Tissue engineering hydrogels with tunable degradability

Tissue Engineering

Min Lee, Soyon Kim, University of California, Los Angeles; Zhong-Kai Cui, University of California, Los Angeles;

Min Lee, University of California, Los Angeles

Corresponding Author: Min Lee, University of California, Los Angeles, leemin@ucla.edu

Abstract

Photopolymerizable hydrogels derived from naturally occurring polymers have a unique opportunity to regenerate

damaged tissues due to their excellent biocompatibility, hydrophilic nature favorable for cell ingrowth, and ability to be

cured in situ through a minimally invasive procedure. We have previously developed injectable hydrogels using

photopolymerizable chitosan (MeGC) and riboflavin (RF), an aqueous initiator from natural vitamin, that allow gelation

in situ upon exposure to visible blue light (VBL). Tissue engineering scaffolds must be designed to degrade to allow

subsequent tissue regeneration or the release of encapsulated bioactive molecules. Although the MeGC hydrogel

supported proliferation and deposition of extracellular matrix (ECM) by encapsulated mesenchymal stem cells, it

showed relatively slow degradation that may hinder cell recruitment and delay tissue regeneration. In this work, we

have developed MeGC hydrogels with tunable degradation kinetics by introduction of hydrogel-specific enzymes.

Lysozyme is existed ubiquitously in human body and could degrade chitosan by cleaving its polysaccharide backbone.

Thus, we investigated whether the degradation rate of MeGC hydrogels can be modulated by introducing lysozyme and

varying the enzyme concentration. Briefly, glycidyl methacrylate was reacted with lysozyme solution to synthesize

photo-reactive lysozyme. Methacrylated lysozyme was mixed with MeGC solutions and hydrogels were formed by VBL-

crosslinking in the presence of RF. The incorporation of lysozyme to MeGC hydrogels significantly increased the

degradation rate of the hydrogels in a dose dependent manner. The lysozyme-functionalized MeGC hydrogels were not

cytotoxic and significantly enhanced proliferation of encapsulated bone marrow stromal cells (BMSCs) as confirmed by

the alamar blue assay. The encapsulation of BMSCs improved hydrogel modulus over time in culture compared with

cell-free hydrogels, indicating deposition of ECM on hydrogels as the hydrogel degrades. In addition, the lysozyme-

functionalized hydrogels significantly upregulated alkaline phosphatase (ALP) expression and osteogenic gene markers in

encapsulated BMSCs and increased mineral deposition. Hydrogel degradation and osteogenic activity was further

confirmed in a mouse calvarial defect model. Histological examination of the in vivo samples demonstrated remarkable

hydrogel degradation with high cell infiltration at day 7. The MeGC hydrogels functionalized with lysozyme significantly

promoted calvarial repair at week 6 as demonstrated by micro-computerized tomography and histology, compared with

unmodified MeGC. These findings suggest a promising approach to modulate the degradation behavior of hydrogels via

enzyme functionalization for improved tissue remodeling.

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Effects of Wettability and Surface Roughness of Hollow Fiber Membranes on Bacterial Adhesion

Biomaterials

Kaitlyn Anderson, Utah State University; David Britt, Utah State University

Corresponding Author: David Britt, Utah State University, Biological Engineering Department, david.britt@usu.edu

Abstract

Hollow fiber membranes (HFMs) have a wide variety of applications, such as for hemodialysis filtration membranes and

as bioreactors, and, depending on the use, cellular adhesion to the HFMs may be discourage, the former, or encouraged,

the latter. Fortunately, HFMs can be created from many different spinmass mixtures of polymers and additives with

each mixture yielding a particular set of HFM characterizations. This research will be focusing on the properties of

wettability and surface roughness of a variety of commercially available HFMs and how they impact the adhesion of two

biofilms forming bacterial species, Pseudomonas chlororapis isolate O6 (PcO6) and Bacillus subtilis isolate 309 (Bs309).

