請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45933
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊台鴻 | |
dc.contributor.author | Yu-Tsang Lee | en |
dc.contributor.author | 李裕滄 | zh_TW |
dc.date.accessioned | 2021-06-15T04:49:12Z | - |
dc.date.available | 2011-08-12 | |
dc.date.copyright | 2010-08-12 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-03 | |
dc.identifier.citation | 1. Lakes R. Materials with structural hierarchy. Nature 1993 Feb;361(6412):511-515.
2. Pekounov Y, Petrov OE. Bone resembling apatite by amorphous-to-crystalline transition driven self-organisation. Journal of Materials Science: Materials in Medicine 2008;19(2):753-759. 3. Deligianni DD, Katsala ND, Koutsoukos PG, Missirlis YF. Effect of surface roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation and detachment strength. Biomaterials: Elsevier Science Ltd, 2001. p. 87-96. 4. Felson DT. Developments in the clinical understanding of osteoarthritis. arthritis research and therapy 2009;11(1):11. 5. Moreland LW. Intra-articular hyaluronan (hyaluronic acid) and hylans for the treatment of osteoarthritis: mechanisms of action. Arthritis Research & Therapy 2003;5(2):54-67. 6. Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000 Dec;21(24):2529-2543. 7. Ge ZG, Yang F, Goh JCH, Ramakrishna S, Lee EH. Biomaterials and scaffolds for ligament tissue engineering. J Biomed Mater Res Part A 2006 Jun;77A(3):639-652. 8. Popat KC, Daniels RH, Dubrow RS, Hardev V, Desai TA. Nanostructured surfaces for bone biotemplating applications. Journal of Orthopaedic Research 2006 Apr;24(4):619-627. 9. dos Santos EA, Farina M, Soares GA, Anselme K. Chemical and topographical influence of hydroxyapatite and beta-tricalcium phosphate surfaces on human osteoblastic cell behavior. J Biomed Mater Res Part A 2009 May;89A(2):510-520. 10. Christenson EM, Anseth KS, van den Beucken L, Chan CK, Ercan B, Jansen JA, et al. Nanobiomaterial applications in orthopedics. Journal of Orthopaedic Research 2007 Jan;25(1):11-22. 11. Bland JH, Cooper SM. Osteoarthritis - A review of the cell biology involved and evidence for resersibility - management rationally related to known genesis and patho-physiology. Seminars in Arthritis and Rheumatism 1984;14(2):106-133. 12. Belcher C, Yaqub R, Fawthrop F, Bayliss M, Doherty M. Synovial fluid chondroitin and keratan sulphate epitopes, glycosaminoglycans, and hyaluronan in arthritic and normal knees. Annals of the Rheumatic Diseases 1997 May;56(5):299-307. 13. Wobig M, Dickhut A, Maier R, Vetter G. Viscosupplementation with hylan G-F 20: A 26-week controlled trial of efficacy and safety in the osteoarthritic knee. Clinical Therapeutics 1998 May-Jun;20(3):410-423. 14. Goldberg VM, Buckwalter JA. Hyaluronans in the treatment of osteoarthritis of the knee: evidence for disease-modifying activity. Osteoarthritis and Cartilage 2005 Mar;13(3):216-224. 15. Stappenbeck TS, Miyoshi H. The Role of Stromal Stem Cells in Tissue Regeneration and Wound Repair. Science 2009 Jun;324(5935):1666-1669. 