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  • UNIVERSIDADE DA BEIRA INTERIOR

    Cincias da Sade

    Production of electrospun nanofibers for tissue engineering and other biotechnologic applications

    Tiago Ruivo Correia

    Master Degree Thesis in

    Biomedical Sciences

    (2nd Cycle of studies)

    Supervisor: Ildio Joaquim Sobreira Correia, Ph. D.

    Covilh, June 2012

  • UNIVERSIDADE DA BEIRA INTERIOR

    Cincias da Sade

    Produo de nanofibras para aplicao na Engenharia de Tecidos e noutras aplicaes

    Biotecnolgicas

    Tiago Ruivo Correia

    Dissertao para obteno do Grau de Mestre em

    Cincias Biomdicas

    (2 ciclo de estudos)

    Orientador: Prof.Doutor Ildio Joaquim Sobreira Correia

    Covilh, Junho de 2012

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    Acknowledgments

    This year was laborious and scientifically rewarding. Nevertheless, this task was carried out

    with the help of some people who I want to thank:

    Firstly, I would like to express my deepest gratitude to my supervisor Professor Ildio Correia

    for his guidance, patience and support during this Master Thesis. I also thank him for all the

    conditions and knowledge provided in this area.

    I also would like to thank Eng. Ana Paula from the Optics department of Universidade da Beira

    Interior for the time spent on the acquisition of the scanning electron microscopy images.

    To my work group for the fellowship and healthy atmosphere felt during all this year. So, I

    wish to thank them for supporting me through the hardest difficulties.

    To my closest friends for all the courage and strength given. Also, I want to thank them for

    their patience and support.

    Definitely, to my parents, who have been supporting me all these years. I deeply want to

    thank them for their unconditional guidance and faith.

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    Try not to become a man of success but a man of value.

    Albert Einstein

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    Abstract

    As a specialized and complex structure, bone is a tissue with the capacity to self-regenerate

    that play different functions in our body. However, when there is a critical bone defect the

    self-regenerative capacity is lost. Currently clinical treatments are based on bone grafts and

    other bone substitutes which possess several limitations. Hereupon, Tissue Engineering arises

    as a new scientific field that combines life sciences and engineering knowledges to create

    biological substitutes capable of restoring defects and lesions of biological tissues. In this

    context, a new strategy to mimic the extracellular matrix of bone and cellular

    microenvironment was developed in this work. Therefore, the electrospinning apparatus was

    used to produce poly(-caprolactone), polyethylene oxide-sodium alginate and

    poly(vinyl)pirrolidone nanofibers. Subsequently, the same procedure was used for coating the

    alginate aggregated microparticle scaffold. In addition, polycaprolactone electrospun

    nanofiber membranes were also produced in order to improve the mechanisms on phase

    separation area. These membranes were subjected to a coating process in order to improve

    specific properties, such as pore size, fibers diameter and surface interactions. The biological

    properties of the coated scaffolds were evaluated through in vitro cytotoxicity assays. The

    results showed that all the coated scaffolds had their biological performance improved when

    compared to the same scaffolds without coating. The membranes showed to be useful for the

    separation of biomolecules.

    Keywords

    Coating of scaffolds, electrospinning, electrospun nanofiber membrane, nanofibers, tissue

    engineering.

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    Resumo

    Como uma estrutura especializada e complexa, o osso um tecido com a capacidade de auto-

