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ELECTROSPUN NANOFIBROUS SCAFFOLDS FOR TISSUE ENGINEERING

A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES

OF MIDDLE EAST TECHNICAL UNIVERSITY

BY

ALBANA NDREU

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR

THE DEGREE OF MASTER OF SCIENCE IN

BIOTECHNOLOGY

JANUARY 2007

Approval of the Graduate School of Natural and Applied Sciences

Prof. Dr. Canan zgen

Director

I certify that this thesis satisfies all the requirements as a thesis for the degree of Master of Science.

Prof. Dr. Fatih Yldz Head of Department

This is to certify that we have read this thesis and that in our opinion it is fully adequate, in scope and quality, as a thesis and for the degree of Master of Science.

Prof. Dr. Nesrin Hasrc

Co-Supervisor

Prof. Dr. Vasf Hasrc

Supervisor

Examining Committee Members

Prof. Dr. Mesude can (METU, BIO)

Prof. Dr. Vasf Hasrc (METU, BIO)

Prof. Dr. Hseyin A. ktem (METU, BIO)

Prof. Dr. skender Ylgr (Ko Unv., CHEM)

Prof. Dr. Tlin Gray (METU, BIO)

iii

I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work. Name, Last name : Albana NDREU

Signature :

iv

ABSTRACT

ELECTROPSUN NANOFIBROUS SCAFFOLDS FOR TISSUE

ENGINEERING

Ndreu, Albana

M.S., Department of Biotechnology

Supervisor : Prof. Dr. Vasf Hasrc

Co-Supervisor : Prof. Dr. Nesrin Hasrc

December 2006, 83 pages

In this study a microbial polyester, poly(3-hydroxybutyrate-co-3-

hydroxyvalerate) (PHBV), and its blends were wet or electrospun into

fibrous scaffolds for tissue engineering.

Wet spun fiber diameters were in the low micrometer range (10-50 m).

The polymer concentration and the stirring rate affected the properties the

most. The optimum concentration was determined as 15% (w/v).

Electrospun fiber diameters, however, were thinner. Solution viscosity,

potential, distance between the syringe tip and the collector, and polymer

type affected the morphology and the thickness of beads formed on the

fibers. Concentration was highly influential; as it increased from 5% to 15%

(w/v) fiber diameter increased from 284 133 nm to 2200 716 nm.

Increase in potential (from 20 to 50 kV) did not lead to the expected

decrease in fiber diameter. The blends of PHBV8 with lactide-based

v

polymers (PLLA, P(L,DL-LA) and PLGA (50:50)) led to fibers with less beads

and more uniform thickness.

In vitro studies using human osteosarcoma cells (SaOs-2) revealed that wet

spun fibers were unsuitable because the cells did not spread on them while

all the electrospun scaffolds promoted cell growth and penetration. The

surface porosities for PHBV10, PHBV15, PHBV-PLLA, PHBV-PLGA (50:50)

and PHBV-P(L,DL)LA were 38.03.8, 40.18.5, 53.84.2, 50.04.2 and

30.82.7%, respectively. Surface modification with oxygen plasma

treatment slightly improved the cell proliferation rates.

Consequently, all scaffolds prepared by electrospinning revealed a significant

potential for use in bone tissue engineering applications; PHBV-PLLA blend

appeared to yield the best results.

Keywords: Tissue Engineering; Extracellular matrix (ECM); Biodegradable;

Nanofibers; Wet spinning; Electrospinning.

vi

Z

DOKU MHENDSL AMALI ELECTROSPN EDLM

NANOFBRL TAIYICILAR

Ndreu, Albana

Yksek Lisans, Biyoteknoloji ABD

Tez Yneticisi : Prof. Dr. Vasf Hasrc

Ortak Tez Yneticisi : Prof. Dr. Nesrin Hasrc

Aralk 2006, 83 sayfa

Bu almada, mikroorganizmalar tarafndan retilen bir polyester olan

poli(3-hidroksibutirat-ko-3-hidroksivalerat) (PHBV) ve bunun karmlar,

doku mhendisliinde kullanlmak zere wet spinning ve electrospinning

yntemleriyle lifsi yapl hcre taycs oluturulmasnda kullanlmtr.

Wet spinning yntemiyle oluturulan lifsi yaplarn aplar 10-50

mikrometre arasnda deimektedir. Boyutu etkileyen en nemli etkenler

arasnda polimer konsantrasyonu ve kartrma hz gelmektedir. En uygun

konsantrasyon %15 (w/v) olarak belirlenmitir.

