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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39285Full metadata record
| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 劉華昌(Hwa-Chang Liu),林峰輝(Feng-Huei Lin) | |
| dc.contributor.author | Chih-Hung Chang | en |
| dc.contributor.author | 張至宏 | zh_TW |
| dc.date.accessioned | 2021-06-13T17:25:24Z | - |
| dc.date.available | 2005-01-31 | |
| dc.date.copyright | 2005-01-31 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-01-24 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39285 | - |
| dc.description.abstract | Abstract
Articular cartilage injury is a great challenge for orthopedic surgeons. Tissue engineering had merged as a new method for cartilage repair. The tissue engineering triads consisted of cells, biomaterial scaffold and bioactive factors and environment (provided by bioreactor). In our study, all these three aspects had been explored. Part I. Cartilage tissue engineering: Hypothesis: Current synthetic scaffolds for cartilage tissue engineering have many shortcomings. We hypothesized that a tri-copolymer formed from gelatin, chondroitin, and hyaluronan might mimic cartilage matrix and provide the necessary information for cartilage tissue engineering. Methods: Porcine chondrocytes seeded tricopolymer were cultured in spinner flasks and Petri dish for 2, 3, 4 and 5 weeks for in vitro cartilage tissue engineering. Large scale animal study with 15 miniature pigs is well designed, with randomized control study to compare the therapeutic effect of allogenous chondrocytes seeded tricopolymer scaffold based tissue engineering, autogenous osteochondral transplantation and spontaneous healing process for full thickness articular defects and osteochondral defects. Another 6 pigs were treated with defects creation for spontaneous healing or filled with scaffold without cell seeding. Results: In culturing results of the tissue-engineered constructs in spinner flask, we found chondrocytes are distributed more evenly than in the scaffold in Petri dish. The constructs were found with new extracellular matrix synthesis with type II collagen contents. And the chondrocytes still retain their phenotype as demonstrated by immunohistochemistry. In animal study, after exclusion of the cases with infection and secondary arthritis, the best results comes from the autogenous osteochondral transplantation except the integration to host cartilage was poor. The results of tissue engineering treated group were satisfactory with repair tissue varied from hyaline cartilage to fibrocartilage. The spontaneous healing response is not enough for good repair. Filling of scaffolds without cell seeding can not offer good repair. For osteochondral defects, the subchondral bone plate was not restored through cartilage tissue engineering, thus osteochondral tissue engineering is necessary. Part II. Osteochondral tissue engineering: Hypothesis: Osteochondral tissue engineering is necessary for the treatment of osteochondral defect. We hypothesized through a biphasic scaffold and double-chamber bioreactor, tissue engineered osteochondral constructs can be developed. Methods: Gelatin sponge was formed on the top of the calcined bovine bone to fabricate a biphasic scaffold. Porcine chondrocytes were seeded on the biphasic scaffold and cultured in specially designed double chamber bioreactor for 2 and 4 weeks. Then the human mesenchymal stem cells (hMSCs) were induced separately for chondrogenesis and osteogenesis, and co-cultured in double chamber bioreactor for fabrication of tissue engineered osteochondral constructs. Results: Cartilage formation with porcine chondrocytes seeded biphasic scaffold on the surface of the calcined bovine bone was successful demonstrated. The induction of hMSC is successful, except that there is a lower efficiency for chondrogenic induction. Cell pellets were too aggregated, which can not cover the whole surface of calcined bovine bone. The calcined bovine bone is easily cracked in injection seeding of cells, further modification of the procedure in fabrication of osteochondral constructs is necessary. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T17:25:24Z (GMT). No. of bitstreams: 1 ntu-94-F89548018-1.pdf: 4467290 bytes, checksum: 16cc9e7f3839aa38fcff8b34f89f896c (MD5) Previous issue date: 2005 | en |
| dc.description.tableofcontents | Contents