    -regenerao responsvel por muitas funes no nosso corpo. No entanto, quando existe um

    defeito sseo critico a capacidade auto-regenerativa no suficiente para reparar a leso em

    causa. Na actualidade, os tratamentos clnicos baseiam-se em enxertos de osso e outros

    substitutos de osso que possuem vrias limitaes. Assim, a engenharia de tecidos surge como

    um novo campo cientfico que combina Cincias da vida e os conhecimentos de engenharia de

    forma a criar um substituto biolgico capaz de resolver os defeitos e leses nos tecidos

    biolgicos. Neste contexto, uma nova estratgia para imitar a matriz extracelular do osso e o

    microambiente celular foi desenvolvida atravs deste trabalho. Um aparelho electrospinning

    foi usado para a produo de fibras de policaprolactona, de alginato de sdio, xido de

    polietileno e polivinilpirrolidona. Este processo foi ainda usado para revestir scaffolds de

    agregados de micropartculas de alginato. Por outro lado, foram tambm desenvolvidas

    membranas base de policaprolactona com o objetivo de serem usadas na purificao de

    diferentes biomolculas. As membranas produzidas foram ainda submetidas a um processo de

    revestimento para melhorar propriedades especficas. A Caracterizao biolgica dos

    scaffolds revestidos foi realizada atravs de ensaios in vitro. Os resultados obtidos mostraram

    que todos os revestimentos efetuados nos scaffolds melhoraram o seu desempenho biolgico,

    relativamente aos scaffolds sem revestimento. As membranas produzidas por electrospinning

    apresentaram boas propriedades, para serem testadas na separao de biomolculas.

    Palavras-Chave

    Electrospinning, engenharia de tecidos, membranas de nanofibras, nanofibras, revestimento de scaffolds.

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    Index

    Chapter I:

    Introduction ....................................................................................... 1

    1.1 Bone structure ......................................................................... 2

    1.2 Composition of bone .................................................................. 4

    1.3 Bone formation and repair ........................................................... 6

    1.4 Bone disorders ......................................................................... 8

    1.5 Bone grafts ............................................................................. 9

    1.6 Cell surface interactions........................................................... 10

    1.7 Electrospinning ....................................................................... 11

    1.7.1 Parameters that influence electrospinning technique ........................ 14

    1.7.2 Polymeric nanofibers ............................................................... 16

    1.7.3 Nanofibers applications for bone tissue engineering .......................... 18

    1.8 Objectives ................................................................................ 19

    Chapter II: ........................................................................................ 20

    Materials and Methods ......................................................................... 20

    2.1 Materials .................................................................................. 21

    2.2 Methods ................................................................................... 21

    2.2.1 Electrospinning setup .............................................................. 21

    2.2.2 Preparation of the polymer solutions ............................................ 21

    2.2.3 Electrospinning setting ............................................................. 22

    2.2.4 Scanning electron microscopy .................................................... 22

    2.2.5 Fourier transform infrared spectroscopy ........................................ 22

    2.2.6 Nanofibers coating of alginate microparticle aggregated scaffolds ......... 23

    2.2.7 Proliferation of human osteoblast cells in the presence of the alginate

    microparticle aggregated scaffolds ..................................................... 23

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    2.2.8 Characterization of the cytotoxicity profile of the alginate microparticle

    aggregated scaffolds ...................................................................... 23

    2.2.9 Statistical Analysis .................................................................. 24

    2.2.10 Production and coating of electrospun nanofiber membranes (ENMs)..... 24

    Chapter III:

    Results and Discussion .......................................................................... 25

    3.1 Coating of the alginate microparticle aggregated scaffolds with different

    polymeric nanofibers......................................................................... 26

    3.1.1 Characterization of scaffolds coated with PCL nanofibers ................... 27

    3.1.2 Characterization of scaffolds coated with PEO-SA nanofibers ............... 28

    3.1.3 Characterization of scaffolds coated with PVP nanofibers ................... 29

    3.2 Characterization of electrospun nanofiber membranes (ENMs) ................... 31

    3.3 Fourier transform infrared spectroscopy analysis of the ENM surfaces ........ 35

    3.4 Evaluation of the cytotoxic profile of the different coatings on the scaffold .. 38

    Chapter IV:

    Conclusions and Future Perspectives ........................................................ 42

    Chapter V:

    References ....................................................................................... 44

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    List of Figures

    Chapter I: Introduction

    Figu