Electrospinning yntemiyle elde edilen polimerik ipliklerin wet spinning

yntemiyle elde edilenlere gre daha ince olduu gzlenmitir. Liflerde

oluabilen polimer boumlarnn biimi ve kalnl zeltinin akkanl,

kullanlan potansiyel, uzaklk ve polimer tipi gibi gelerden etkilenmektedir.

Liflerin aplar zellikle polimer konsantrasyonundan byk lde

etkilenmektedir. Konsantrasyonun %5 (w/v)den %15 (w/v)e ykseltilmesi

vii

liflerin apn 284 133 nmden 2200 716 nmye ykseltmitir. Uygulanan

potansiyeldeki art (20 kVdan 50 kVa) lif apnda beklenen azalmay

gstermemitir. PHBV8 ile laktid kkenli polimerlerin (PLLA, P(L,DL-LA) ve

PLGA (50:50)) karmlarnn kullanm daha az boumlu ve genel olarak tek

dze kalnlkl liflerin olumasn salamtr.

nsan osteosarkoma hcreleri (SaOs-2) kullanlarak gerekletirilen in vitro

almalar wet spinning tekniiyle oluturulan liflerin hcrelerin yaylmas

asndan uygun olmadn, electrospinning yntemiyle yaplan tayclarn

ise hcrelerin bymesi ve tayc iinde yaylmas bakmndan uygun

olduunu gstermitir. PHBV10, PHBV15, PHBV-PLLA, PHBV-PLGA (50:50)

ve PHBV-P(L,DL)LA ile elde edilen yaplarn yzey gzeneklilii srasyla %

38.0 3.8, % 40.1 8.5, % 53.8 4.2, % 50.0 4.2 ve % 30.8 2.7

olarak saptanmtr. Oksijen plazma tekniiyle yaplan yzey deiikliklerinin

hcre oalma hzn ok az arttrmtr.

Sonu olarak, electrospinning yntemiyle elde edilen btn hcre

tayclarnn kemik doku mhendisliinde kullanlma potansiyeline sahip

olduu gsterilmi ve PHBV-PLLA polimer karmlar kullanlarak hazrlanan

tayclarn en iyi sonucu verdii belirlenmitir.

Anahtar kelimeler: Doku Mhendislii; Hcre D Matriks (ECM);

Biyobozunur; Nanolifler; Wet spinning; Electrospinning.

viii

To My Family

ix

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to my supervisor Prof. Dr. Vasf

Hasrc, for his continuous guidance, support, and help throughout this

study.

I would also like to thank Prof. Dr. Nesrin Hasrc for her contributions to this

study as my co-supervisor.

I am very grateful to Prof. Nureddin Ashammakhi and his students Lila

Nikkola and Hanna Ylikauppila for their hospitality and kindness during my

insightful visit to Institute of Biomaterials (Tampere University of

Technology, Finland). It was a very good and fruitful experience for me and

I would like to thank their staff for supporting all the necessary equipments

required to handle my experiments.

I am grateful to my husband Dr. Ismail Halili for his help, support and

kindness during all my MSc. studies.

I would like to thank my labmates for their help, support and understanding

especially in my hard days; Pnar Ylgr, Nihan ztrk, Engin Vrana, Halime

Kenar, Deniz Ycel, Buket Bamanav, Pnar Zorlutuna, Erkin Aydn and Dr.

Mathilde Hindie; they have all contributed to this study in some way.

I am grateful to the EU FP6 Expertissues Project, through which the

research was funded.

I would finally like to acknowledge METU Central Laboratory for analyses

done in their facilities.

x

TABLE OF CONTENTS

ABSTRACT ........................................................................................iv

Z...................................................................................................vi

DEDICATION................................................................................... viii

ACKNOWLEDGEMENTS .......................................................................ix

TABLE OF CONTENTS ......................................................................... x

LIST OF TABLES .............................................................................. xiv

LIST OF FIGURES .............................................................................xv

NOMENCLATURE.............................................................................xviii

CHAPTERS

1. INTRODUCTION ............................................................................. 1

1.1 Nanotechnology ........................................................................... 1

1.2 Tissue Engineering ....................................................................... 1

1.2.1 Scaffold Materials ............................................................ 3

1.2.2 Scaffold Characteristics and Types ..................................... 6

1.3 Micro and Nanofib