PcO6 produces a biofilm that is orange, thick, and slimy and, in stark contrast, Bs309 produces a biofilm that is white,

dry, and wrinkly and this difference will provide insight to bacterial preference in HFMs. In order to encourage the

growth of the bacterial biofilms on the external surface of the HFM, a lysogeny broth (LB), a nutritionally rich medium,

will be flowed continuously through the lumen of the HFM that will then wick the outer surface. Wettability of the HFMs

will be assessed using a tensiometer that will measure the amount of saturation and subsequent evaporation the HFMs

are capable of and the surface roughness of the HFMs will be observed through SEM imaging. The bacterial biofilms will

be imaged using a fluorescence microscope on a day to day basis in order to observe any visual changes that occur in the

growth between the different HFMs.

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Fabricating Bioinspired Materials through Intrinsic and Extrinsic Control of Freeze Casting

Biomaterials

Steven Naleway, University of Utah

Corresponding Author: Steven Naleway, University of Utah, steven.naleway@mech.utah.edu

Abstract

Freeze casting is a bioinspired technique for the fabrication of tailored, porous ceramic materials. Mimetic of the growth

of mammalian bone and other biomaterials where biopolymers template the deposit of biominerals to create complex

composites, freeze casting employs a template of growing ice crystals to create a complex porous microstructure in any

ceramic. I have proposed that this bioinspired technique can be controlled through either intrinsic (those that modify

from within by altering the constituents) or extrinsic (those that apply external forces or templates) means. Through

these classifications, I present novel examples of both intrinsic and extrinsic freeze cast, bioinspired structures with a

focus on providing advanced control of the final material structure and properties. These new materials and techniques

are proposed for structural, biomedical, and green material applications.

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Antimicrobial Carbon-based Nanomaterials

Biomaterials

Liju Yang, North Carolina Central University

Corresponding Author: Liju Yang, North Carolina Central University, lyang@nccu.edu

Abstract

Infectious diseases caused by bacterial pathogens have been a serious threat to public health for decades and remain

one of the major concerns of our society. Control and prevention of pathogen contamination are effective ways to

reduce the risk of such disease. The advancement of nanotechnology has brought various nanomaterials with

remarkable properties for a wide range of applications, including the antimicrobial fields. Carbon-based nanomaterials

represent a class of relatively safe and cost-effective materials yet with desirable antimicrobial properties.

Our group has been exploring the antimicrobial activity of carbon-based nanomaterials and their composites, mainly

including carbon nanotubes (CNTs) and carbon “quantum” dots. We investigated the applications of single-walled CNTs

(SWNTs), multi-walled CNTs (MWNTs), CNTs-Ag, CNTs-chemicals/natural peptides, CNTs-NIR, CNTs-coated surfaces, and

various polymer-modified carbon dots for inactivation of bacterial pathogens, bacterial spores, and viruses, inhibition of

biofilms, as well as for capturing and concentrating bacterial pathogens. The results indicated that SWNTs, their

composites or combination with other chemical and physical methods were very effectively inactivated various bacterial

pathogens including common Salmonella, E. coli cells, Bacillus anthracis cells in suspensions. Whereas MWNTs were not

effective in inactivating bacterial cells in suspension, but MWNTs modified surfaces, including MWNT forest on silicon

wafer and MWNT sheet on poly(methyl methacrylate) (PMMA) film, significantly increased surface hydrophobicity and

enhanced the attachment of spores on their surfaces compared to the uncoated substrates, respectively, showing their

potential as adsorbents for removal of pathogens from fluids.

Another class of notable antimicrobial carbon-based nanomaterials is carbon dots (CDots). CDots defined as small

carbon nanoparticles with various surface passivation schemes, with their optical properties and photocatalytic

functions. They have been evaluated for their photoinduced bactericidal functions, with the results suggesting that the

dots were highly effective in bacteria-killing with visible light illumination. Mechanistic implications of the results will be

discussed. Challenges and opportunities in further development of CDots into a new class of effective visible/natural

light-responsible bactericidal agents for bacteria control and other potential antimicrobial applications will be discussed.

Acknowledgement: This research was supported partially by the USDA grant 2011-68003-30395 and the NIH grant

R15GM114752.

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The approaching renaissance for calcium phosphates in biomedicine

Biomaterials

Vuk Uskokovic, Chapman University, University of Illinois at Chicago

Corresponding Author: Vuk Uskokovic, Department of Bioengineering, University of Illinois, Department of Biomedical and Pharmaceutical Sciences, Chapman University, uskokovi@chapman.edu

Abstract

Calcium phosphate was selected by Nature to be the firm foundations of our bodies. This has served as an invaluable

inspiration for materials scientists who have attempted to discover in this material more potential than meets the eye.

