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형태 및 상 변형에 따른 연골유래 세포외기질 생체소재의 조직재생 응용

Application of Cartilage Extracellular Matrix Biomaterials on Tissue Repair

초록/요약

Tissue derived biological extracellular matrix (ECM) is widely used in tissue engineering. ECM consists of the structural and functional molecules secreted by the cells. Therefore, the specific composition of the ECM constituents will vary depending on the tissue source. Biocompatible ECM is effect to be cell adhesion, proliferation and differentiation in many studies. The aim of this study has focused on developing novel various type of cartilage ECM biomaterials (morphologic and phase modification) for tissue engineering application. In chapter ?, I have developed a novel drug delivery system that continuously releases rhTGF-β3 using a multilayered extracellular matrix (ECM) membrane. We hypothesize that the sustained release of rhTGF-β3 could activate stem cells and result in enhanced repair of cartilage defects. The properties and efficacy of the ECM multilayer-based delivery system (EMLDS) are investigated using rhTGF-β3 as a candidate drug. The bioactivity of the released rhTGF-β3 is evaluated through chondrogenic differentiation of mesenchymal stem cells (MSCs) using western blot and circular dichroism (CD) analyses in vitro. The cartilage reparability is evaluated through implanting EMLDS with endogenous and exogenous MSC in in vivo and ex vivo models, respectively. In the results, the sustained release of rhTGF-β3 is clearly observed over a prolonged period of time in vitro and the released rhTGF-β3 maintains its structural stability and biological activity. This chapter developed a novel drug delivery system (DDS) using chondrocyte derived ECM biomaterial for cartilage repair. In chapterⅡ, I prepared natural scaffold using the cartilage ECM (PCP) and investigated its potential to support osteogenesis of MSCs compared with hydroxyapatite (HA) and poly lactic-co-glycolic acid (PLGA). MSCs-seeded cartilage ECM scaffolds were induced osteogenesis in vitro and transplanted in rat calvarias defect to see in vivo bone formation. In the results, ALP activity and osteogenic gene expressions were significantly higher in cartilage ECM and HA scaffold than in PLGA scaffold. The mineral deposition into the matrix was superior in the ECM scaffold group to other groups. The gene expressions of transcriptional level for osteogenic markers were significantly increased in the ECM scaffold group. 3D porous scaffold using porcine cartilage-derived ECM powder (PCP) successfully support osteogenic differentiation of MSCs to form high quality bone tissue formation in vitro and in vivo. In chapter Ⅲ, this study aimed to develop a 3D plotting system to enable the manufacturing of a biphasic graft consisting cartilage and subchondral bone for application to osteochondral defects. The material advantages of both synthetic (PLGA) and natural (alginate) polymers were combined for a supporting frame and cell printing. Specifically, in order to promote the maturity of the osteochondral graft in our study, cartilage-derived ECM (cECM) or hydroxyapatate (HA) substances blended with alginate was plotted together with human fetal-derived stem cells (HFCPCs) in the cartilage or subchondral bone layer under a multi-nozzle deposition system. The utilized HFCPCs have shown the excellent ability of mesodermal differentiation (adipocyte, osteocyte and chondrocyte) as a plotting cell source. Notably, a plotted biphasic graft shows good integration between cartilage and subchondral bone layers without structural separation. Furthermore, structural collapse of the scaffolds was not observed during the tissue culturing process. The non-toxicity of the cECM and HA substances were proved from a live/dead assay of plotted cell-laden alginate. A fabricated osteochondral graft with cECM and HA substances showed dominant cartilage and bone tissue formation in a differentiation assay. This study identified the plotting feasibility and tissue formation of 3D plotted osteochondral biphasic grafts using PLGA and certain substances (cECM or HA) blended with alginate for the regeneration of osteochondral defects In conclusion, we developed various forms of novel cartilage ECM biomaterials for tissue engineering application. Cartilage ECM’s biological function is maintained in morphologic change and phase modification into powder form and 3D porous scaffold. Our results suggest that developed various types of biological ECM materials could be a useful source for various therapeutic applications on regenerative medicine field.