16. Mobasheri A, Csaki C, Clutterbuck AL, Rahmanzadeh M, Shakibaei M. Mesenchymal stem cells in connective tissue engineering and regenerative medicine: Applications in cartilage repair and osteoarthritis therapy. Histol Histopath 2009 Mar;24(3):347-366. 17. Toole BP, Zoltan-Jones A, Misra S, Ghatak S. Hyaluronan: A Critical Component of Epithelial-Mesenchymal and Epithelial-Carcinoma Transitions. Cells Tissues Organs: Karger AG, 2005. p. 66-72. 18. Di Martino A, Sittinger M, Risbud MV. Chitosan: A versatile biopolymer for orthopaedic tissue-engineering. Biomaterials 2005;26(30):5983-5990. 19. Chesnutt BM, Yuan Y, Brahmandam N, Yang Y, Ong JL, Haggard WO, et al. Characterization of biomimetic calcium phosphate on phosphorylated chitosan films. Journal of Biomedical Materials Research - Part A 2007;82(2):343-353. 20. Sendemir-Urkmez A, Jamison RD. The addition of biphasic calcium phosphate to porous chitosan scaffolds enhances bone tissue development in vitro. Journal of Biomedical Materials Research - Part A 2007;81(3):624-633. 21. LeGeros RZ. Properties of osteoconductive biomaterials: Calcium phosphates. Clinical Orthopaedics and Related Research 2002 Feb(395):81-98. 22. Kumta PN, Sfeir C, Lee DH, Olton D, Choi D. Nanostructured calcium phosphates for biomedical applications: Novel synthesis and characterization. Acta Biomaterialia 2005;1(1):65-83. 23. Ehara A, Ogata K, Imazato S, Ebisu S, Nakano T, Umakoshi Y. Effects of α-TCP and TetCP on MC3T3-E1 proliferation, differentiation and mineralization. Biomaterials 2003;24(5):831-836. 24. Gravel M, Gross T, Vago R, Tabrizian M. Responses of mesenchymal stem cell to chitosan-coralline composites microstructured using coralline as gas forming agent. Biomaterials 2006;27(9):1899-1906. 25. Manjubala I, Ponomarev I, Wilke I, Jandt KD. Growth of osteoblast-like cells on biomimetic apatite-coated chitosan scaffolds. Journal of Biomedical Materials Research - Part B Applied Biomaterials 2008;84(1):7-16. 26. Abraham LC, Zuena E, Perez-Ramirez B, Kaplan DL. Guide to collagen characterization for biomaterial studies. Journal of Biomedical Materials Research Part B-Applied Biomaterials 2008 Oct;87B(1):264-285. 27. Shoulders MD, Raines RT. Collagen Structure and Stability. Annual Review of Biochemistry 2009;78:929-958. 28. Tzaphlidou M. Bone Architecture: Collagen Structure and Calcium/Phosphorus Maps. Journal of Biological Physics 2008 Apr;34(1-2):39-49. 29. Abreu JG, Ketpura NI, Reversade B, De Robertis EM. Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nature Cell Biology 2002 Aug;4(8):599-604. 30. Charles-Harris M, Koch MA, Navarro M, Lacroix D, Engel E, Planell JA. A PLA/calcium phosphate degradable composite material for bone tissue engineering: An in vitro study. Journal of Materials Science: Materials in Medicine 2008;19(4):1503-1513. 31. Davidson ENB, Vitters EL, Mooren FM, Oliver N, van den Berg WB, van der Kraan PM. Connective tissue growth factor/CCN2 overexpression in mouse synovial lining results in transient fibrosis and cartilage damage. Arthritis and Rheumatism 2006 May;54(5):1653-1661. 