Efforts are currently being made to expand the application repertoire of calcium phosphates beyond their use as

traditional bone fillers or tissue engineering construct components that impart osteoconductivity and high compressive

strength. The application of calcium phosphates for sustained drug delivery, gene and anticancer therapies, antibiofilm

coatings and hard tissue regeneration has been intensely explored recently. All this plethora of applications for calcium

phosphates that are now in the R&D stage are the consequence of the immense structural complexity of this material,

which is being directly reflected in its ability to display an array of exciting properties under precise synthesis regimens.

Like water, the princess of peculiarities in the realm of liquids, calcium phosphate deserves the epithet of the prince of

peculiarities in the realm of solids. Its protean nature and the applicative potentials arising from this peculiar nature will

be described in this lecture. Foreseen on the horizon will be a new generation of materials for therapeutic and

regenerative applications, containing only precisely designed calcium phosphates and substituting for the role of

expensive bone growth factors, antibiotics, viral vectors and polymers.

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Vitamin E-coated hernia repair meshes for the mitigation of the oxidative stress

Biomaterials

Dmitry Gil, Vladimir Reukov, Clemson University; William Cobb, Greenville Health System; Alexey Vertegel, Clemson University

Corresponding Author: Dmitry Gil, Clemson University, dgil@clemson.edu

Abstract

Hernia repair surgeries are among the most common operations with over 20 million procedures performed annually.

Recent advancements in biomedical engineering made this type of surgeries safer than ever. However, multiple

evidence suggests that almost 30% of patients who underwent hernioplasty experience pain, and more than half of

those patients are unable to participate in daily, physical or athletic activities due to the severity of the discomfort.

Although the actual mechanism of the development of post-hernioplasty complications is still unknown, most of the

researchers lean towards an oxidative stress as a key factor that leads to mesh deterioration. In an attempt to address

this problem, polypropylene hernia repair meshes were coated with a well-known antioxidant – -tocopherol (vitamin E).

The goal of this study was to evaluate in vivo anti-inflammatory properties of Vitamin E-coated polypropylene hernia

repair meshes. A rabbit model was used to evaluate the efficacy of the antioxidant coating. 5 weeks after the surgery,

the samples were explanted and histopathological evaluation was performed. Plain polypropylene mesh was used as a

reference. It was found that implantation of vitamin E-coated mesh reduced the inflammatory response when compared

to a plain polypropylene implant. Subsequently, the reduced inflammatory response positively affected the healing

process, leading to an improved connective tissue architecture. In particular, the uncoated implant was surrounded by

heavier fibrosis and collagen encapsulation when compared to the vitamin E coated mesh. Highly organized collagen and

heavy fibrosis encompassing hernia meshes are undesirable as it may lead to implant shrinkage and could also be

correlated with the incidence of adhesions. Analysis of the collagen framework at the surgical site was of particular

interest since the biomechanical strength in wound tissue, in many ways, is defined by collagen architecture and

organization. An increased content of immature collagen in the vicinity of the plain mesh was noticed, when compared

to the vitamin E coated mesh. Thus, data obtained in this work show the efficacy of the vitamin E as an antibacterial

coating on hernia repair meshes. These results serve as a foundation for further animal and clinical studies of Vitamin E

coated hernia repair meshes.

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Decreased Macrophage Attachment on Biomimetic Surfaces

Biomaterials

Ching-An Peng, Jun Zhang, Department of Biological Engineering, University of Idaho

Corresponding Author: Ching-An Peng, University of Idaho, capeng@uidaho.edu

Abstract

CD200, or OX-200, is a membrane glycoprotein encoded by the CD200 gene. CD200 interacts with its receptor CD200R

expressed on the surface of myeloid cells such as macrophages and neutrophils. CD200 has shown to decrease the

activation of inflammatory cells and reduce inflammatory responses to implanted biomaterials. Polydopamine is an

ultrathin film capable of further surface functionalization. The purpose of this study is to immobilize CD200 on

polydopamine coated surface to prevent macrophage attachment. Briefly, the surface of a 6-well tissue culture plate

was coated with polydopamine and then functionalized with biotin. CD200-streptavidin fusion protein was appended on

the surface through biotin-streptavidin binding. J774A.1 macrophages were inoculated onto control and CD200 coated

surface. As a result, CD200 modified surface showed decreased macrophage attachment for 8 hours compared to

control surfaces. The CD200-CD200R signal was blocked by treating CD200 coated surface with anti-CD200, as well as

treating macrophages with anti-CD200R. After blocking the signal, the control experiments showed no delay in cell

attachment. It was confirmed that the CD200-CD200R signal effectively reduced macrophage attachment.

