possível observar células epiteliais respiratórias e condrócitos remanescentes nas traqueias. Porém, a repetição de 10 ciclos do mesmo protocolo de descelularização remove completa-mente as células epiteliais e reduz a quantidade de condrócitos. Os animais que foram transplantados com as traqueias descelularizadas com 5 ciclos vieram a óbito após 10-12 dias devido à rejeição pela presença das células epiteliais dos coelhos doadores. Os animais que foram transplantados com as traqueias descelularizadas com 10 ciclos não apresentaram sinais de rejeição, porém, vieram a óbito após 40-90 dias devido à falta de regeneração epitelial. O epitélio traqueal é a área mais imunogênica da traqueia, ou seja, a área capaz de provocar uma resposta imunológica, o que ocorreu na primeira etapa. Entretanto, sua ausência também apresenta algumas desvantagens como ocorrência de infec-ções e formação de supuração aguda na luz traqueal, dado observado na segunda etapa do trabalho.Não foi possível a realização do cultivo das células epiteliais de todos os animais devido à frequência de contaminações de difícil controle. A regeneração epitelial é essencial para que não ocorra a prolife-ração fibroblástica. A luz traqueal apresenta células produtoras de muco e células ciliadas responsáveis pela limpeza; se as células ciliadas não se apresentarem de forma viável podem ocorrer episódios de pneumonia e obstrução traqueal recorrente. O processo de descelularização utilizado é capaz de re-mover as células epiteliais da traqueia de coelhos após 10 ciclos, o que evita o processo de rejeição pós-transplante, porém, posteriormente, a presença do epitélio respiratório é essencial para que não ocorra a obstrução do enxerto durante a cicatrização. Sendo assim, será necessário realizar outras investigações na área, a fim de se prolongar a vida útil desses enxertos in vivo.

Keywords: Transplante; Scaffold; Traquéia; Engenharia De Tecidos.

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Abstract - Área Tissue engineering

DEVELOPMENT OF A CONDUIT OF PLGA-GELATIN ALIGNED NANO-FIBERS PRODUCED BY ELECTROSPINNING SEEDED WITH STEM

CELLS FOR NERVE REGENERATION.

Pozzobon, L G 1, Sperling, L. E.1,2, Pranke, P.1,2,3

1Laboratório De Hematologia E Células-Tronco, Universidade Federal Do Rio Grande Do Sul, Brasil2Programa De Pós-Graduação Em Fisiologia, Universidade Federal Do Rio Grande Do Sul, Brasil3Instituto De Pesquisa Com Células-Tronco, Brasil

A promising alternative to conventional nerve grafting is the use of artificial grafts made from biodegradable and biocompatible materials and supportive cells. In this study, a poly (lactic--co-glycolic acid) (PLGA) conduit of aligned nanofibers was produced by the electrospinning method and seeded with mouse embryonic stem cells (mESCs) that were subsequently dif-ferentiated into neural precursor cells. Scaffolds of aligned fibers with an average diameter of 0.90±0.36 µm, an alignment coefficient of 0.817±0.07 and an 112.5±0.12º contact angle were produced. A treatment with gelatin increased the fiber diameter to 1.05±0.32 µm, reduced the alignment coefficient to 0.655±0.045 and made the scaffold very hydrophilic with a contact an-gle of 0°. The conduits seeded with murine embryonic stem cells (mESCs) were submitted to a neural differentiation protocol and the cell proliferation, differentiation and viability were analy-zed. The real time PCR analysis showed that the cells were upregulating the expression of neural markers nestin and beta3 tubulin. The MTT assay showed that the cells maintained via-ble and proliferated after 7 days in culture, showing better viability on gelatin treated conduits. Moreover, the Live/Dead assay indicated high cell viability, with almost no dead cells. Confocal images of phalloidin/DAPI staining showed that the cells adhered and proliferated widely, in fully adaptation with the biomaterial. In conclusion, a method is presented here for producing conduits made of aligned biocompatible and biodegradable PLGA nanofibers with a very good cell-conduit interaction as promising regenerative product for nerve grafting.

Keywords: Biomaterial; Stem Cells; Nerve Graft; Conduit; Aligned Nanofibers; Tissue Engene-ering; Regenerative Medicine.