32. Haubeck HD, Kock R, Fischer DC, Vandeleur E, Hoffmeister K, Greiling H. Transforming growth factor beta-1, a major stimulator of hyaluronan synthesis in human synovial lining cells. Arthritis and Rheumatism 1995 May;38(5):669-677. 33. Bakker AC, van de Loo FAJ, van Beuningen HM, Sime P, van Lent P, van der Kraan PM, et al. Overexpression of active TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation. Osteoarthritis and Cartilage 2001 Feb;9(2):128-136. 34. Murata M, Yudoh K, Masuko K. The potential role of vascular endothelial growth factor (VEGF) in cartilage - How the angiogenic factor could be involved in the pathogenesis of osteoarthritis? Osteoarthritis and Cartilage 2008 Mar;16(3):279-286. 35. Enomoto H, Inoki I, Komiya K, Shiomi T, Ikeda E, Obata K, et al. Vascular endothelial growth factor isoforms and their receptors are expressed in human osteoarthritic cartilage. American Journal of Pathology 2003 Jan;162(1):171-181. 36. Lee IC, Lee YT, Yu BY, Lai JY, Young TH. The behavior of mesenchymal Stem cells on micropatterned PLLA membranes. J Biomed Mater Res Part A 2009 Dec;91A(3):929-938. 37. Lee IC, Wang JH, Lee YT, Young TH. The differentiation of mesenchymal stem cells by mechanical stress or/and co-culture system. Biochem Biophys Res Commun 2007 Jan;352(1):147-152. 38. Yu BY, Chou PH, Sun YM, Lee YT, Young TH. Topological micropatterned membranes and its effect on the morphology and growth of human mesenchymal stem cells (hMSCs). Journal of Membrane Science 2006;273(1-2):31-37. 39. Yu BY, Chen PY, Sun YM, Lee YT, Young TH. The behaviors of human mesenchymal stem cells on the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) membranes. Desalination 2008 Dec;234(1-3):204-211. 40. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999 Apr;284(5411):143-147. 41. Lee IC, Wang JH, Lee YT, Young TH. Development of a useful technique to discriminate anterior cruciate ligament cells and mesenchymal stem cells - The application of cell electrophoresis. J Biomed Mater Res Part A 2007 Jul;82A(1):230-237. 42. Caplan AI. Mesenchymal stem cells. Journal of Orthopaedic Research 1991;9(5):641-650. 43. Deans RJ, Moseley AB. Mesenchymal stem cells: Biology and potential clinical uses. Experimental Hematology 2000;28(8):875-884. 44. Chen YJ, Huang CH, Lee IC, Lee YT, Chen MH, Young TH. Effects of cyclic mechanical stretching on the mRNA expression of tendon/ligament-related and osteoblast-specific genes in human mesenchymal stem cells. Connect Tissue Res 2008;49(1):7-14. 45. Damsky CH, Werb Z. Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information. Current Opinion in Cell Biology 1992;4(5):772-781. 46. Petite H, Viateau V, Bensaid W, Meunier A, de Pollak C, Bourguignon M, et al. Tissue-engineered bone regeneration. Nature Biotechnology 2000 Sep;18(9):959-963. 47. Caplan AI. Mesenchymal stem cells: Cell-based reconstructive therapy in orthopedics. Tissue Engineering 2005 Jul;11(7-8):1198-1211. 