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Organ-on-a-chip for assessing environmental toxicants

Tissue Engineering

Soohee Cho, University of Arizona; Kattika Kaarj, University of Arizona; Jeong-Yeol Yoon, University of Arizona

Corresponding Author: Jeong-Yeol Yoon, University of Arizona, jyyoon@email.arizona.edu

Abstract

Man-made xenobiotics, whose potential toxicological effects are not fully understood, are oversaturating the already-

contaminated environment. Due to the rate of toxicant accumulation, unmanaged disposal, and unknown adverse

effects to the environment and the human population, there is a crucial need to screen for environmental toxicants.

Animal models and in vitro models are ineffective models in predicting in vivo responses due to inter-species difference

and/or lack of physiologically-relevant 3D tissue environment. Such conventional screening assays possess limitations

that prevent dynamic understanding of toxicants and their metabolites produced in the human body. Organ-on-a-chip

systems can recapitulate in vivo like environment and subsequently in vivo like responses generating a realistic mock-up

of human organs of interest, which can potentially provide human physiology-relevant models for studying

environmental toxicology. Feasibility, tunability, and low-maintenance features of organ-on-chips can also make possible

to construct an interconnected network of multiple-organs-on-chip towards a realistic human-on-a-chip system. Such

interconnected organ-on-a-chip network can be efficiently utilized for toxicological studies by enabling the study of

metabolism, collective response, and fate of toxicants through its journey in the human body. Further advancements

can address the challenges of this technology, which potentiates high predictive power for environmental toxicology

studies.

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Silicon nanowire synergize with electrical stimulation to promote ventricular maturation of hiPSC-derived cardiomyocytes

Tissue Engineering

Ying Mei, Dylan Richards, Clemson-MUSC Bioengineering Program, Charleston, SC 29425

Corresponding Author: Ying Mei, Clemson University, mei@clemson.edu

Abstract

Cardiovascular disease is the leading cause of death and disability worldwide. Due to the limited regeneration capacity

of adult human hearts, human induced pluripotent stem cell (hiPSC) has emerged as a powerful cell source for cardiac

repair. However, the current hiPSC-derived cardiomyocytes (hiPSC-CMs) poses arrhythmogenic risk after transplantation

due to their unspecific and immature phenotype. In addition, the hiPSC-CMs are quickly redistributed to other organs

after transplantation due to the shear stress in the beating hearts. To address this, my lab recently pioneered the use of

the electrical conductive silicone nanowire to facilitate the self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac

spheroids. The addition of e-SiNWs into human cardiac spheroids creates an electrically conductive microenvironment

and improves the functions of hiPSC-CMs. In addition, 3D configuration of the nanowired spheroids can improve their

engraftment after transplantation. Further, our recent research showed electrical stimulation can synergize with silicon

nanowire to facilitate the ventricular lineage specification and cellular maturation of hiPSC-CMs. Here, I will discuss the

recent development of my lab in the use of the electrical stimulation to derive hiPSC-CMs with enhanced ventricular

maturity for transplantation applications.

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Sulfonated chitosan hydrogel to stabilize bone morphogenetic protein activity for enhanced osteogenesis

Tissue Engineering

Min Lee, Soyon Kim, University of California, Los Angeles; Zhong-Kai Cui, University of California, Los Angeles;

Min Lee, University of California, Los Angeles

Corresponding Author: Min Lee, University of California, Los Angeles, leemin@ucla.edu