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목차

TABLE OF CONTENTS………………………………….………………i
LIST OF FIGURES……………………………………….……………v
LIST OF TABLES……………………………….…………………… vii
ABBREVITATIONS…………………………………………………. viii
ABSTRACT……………………………………………………………... ix

BACKGROUND
1. Scaffolds for Tissue Engineering……...…………………...…...……….…………02
1.1 Biomaterials for scaffold fabrication………………………….....………..………02
2. Extracelluar matirx ……………..……………………….…….………………..…04
2.1Tissue derived ECM scaffolds for tissue repair……………….…….……...…..…04
3. Composition of ECM in cartilage ……..…………………….……………………06
3.1Collagen ………………………………..………………………...………..………06
3.2 Proteoglycans …….....…………..……………………...………..…….….………07
3.3 Cartilage tissue engineering …………………..…………………..………………08
4. Motivation of this study……………………...…………………….………………09

CHAPTERⅠ
1. Introduction…….....………………………………………………………………...11
2. Materials and Methods.……………………………….…………………………...14
2.1 Preparation of rhTGF-β3-loaded EMLDS…………….…………..……………...14
2.2 Pattern and profile of release of rhTGF-β3 through the ECM multilayer………16
2.3 Cell isolation and culture ………………….……………………………………...16
2.4 Circular dichroism (CD) analysis ………..……….……………….……………...17
2.5 Western blot analyses …………..……………….……………………………...17
2.6 In vitro chondrogenesis .………..……………….……………………………...18
2.7 Ex vivo implantation for cartilage repair..…………….………………………….18
2.8 Histologic evaluation….………..……………….………………………………...19
2.9 Statistical analysis …….…………..…………….……………………………….19
3. Results………………….……………....…………………………………………... 20
3.1 Release profile for rhTGF-β3…………………….………………….…………...20
3.2 Circular dichroism (CD) analysis for protein structure.…………………..……...23
3.3 Bioactivity assay for rhTGF-β3.…..…………….……………………...24
3.4 In vitro chondrogenesis ………...……………….………..…………………...25
3.5 Ex vivo implantation for human cartilage repair ….….………………………....26
4. Discussion……………....………....………………………………………………...29
5. Summary……………...……..…....………………………………………………...34

CHAPTERⅡ
1. Introduction……………….....…………………………………………………..36
2. Materials and Methods...……....……………………………………………39
2.1 Scaffold preparation………………….....……….………….…………………...39
2.2 Cell seeding into scaffold and differentiation…………………………………...40
2.3 Evaluation of differentiation by RT-PCR analysis ……..……………………….40
2.4 Biochemical analysis ..…………...………..…………….……………………...42
2.5 Animal preparation …...…………..…………….…..…………………………...42
2.6 Micro-CT analysis……....………..…………….……………………………...43
2.7 Histologic evaluation….………..……………….……………………………...43
2.8 Statistical analysis …….………..………..……………………………………...43
3. Results………………………....………………………………………………...44
3.1 Characterization of scaffold .……………….……………….………………...44
3.2 In vitro evaluation of osteogenic differentiation………………………………...45
3.2.1 ALP activity …......……..………………….………………………………...45
3.2 2 RT-PCR analysis .………..….……….……………….……………………...46
3.2.3 Histology ….…..………..………….………………...……………………...48
3.3 In vivo bone formation………..…..……….….………………………………...53
3.3.1 Micro-CT analysis..……....…..……..…………….……...………………...53
3.3.2 Histology….…………..……..…………….………...……………………...53
4. Discussion…………….……....…………………………………………………...56
5. Summary……………...……..…....…………………………………………….....59

CHAPTERⅢ
1. Introduction………...…………………………………………………………….....61
2. Materials and Methods...…………....…………..…………………………….....64
2.1 Cell culture .……….………….………………………………………...………...64
2.2 Multi-head 3D plotting system …………..……………..………………………...64
2.3 Plotting of biphasic scaffolds for osteochondral grafts.………..………..……...66
2.4 SEM analysis and viability analysis.…………….……………………………...66
2.5 Evaluation of differentiation with RT-PCR analysis..…………...……………...67
2.6 Biochemical analysis ….………..………..……...……………………………...69
2.7 Statistical analysis …….………..………..……...……………………………...69
3. Results…….………….….……....………………………………………………...70
3.1 Characterization of scaffolds and cell viability....……..……………………..…...70
3.2 cECM blending effects on chondrogenic differentiation in the cartilage layer scaffold…………………………………………………………………………...73
3.3 HA blending effects on osteogenic differentiation in a subchondral bone layer scaffold…………….…………………………………………………………….73
3.4 Fabrication of hybrid scaffold ……………….….…………………………...75
4. Discussion…....……...………....………..………………………………………...76
5. Summary…………...……..…....………………………………………………...79

CONCLUSION………..…..…………………………………………………………80
REFERENCE…………………...…………………………………………………...81
국문요약…………….……………..…………………………………………………91

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