Abstract - Área Tissue engineering

HIDROGEL INJETÁVEL E TERMOSSENSÍVEL DE POLI(NIPAAM-CO-A-AC-CO-HEMAPLDLA-CO-TMC) DIRECIONADO À ENGENHARIA TECI-

DUAL - ANÁLISE IN VIVO.

Roversi, I. O.1, Sales, D. M.1, Mistura, D. V.1, Oliveira, N. M.1, Hausen, M. A.1, Duek, E. A. R.1

1Pontifícia Universidade Católica de São Paulo, Faculdade de Ciências Médicas e da Saúde, Labora-tório de Biomateriais, Brasil

A Medicina Regenerativa e Reparativa emprega materiais biocompatíveis criados a partir de Engenha-ria Tecidual objetivando promover a recuperação ou substituição de tecidos lesionados. Hidrogéis são sistemas de redes tridimensionais compostas de cadeias poliméricas que apresentam a capacidade de absorver e reter àgua preenchendo os espaços entre as cadeias. A biocompatibilidade destaca-se por essa característica hidrofílica que propicia o microambiente semelhante bioquimicamente à ma-triz extracelular e fornece, com seu sistema tridimensional polimérico, o arcabouço para proliferação celular e consequente reparação tecidual. A biodegradação de hidrogéis ocorre pela solubilização por erosão ou pelo aumento do pH induzindo aumento de cargas iônicas que promovem hidrólise segui-da de dissolução e degradação catalisada por enzimas provenientes do tecido local. A reticulação (reação de gelificação) de alguns hidrogéis ocorre por termossensibilidade dentro do tecido animal aplicado, desse modo é possível injetá-los com técnicas minimamente invasivas em suas formas líqui-das ou pastosas. Considerando as características fisico-quimico-biológicas dessa classe de materiais biocompatíveis e a via minimamente invasiva de implantação, esse estudo visa compreender as al-terações in vivo geradas pelo hidrogel injetável e termossensível de Poli(NIPAAm-co-AAc-co-HEMA-PLDLA-co-TMC) no tecido subcutâneo de ratos Wistar. Foi implantado, via injetável, 3 mL do hidrogel (12% m/v) marcado com nanquim e contraste iodado não-iônico (Iopamiron 300) no tecido subcutâneo do dorso de 20 ratos Wistar divididos em grupos de 4 animais. Realizou-se Tomografias Computado-rizadas de Alta Resolução (TCAR - Semp Toshiba Alexion) nos animais imediatamente à injeção do hidrogel e após 24 horas. Nos períodos de 1, 3, 5, 7 e 14 dias fez-se o sacrifício, extração cirúrgica do tecido e processamento para análise histológica à Microscopia Óptica. À análise em TCAR o im-plante formou, em 24 horas, cavidades no subcutâneo dos animais e liberou a água contida em sua rede polimérica, distribuindo-se na área implantada, evidenciando, por reconstruções tridimensionais e dimensionamento das cavidades, a termossensibilidade e integração tecidual. À análise histológica observou-se invasão de macrófagos e monócitos em diferenciação na matriz do hidrogel como pro-cesso inflamatório agudo, formação poros decorrentes da fagocitose e migração celular a esses sítios formando lâmina inflamatório-cicatricial, aparecimento de microcavidades estruturadas no local de implante, ausência apoptose e necrose, neovascularização aos 7 dias possibilitando distribuição local de substâncias, integração tecidual gerando lâmina inflamatório-cicatricial e alterações da morfologia local que possibilitam aplicações para regeneração e reparação teciduais. A indução da reorganiza-ção tecidual evidenciada promovida pelo hidrogel de Poli(NIPAAm-co-AAc-co-HEMAPLDLA-co-TMC) possibilita aplicações em Engenharia Tecidual, mais especificamente em “Drug Delivery” (liberação controlada e localizada de drogas), considerando a contenção de substâncias ativas pelas microcavi-dades formadas no subcutâneo. Pode-se empregar fatores de crescimento, fatores de diferenciação celular, fármacos e moduladores associados à rede tridimensional polimérica promovendo sua libera-ção gradativa na matriz extracelular e distribuição pela neovascularização local.

Keywords: Hidrogel, Tecido Conjuntivo, Engenharia Tecidual.

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