Abstract

Although bone morphogenetic protein-2 (BMP-2) has demonstrated extraordinary potential in bone formation, its

clinical applications require supraphysiological milligram-level doses. The premature release of such high dose BMP-2

from conventional collagen carriers may lead to ectopic bone formation and numerous side effects such as well-

documented life-threatening cervical swelling and osteoclastic bone resorption. Heparin is widely used in controlled

release systems for many heparin binding growth factors including BMP-2 due to its strong binding property and

protective effect for these proteins. However, heparin suffers from natural variability in structure, difficult modification,

and many non-targeting bioactivities. Similar biological activities to heparin have been observed in small compounds

that contain groups similar to those in the heparin, in particular, sulfates or sulfonates. In this work, we developed a

hydrogel surface that can mimic heparin to stabilize BMP-2 and enhance osteogenesis by incorporating two heparin-

mimicking polysulfonates, Poly-vinylsulfonic acid (PVSA) and Poly-4-styrenesulfonic acid (PSS) into photo-crosslinkable

hydrogel already developed in our previous study using methacrylated glycol chitosan (MeGC) and riboflavin initiators

Incorporation of heparin-mimicking molecules was performed by dissolving PVSA or PSS in MeGC solution and

subsequently crosslinking MeGC to form a gel. The homogeneous distribution and high entrapment efficiency (>95%) of

PVSA or PSS was verified by toluidine blue staining. The protective effect of PVSA or PSS on BMP-2 stability was

confirmed in various therapeutically relevant environments presenting thermal, acidic and enzymatic stressors in

comparison to natural heparin by assessing the ability of BMP-2 to increase alkaline phosphatase (ALP) expression in

bone marrow stromal cells (BMSCs). Ability of the developed hydrogels to enhance BMP-2 signaling and osteogenic

activity was determined with or without the addition of exogenous BMP-2. The sulfonated MeGC hydrogels significantly

increased osteogenic differentiation of encapsulated BMSCs without BMP-2 supplementation as observed by increased

mineralization as well as upregulated osteogenic gene markers compared to unmodified hydrogel. This is mostly due to

the sulfonated hydrogel surface that sequestered and augmented endogenous BMP-2 activity as validated by

immunostaining for BMP-2. Moreover, the sulfonated hydrogels supported sustained release of loaded BMP-2 with

reduced initial burst and significantly increased osteogenesis of encapsulated BMSCs compared to unmodified MeGC.

These findings suggest a novel hydrogel platform that could stabilize and enhance BMP-2 in bone tissue engineering.

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Understanding the Role of Chain Flexibility in Amyloid Protein Aggregation through Rationally Designed Protein Sequences

Biomaterials

S. Zeb Vance, University of South Carolina; Xavier Redmon, University of Arkansas; Rachel Hall, University of South Carolina, Jamie Crawford, University of South Carolina, Christa Hestekin, University of Arkansas; Melissa Moss, University

of South Carolina

Corresponding Author: Melissa Moss, University of South Carolina, mossme@cec.sc.edu

Abstract

The aggregation of amyloid proteins is associated with a myriad of medical conditions including Alzheimer’s disease

(AD), diabetes, and Parkinson’s disease. While attributable to different amyloid proteins, these proteins all share a

common feature: a periodic glycine motif (GxxxG). This glycine motif, associated with increased backbone flexibility, is

extended in a number of familial mutations that significantly increase severity of AD. A better understanding of the role

that chain flexibility plays in protein aggregation will allow for new and innovative protein engineering platforms for

future nanotech development as well as give insight into therapeutic strategies.

In this study, the glycine motif is targeted via either extension, by introduction of additional periodic glycine, or

contraction, by replacement of glycine with a bulky or constrained amino acid. Modifications that extend periodic

glycine include those that align with familial AD mutations. To examine how these alterations to the glycine repeat motif

impact aggregation kinetics, aggregation was monitored via thioflavin-T fluorescence and fit using a novel kinetic

equation that accounts for unique features observed at late stages of aggregation. Aggregation products were visualized

using transmission electron microscopy to examine morphological features. In addition, aggregates were fractionated by

size exclusion chromatography for size analysis via light scattering and morphology analysis via surface hydrophobicity.

Results indicate that increased chain flexibility correlates with faster nucleation as well as a larger quantity of small,

intermediate aggregates that exhibit reduced fibril morphology with unchanged surface hydrophobicity. Taken together

with observations that smaller aggregates are more physiologically active, these results support the hypothesis that

increases in protein chain flexibility, including that associated with familial mutations, may contribute to disease

progression.

Future work will extend studies to other amyloid proteins, such as amylin and chaplin H.

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