48. Coelho MJ, Fernandes MH. Human bone cell cultures in biocompatibility testing. Part II: Effect of ascorbic acid, β-glycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials 2000;21(11):1095-1102. 49. Ogata K, Imazato S, Ehara A, Ebisu S, Kinomoto Y, Nakano T, et al. Comparison of osteoblast responses to hydroxyapatite and hydroxyapatite/soluble calcium phosphate composites. J Biomed Mater Res Part A 2005 Feb;72A(2):127-135. 50. Toworfe GK, Bhattacharyya S, Composto RJ, Adams CS, Shapiro IM, Ducheyne P. Effect of functional end groups of silane self-assembled monolayer surfaces on apatite formation, fibronectin adsorption and osteoblast cell function. J Tissue Eng Regen Med 2009 Jan;3(1):26-36. 51. Nelson M, Balasundaram G, Webster TJ. Increased osteoblast adhesion on nanoparticulate crystalline hydroxyapatite functionalized with KRSR. International journal of nanomedicine 2006;1(3):339-349. 52. Thein-Han WW, Misra RDK. Biomimetic chitosan-nanohydroxyapatite composite scaffolds for bone tissue engineering. Acta Biomaterialia 2009 May;5(4):1182-1197. 53. Kawasaki K, Ochi M, Uchio Y, Adachi N, Matsusaki M. Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels. Journal of Cellular Physiology 1999 May;179(2):142-148. 54. Ghosh P, Holbert C, Read R, Armstrong S. Hyaluronic acid (hyaluronan) in experimental osteoarthritis. 3rd International Symposium on Osteoarthritis - Challenges for the 21st-Century; 1994 Oct 04-07; Val David, Canada; 1994. p. 155-157. 55. Li J, Chen Y, Yin Y, Yao F, Yao K. Modulation of nano-hydroxyapatite size via formation on chitosan-gelatin network film in situ. Biomaterials 2007;28(5):781-790. 56. Maniwa S, Ochi M, Motomura T, Nishikori T, Chen J, Naora H. Effects of hyaluronic acid and basic fibroblast growth factor on motility of chondrocytes and synovial cells in culture. Acta Orthopaedica Scandinavica 2001 Jun;72(3):299-303. 57. Barland P, Novikoff AB, Hamerman D. Electron microscopy of human synovial membrane Journal of Cell Biology 1962;14(2):207-220. 58. Varedi M, Tredget EE, Scott PG, Shen YJ, Ghahary A. Alteration in cell morphology triggers transforming growth factor beta-1, collagenase and tissue inhibitor of metalloproteinases-1 expression in normal and hypertropic scar fibrobalasts. Journal of Investigative Dermatology 1995 Jan;104(1):118-123. 59. Varedi M, Ghahary A, Scott PG, Tredget EE. Cytoskeleton regulates expression of genes for transforming growth factor-beta 1 and extracellular matrix proteins in dermal fibroblasts. Journal of Cellular Physiology 1997 Aug;172(2):192-199. 60. Lesley J, Hascall VC, Tammi M, Hyman R. Hyaluronan binding by cell surface CD44. Journal of Biological Chemistry 2000 Sep;275(35):26967-26975. 61. Ohkawara Y, Tamura G, Iwasaki T, Tanaka A, Kikuchi T, Shirato K. Activation and transforming growth factor-beta production in eosinophils by hyaluronan. American Journal of Respiratory Cell and Molecular Biology 2000 Oct;23(4):444-451. 