Encapsulating chondrocyte using injectable and thermosensitive mPEG-polyester hydrogels

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研究生：	彭心怡 Peng, Sydney
論文名稱：	Encapsulating chondrocyte using injectable and thermosensitive mPEG-polyester hydrogels
指導教授：	朱一民 Chu, I-Ming
口試委員:	湯學成 Tang, Shiue-Cheng 姚少凌 Yao, Chao-Ling
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	77
中文關鍵詞：	水膠、細胞包埋、溫度敏感型水膠
外文關鍵詞：	thermosensitive, hydrogel, mPEG-PLGA, mPEG-PVLA, chondrocytes, cell encapsulation
相關次數：	點閱：2 下載：0
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The objective of this study was to evaluate thermosensitive mPEG-polyester hydrogels prepared from diblock copolymers methoxy poly(ethylene glycol)-(polylactide-co-glycolide) (mPEG-PLGA) and methoxy poly(ethylene glycol)-(valerolactone-co-lactide) (mPEG-PVLA) for chondrocyte encapsulation in cartilage tissue engineering applications. Different block lengths of mPEG-polyester copolymers were prepared by ring-opening polymerization and preliminary results showed that hydrogels prepared from mPEG-PLGA sized (550-1405) and mPEG-PVLA sized (550-1405) and (750-1916) were capable of gelation under physiological temperature. The swelling ratio of mPEG-PVLA (750-1916) hydrogel was significantly higher than those compared by at least 2-folds. Copolymer solutions prepared in phosphate buffer saline (PBS) resulted in acidic pH environments of 4.1~4.9 within the hydrogel which is hostile to encapsulated cells. Substituting PBS with Dulbecco’s modified eagle medium (DMEM) resulted in neutral hydrogels with pH ~7. Degradation study showed that hydrogels underwent incomplete degradation within a 30 day period with mPEG-PVLA exhibiting slower degradation compared to mPEG-PLGA due to greater hydrophobicity.
Viability of chondrocytes encapsulated was confirmed by thiazolyl blue tetrazolium bromide (MTT) and LIVE/DEAD staining which demonstrated homogenous encapsulation as well as over 50% viability immediately post-encapsulation. Taken together with DNA quantification results, we concluded that the hydrogels were capable of supporting proliferation of chondrocytes within 7 days of culturing in vitro. In addition to proliferation, extracellular matrix (ECM) proteins namely glycosaminoglycans (GAGs) increased most significantly in mPEG-PVLA (750-1916) hydrogel with a 11.8-fold increase from day 1 to day 21. On day 14, the collagen content mPEG-PVLA (750-1916) was higher than all other groups (p<0.05). The addition of 5% collagen type I did not significantly affect proliferation and GAG or collagen secretion as was expected. Results of this study indicate that mPEG-polyester hydrogels are capable of supporting chondrogenesis in vitro and that their application as injectable cartilage tissue engineering systems is promising.

ABSTRACT    I
TABLE OF CONTENTS    III
LIST OF FIGURES    V
LIST OF TABLES    VII
LIST OF ABBREVIATION    VIII
INTRODUCTION    1
REVIEW OF LITERATURE    2
1 CARTILAGE TISSUE ENGINEERING    2
1.1 Choice of Cells    6
2. HYDROGEL SYSTEM    10
2.1 Types of Hydrogels (Natural vs. Synthetic)    10
2.2 Stimuli-response Hydrogel (pH and Light Sensitive)    12
2.3 Thermosensitive Hydrogels    14
2.4 Poly(lactide-co-glycolide) (PLGA)    18
2.5 Poly(valerolactone-co-lactide) (PVLA)    20
3 TISSUE ENGINEERING WITH HYDROGEL    22
AIM AND HYPOTHESIS    25
MATERIAL AND METHODOLOGY    27
1 MATERIALS AND EQUIPMENTS    28
2 SYNTHESIS OF MPEG-PLGA AND MPEG-PVLA COPOLYMERS    30
3 CHARACTERIZATION OF DIBLOCK COPOLYMERS    31
3.1 Proton Nuclear Magnetic Resonance (1H NMR)    31
3.2 Gel Permeation Chromatography (GPC)    31
3.3 Preparation of Copolymer Solution    31
3.4 Sol-gel Transition Profile    32
3.5 Degradation of Polymer in vitro    32
3.6 Swelling Ratio    33
4 CELL ENCAPSULATION    34
4.1 Isolation and Culturing of Cells    34
4.2 Encapsulation of Cells in 3D Hydrogel Model    34
4.3 Induced Chondrogenesis    35
4.4 Cell Viability    35
4.5 LIVE/DEAD Staining    35
5 BIOCHEMICAL ASSAYS    37
5.1 DNA Content    37
5.2 GAG Content    37
5.3 Collagen Content    38
5.4 Statistical Analysis    38
RESULTS AND DISCUSSION    39
1 SYNTHESIS OF MPEG-PLGA AND MPEG-PVLA COPOLYMERS    39
2 CHARACTERIZATION OF DIBLOCK COPOLYMERS    45
2.1 Preparation of Polymer Solution    45
2.2 Sol-gel Transition Profile    47
2.3 Swelling Ratio    54
2.4 Degradation    55
3 CELL ENCAPSULATION AND BIOCHEMICAL ANALYSIS    59
3.1 MTT Assay    59
3.2 LIVE/DEAD Stain    63
3.3 DNA Content    65
3.4 GAG Content    67
3.5 Collagen Content    69
CONCLUSIONS    70
REFERENCE    73

                                

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