Chapter 1 Introduction 1-1 Foreword…………………………………………………………………….................1 1-2 Physiology of spontaneous repair of articular cartilage injury……………………..…3 1-3 Current treatment modalities for articular cartilage injury…………………................5 1-3.1 Bone marrow stimulating techniques………………………………..…5 1-3.2 Autogenous and allogenous osteochondral transplantation…................8 1-3.3 Autogenous chondrocytes implantation………………………............11 1-4 Future trends and considerations in repair of cartilage injury……………….............14 1-4.1 Tissue Engineering and its current status in researches………............14 1-4.2 Animal Model: Preclinical study………………………………….….20 1-5 Purpose of this study…………………………………………………………...........22 Chapter 2 Theoretical Basis 2-1 Cells in cartilage tissue engineering………………………………………................23 2-1.1 Chondrocytes………………................................................................23 2-1.2 Mesenchymal stem cells……………………………………….……..24 2-1.3 Periosteal cells…………………………………………………..........26 2-1.4 Synovial cells…………………………………………………………26 2-2 Biomaterials in cartilage tissue engineering…………………………………………26 2-2.1 Scaffold made from synthetic polymer……………………………….27 2-2.2 Scaffold made from natural substance…………………………..........28 2-2.3 Cartilage composition and tricopolymer to mimic natural matrix........31 2-3 Bioreactors in cartilage tissue engineering……………………………………..........37 2-3.1 The Role of Bioreactors in Cartilage Tissue Engineering…………….37 2-3.2 Design of, and rationale for, the double chamber bioreactor………….40 Chapter 3 Materials and Methods 3-1 Cartilage Tissue Engineering……………………………………………………............43 3-1.1 In vitro study……………………………………………………...........43 3-1.1.1 Fabrication of tri-copolymer scaffold…………………..................43 3-1.1.2 Culture expansion and seeding of chondrocytes…………………..44 3-1.1.3 Evaluation of in vitro study…………………………………..........44 3-1.2 Animal Study……………………………………………………...........45 3-1.2.1 Study design………………………………………………….…….45 3-1.2.2 Evaluation of animal study…………………………………...........50 3-2 Osteochondral Tissue Engineering 3-2.1 In vitro study with chondrocytes and biphasic scaffold…………. ….53 3-2.1.1 Isolation and culture expansion of chondrocytes………………..53 3-2.1.2 Fabrication of biphasic scaffold……………………………........53 3-2.1.3 Culture in double chamber bioreactor……………………….......56 3-2.1.4 Evaluation of in vitro study………………………………...........58 3-2.2 In vitro study with mesenchymal stem cells……………………….....59 3-2.2.1 Culture expansion and induction of mesenchymal stem cells…...59 3-2.2.2 Culture in modified double chamber bioreactor………………....62 3-2.2.3 Evaluation of in vitro study……………………………………...64 Chapter 4 Results and Discussion 4-1 Cartilage Tissue Engineering 4-1.1 Results of in vitro study……………………………………………….65 4-1.1.1 Scanning Electron Microscopy and Light Microscopy………......65 4-1.1.2 Culture in Petri dishes and spinner flasks…………………….......65 4-1.2 Discussion of in vitro study……………………………………….......73 4-1.3 Results and discussion of animal study…………………………….....75 4-1.3.1 Age and body weight………………………………………….….75 4-1.3.2 Gross appearance………………………………………………....82 4-1.3.3 Summary of histology examination……………………................90 4-1.3.4 Analysis of Pineda score for histology examination……….........106 4-1.3.5 Limitations in animal study……………………………………...123 4-2 Osteochondral Tissue Engineering 4-2.1 Results of in vitro study with chondrocytes and biphasic scaffold…….126 4-2.1.1 Light, scanning electron, and confocal microscopy…………….....126 4-2.1.2 Culture results in the Double-Chamber bioreactor……..................127 4-2.2 Discussion of in vitro study with chondrocytes and biphasic scaffold…132 4-2.3 Results and Discussion of in vitro study with mesenchymal stem cells..137 4-2.3.1 Chondrogenic induction of hMSC…………………………….........137 4-2.3.2 Osteogenic induction of hMSC………………………...……..........140 4-2.3.3 Osteochondral tissue engineered construct………………...............142 Chapter 5 Conclusion and Future Suggestion..................................................................144 Reference…………………………………………………………………………….............147 Figure index Chapter 1 Fig. 1-1: Example of bone marrow stimulating technique: microfracture procedure……….7 Fig. 1-2: Autogenous osteochondral transplantation (Mosaicplasty)………………………..10 Fig. 1-3: Autogenous chondrocytes implantations…………………………………………..13 Fig. 1-4: The uneven distribution of the chondrocytes in suspension ……............................13 Fig. 1-5: Tissue Engineering Triads: three key constituents………………………………....15 Chapter 2 Fig. 2-1: Constituents of articular cartilage………………………………………….............34 Fig. 2-2: Diagram of an aggrecan molecule…………………………………………............35 Fig. 2-3: Proteoglycan aggregates ………………………………..