62. Leask A, Abraham DJ. The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology. Biochemistry and Cell Biology-Biochimie Et Biologie Cellulaire 2003 Dec;81(6):355-363. 63. Aulthouse AL, Beck M, Griffey E, Sanford J, Arden K, Machado MA, et al. Expression of the human chodrocyte phenotype in vitro. In Vitro Cellular & Developmental Biology 1989 Jul;25(7):659-668. 64. Balasundaram G, Sato M, Webster TJ. Using hydroxyapatite nanoparticles and decreased crystallinity to promote osteoblast adhesion similar to functionalizing with RGD. Biomaterials 2006;27(14):2798-2805. 65. Morales TI, Roberts AB. Transforming growth factor-beta regulates the metabolism of proteoglycans in bovine cartilage organ-cultures. Journal of Biological Chemistry 1988 Sep;263(26):12828-12831. 66. Frenkel SR, Saadeh PB, Mehrara BJ, Chin GS, Steinbrech DS, Brent B, et al. Transforming growth factor beta superfamily members: Role in cartilage modeling. Plastic and Reconstructive Surgery 2000 Mar;105(3):980-990. 67. Omoto S, Nishida K, Yamaai Y, Shibahara M, Nishida T, Doi T, et al. Expression and localization of connective tissue growth factor (CTGF/Hcs24/CCN2) in osteoarthritic cartilage. Osteoarthritis and Cartilage 2004 Oct;12(10):771-778. 68. Pfander D, Kortje D, Zimmermann R, Weseloh G, Kirsch T, Gesslein M, et al. Vascular endothelial growth factor in articular cartilage of healthy and osteoarthritic human knee joints. Annals of the Rheumatic Diseases 2001 Nov;60(11):1070-1073. 69. Exposito JY, Valcourt U, Cluzel C, Lethias C. The Fibrillar Collagen Family. International Journal of Molecular Sciences 2010 Feb;11(2):407-426. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45933 | - |
dc.description.abstract | 本研究的目的是探討人類間葉細胞在生醫材料上的行為調控和基因型表現,研究內容分為兩大部分,第一部分是有關骨組織生成的相關研究,第二部分是有關退化性關節炎的研究。所取用的間葉細胞來自於人體骨髓以及結締組織,包括間葉幹細胞、滑囊細胞、軟骨細胞。
第一部分研究重點在骨生成的組織工程。骨骼成份中無機物主要是磷酸鈣,和第二型膠原蛋白共同組成骨組織的細胞外間質。於實驗室用化合沈澱法方式來製成磷酸鈣的微晶體,並混摻天然有機化合物幾丁聚醣來製成複合型膜狀材料,材料表面因磷酸鈣的結晶性不同而形成奈米至微米大小不同的顆粒。取人體骨盆腸骨骨髓在體外培養間葉幹細胞,並將人類間葉幹細胞培養在不同的幾丁聚醣/磷酸鈣複合材料上,檢測細胞的行為調控,包括細胞生長增殖、細胞貼附分佈,以及在骨誘導培養基下,對鹼性磷酸酵素分泌的情形。研究的結果顯示,在幾丁聚醣/磷酸鈣複合膜狀材料的表面,磷酸鈣的結晶性和表面構形,會影響間葉幹細胞的行為,其中奈米結晶磷酸鈣有明顯提高細胞的增殖,微米結晶磷酸鈣有助細胞的骨系分化。研究的結果,說明材料表面磷酸鈣的結晶體,會增加人類間葉幹細胞的生長增殖和骨系分化,所製作材料可應用於骨骼組織工程支架的改良和表面處理。 第二部分研究重點在探討玻尿酸對膝關節退化症治療的機制。取人體退化性膝關節內的滑囊組織和軟骨組織,在實驗室培養滑囊細胞和軟骨細胞,先用無血清的培養基培養細胞二十四小時,以排除血清內生長因子的作用,選用不同分子量和濃度的玻尿酸,用來刺激細胞四小時及二十四小時,觀察細胞的形態和生存能力的情形,並檢測細胞對退化性關節炎相關生長因子基因型表現的反應,包括結締組織生長因子、轉型生長因子乙型之一、血管內皮生長因子,同時檢測細胞對第一型膠原蛋白和第二型膠原蛋白的基因型表現反應。研究的結果顯示,不同分子量玻尿酸在不同濃度下,對人類滑囊細胞和軟骨細胞作刺激二十四小時,不改變細胞形態,也不會對細胞產生毒性作用,對生長因子的基因型表現有不同的影響,以較高濃度的玻尿酸刺激細胞,會增加生長因子的基因型表現,在同一濃度之下,較高分子量玻尿酸對滑囊細胞作用時,結締組織生長因子和血管內皮生長因子的基因型表現會有下降。研究的結果,對以玻尿酸來治療退化性膝關節炎的機制有進一步的了解,也有助玻尿酸在生醫材料的應用。 | zh_TW |
dc.description.abstract | Osteogenesis and Osteoarthritis are two big issues in orthopaedic diseases. The main purpose for this study is to understand how to modulate cellular behaviors and phenotypic responses of human mesenchymal cells on different biomaterials. The cells are isolated primarily from human bone marrow and connective tissues, including synovium and cartilage of knee joints.