…………………............36 Fig. 2-4: Structure of articular cartilage……………………………………………………...36 Fig. 2-5: Design of the double-chamber bioreactor………………………………….............42 Chapter 3 Fig. 3-1: Study design of study group, internal control group and external control group…..48 Fig. 3-2: Fabrication of biphasic scaffold.…………………………………………….............55 Fig. 3-3: Gross appearance of biphasic scaffold………………………………………………57 Fig. 3-4: Morphology of hMSC………………………………………………………….........60 Fig. 3-5: Fabrication of engineered osteochondral constructs…………………………...........63 Chapter 4 Fig. 4-1: S.E.M of tricopolymer scaffold…………………...…………………………………68 Fig. 4-2: H.E stain of tricopolymer scaffold……… …………………….………………........68 Fig. 4-3: Chondrocyte distribution in tricopolymer after 3 weeks of culture…………………69 Fig. 4-4: Alcian blue staining of 4 weeks’ cultivation in spinner flask……………………......70 Fig. 4-5: Alcian blue staining of 5 weeks’ cultivation in spinner flask………………………..70 Fig. 4-6: Polarized microscopic examination after 5 weeks’ cultivation in spinner flask…......71 Fig. 4-7: S-100 protein staining of 5 weeks’ cultivation in spinner flask……………….……..71 Fig. 4-8: Anti-type II collagen staining of 5 weeks’ cultivation in spinner flask………………72 Fig. 4-9: Age and weight at implantation in study/internal control group……………………..78 Fig. 4-10: Age and body weight at implantation between study/internal control groups and external control groups………………………………………………....79 Fig. 4-11: Good gross appearance……………………………………………………………...84 Fig. 4-12: Fair gross appearance………...……………………………………………………..85 Fig. 4-13: Poor gross appearance…………………………………………………………..….86 Fig. 4-14: The distribution of the result of gross appearance in study/internal control group..87 Fig. 4-15: Gross appearance of external control groups…………………………...................89 Fig. 4-16: 2 mm deep tissue engineering repaired with hyaline cartilage……………………91 Fig.4-17: 2 mm deep tissue engineering repaired with fibrocartilage………………………..91 Fig. 4-18: 2 mm deep tissue engineering in poor result condyle……………………………..92 Fig. 4-19: 5 mm deep tissue engineering with hyaline cartilage formation………………….94 Fig. 4-20: 5 mm deep tissue engineering with fibrocartilage formation……………………..94 Fig. 4-21: 5 mm deep tissue engineering with central fibrous tissue………………………...95 Fig. 4-22: 5 mm deep tissue engineering in condyle with poor result………………………..95 Fig. 4-23: Condyle treated with auto-transplantation…………………………………………97 Fig. 4-24: Condyle treated with auto-transplantation…………………………………………97 Fig. 4-25: Condyle treated with auto-transplantation…………………………………………97 Fig. 4-26: Osteochondral defect (5 mm deep) for spontaneous repair………………………..99 Fig. 4-27: Osteochondral defect (5 mm deep) for spontaneous repair………………………..99 Fig.4-28: Osteochondral defect (5 mm deep) for spontaneous repair…………………………99 Fig.4-29: Osteochondral defect (5 mm deep) for spontaneous repair………………………..100 Fig.4-30: Osteochondral defect (5 mm deep) for spontaneous repair………………………..100 Fig.4-31: Osteochondral defect (5 mm deep) for spontaneous repair………………………..100 Fig. 4-32: Full thickness defect (2 mm deep) for spontaneous repair………………..….…...102 Fig. 4-33: Full thickness defect (2 mm deep) for spontaneous repair………………...……...102 Fig. 4-34: Osteochondral defects (5 mm deep) filled with scaffold without cell seeding and covered with periosteum………………………………………………………..……………103 Fig. 4-35: Osteochondral defects (5 mm deep) filled with scaffold without cell seeding and covered with periosteum………………………………………………………..……………103 Fig. 4-36: Full thickness defects (2 mm deep) filled with scaffold without cell seeding and covered with periosteum……………………………………………………………………..105 Fig. 4-37: Full thickness defects (2 mm deep) filled with scaffold without cell seeding and covered with periosteum……………………………………………………………………..105 Fig. 4-38: Comparison of mean of “total score” between study and internal control groups..109 Fig. 4-39: Comparison of mean of “filling score” between study and internal control groups………………………………………………………………………………………...110 Fig. 4-40: Comparison of mean of “subchondral score” between study and internal control groups………………………………………………………………………………………...113 Fig. 4-41: Alcian Blue stain in a specimen with 5 mm deep osteochondral defect………….114 Fig. 4-42: Comparison of mean of “matrix score” between study and internal control groups………………………………………………………………………………………...115 Fig. 4-43: Comparison of mean of “cell score” between study and internal control groups…117 Fig. 4-44: Comparison of mean of “integration score” between study and internal control groups…………………………………………………………………………………………119 Fig. 4-45: Comparison between external control group and study group……………………122 Fig. 4-46: A sequel after subcutaneous infection manifested as subcutaneous pouch near knee joint……………………………………………………………………………………..123 Fig. 4-47: SEM, confocal microscopic and histology of biphasic scaffold…………………..129 Fig. 4-48: Histology examination of cartilage growing on biphasic scaffold………………...130 Fig. 4-49: Immunohistochemical examination of cartilage growing on biphasic scaffold…...131 Fig. 4-50: Pellets of chondrogenic induced hMSC…………………………………………...138 Fig. 4-51: Pellets of chondrogenic induced hMSC…………………………………………...138 Fig.4-52: Pellets of chondrogenic induced hMSC with immunohistochemistry stain against S-100 protein………………………………………………………………………………….139 Fig.4-53: Pellets of chondrogenic induced hMSC with immunohistochemistry stain against type I collagen………………………………………………………………………………...139 Fig. 4-54: Von Kossa Stain of osteoinducted hMSCs………………………………………...141 Fig. 4-55: Alkaline-phosphatase stain of osteoinducted hMSCs……………………………..141 Fig. 4-56: Gross appearance of osteochondral constructs……………………………………143 Fig. 4-57: Cartilage cap on the top on calcined bovine bone…………………………………143 Table index Chapter 3 Table. 3-1: Tissue engineering and internal control ………………………………………….49 Table. 3-2: External control groups……………………………………………………………49 Table. 3-3: Modified Pineda score ……………………………………………………………52 Chapter 4 Table. 4-1: Age and weight of study and internal control groups……………………………..76 Table. 4-2: Age and weight of external control groups………………………………………..77 Table. 4-3: The influence of body weight in the study/internal group………………………...81 Table. 4-4: Statistic analysis for gross appearance…………………………….………………87 Table. 4-5: The effect of time on the repair…………………………………………………..107 Table. 4-6: The mean + S.D. of total score and sub-score parameters in study/internal control group………………………………………………………………………………………….108 Table. 4-7: Statistic analysis for total score………………………………………...…….…..109 Table. 4-8: Statistic analysis for filling score…………………………………………….…...111 Table. 4-9: Statistic analysis for subchondral score……………………………………….….113 Table. 4-10: Statistic analysis for matrix score………………………………………….……115 Table. 4-11: Statistic analysis for cell score…………………………………………………..117 Table. 4-12: Statistic analysis for integration score…………………………………………..119 Table. 4-13: Comparison between external control group and study group………………….122 | |
| dc.language.iso | en | |
| dc.subject | 骨軟骨組織工程 | zh_TW |
| dc.subject | 動物實驗 | zh_TW |
| dc.subject | 三重聚合物 | zh_TW |
| dc.subject | 雙腔式生物反應器 | zh_TW |
| dc.subject | 軟骨組織工程 | zh_TW |
| dc.subject | tricopolymer | en |
| dc.subject | animal study | en |
| dc.subject | osteochondral tissue engineering | en |
| dc.subject | double-chamber bioreactor | en |
| dc.subject | cartilage tissue engineering | en |
| dc.title | 以生物活性基質進行軟骨及骨軟骨之組織工程 | zh_TW |
| dc.title | Using Bioactive Matrix Scaffold for Cartilage and Osteochondral Tissue Engineering | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.advisor-orcid | ,林峰輝(double@ha.mc.ntu.edu.tw) | |
| dc.contributor.oralexamcommittee | 楊俊佑(Chyun-Yu Yang),翁文能(Steve Wen-Neng Ueng),楊榮森(Rong-Sen Yang),王盈錦(Yng-Jiin Wang),宋信文(Hsing-Wen Sung) | |
| dc.subject.keyword | 動物實驗,三重聚合物,雙腔式生物反應器,軟骨組織工程,骨軟骨組織工程, | zh_TW |
| dc.subject.keyword | osteochondral tissue engineering,animal study,cartilage tissue engineering,tricopolymer,double-chamber bioreactor, | en |
| dc.relation.page | 164 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2005-01-25 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
| Appears in Collections: | 醫學工程學研究所 | |
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