Human mesenchymal stem cells (hMSCs) have great potential to differentiate to lineages of mesenchymal tissues. Calcium phosphate (CaP) apatite, the main inorganic constituent of mammalian bone tissues, is believed to support hMSCs growth and osteogenic differentiation. Chitosan, a deacetylated derivative of chitin, is a versatile biopolymer to offer broad possibilities for cell-based tissue engineering. In the first part, we have applied a simple and quick method to prepare micro- and nano-scale of calcium phosphate (CaP) crystals mixed in chitosan membranes. The different concentrations of aqueous CaP suspension were mixed with chitosan in acetic acid solution and chitosan/calcium phosphate (C/CaP) films were fabricated by the solvent-casting method. The hMSCs behaviors including cell spreading, proliferation and osteogenic differentiation were examined. In basal culture medium, the addition of CaP in chitosan films could promote the proliferation of hMSCs. The films with nano-crystalline CaP significantly improved cell proliferation. In osteogenic medium, the increased alkaline phosphatase (ALP) level showed the process of osteogenic differentiation of hMSCs on the C/CaP films. The hMSCs on discrete micro-crystalline CaP films revealed higher ALP level. These results demonstrate that the crystallinity and topography of CaP apatite on chitosan membrane scaffolds modulates the behaviors of hMSCs. Intra-articular injection of hyaluronan (hyaluronic acid; HA) is a common way to treat knee osteoarthritis (OA). This treatment can not only maintain the viscoelastic properties of knee but also release the OA pain. However, the exact molecular mechanism is still unknown. In the second part, human synovial cells were stimulated with HA (Sigma) and Hylan (Synvisc) for 24 hours. The human synovial cells were isolated from synovium tissue of advanced-staged osteoarthritic knee. Real-time polymerase chain reaction (real-time PCR) was used to detect the alteration of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) gene expression, which were specific genes related to pathogenesis of OA knees. The gene expressions of matrix-related proteins, collagen I and collagen II, were also studied. Our results illustrated that both HA and Hylan might not cause cytotoxicity or apoptosis in serum deprivation environment. For synovial cells, the cell shapes were not changed after HA and Hylan stimulation for 24 hours. The gene expressions of TGF-β1 and VEGF were significantly increased at the concentration of 0.1mg/ml HA and 0.1mg/ml Hylan, respectively. The synovial cells with treatment of 0.1mg/ml Hylan decreased the CTGF gene expression (0.66-fold) and VEGF (0.78-fold) compared to 0.1mg/ml HA. The type I collagen expressed significantly higher as treated with 0.1mg/ml HA and 0.1mg/ml Hylan. We further isolated human cartilage cells from healthy cartilage of knee. The cartilage cells were treated with three kinds of HA, including Sigma HA, Synvisc Hylan and Artz HA, with 0.1 mg/ml and 0.01mg/ml under serum deprivation condition for 24 hours. With treatment of Synvisc Hylan, the gene expressions of CTGF, TGF-β1,VEGF, collagne I and collage II increased in 0.1mg/ml compared with 0.01mg/ml. However, the gene expression of CTGF, TGF-β1, VEGF, collagne I and collage II decreased in 0.1mg/ml relative to 0.01mg/ml with the treatment of Artz HA. Under the condition of 0.1mg/ml, Artz HA decreased the gene expressions of CTGF(0.8-fold), TGF-β1(0.8-fold) and VEGF(0.5-fold) as compared to Synvisc Hylan. Synvsic Hylan increased the gene expressions of collagen I (1.7-fold) and collagen II (4.9-fold) as compared to Artz HA. As a result, the profile of osteoarthritis-related factors of CTGF, TGF-β1 and VEGF and matrix proteins of collagen I and II might provide the rational mechanism for the therapeutic effects of hyaluronic acid on OA knees. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T04:49:12Z (GMT). No. of bitstreams: 1 ntu-99-F91548044-1.pdf: 2506298 bytes, checksum: d7989a76ab3f1ab58ee11e296cddafa4 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 中文摘要 i
Abstract iii Content vi Tables ix Figures x Chapter 1 Introduction 1 1.1 Motivation 1 1.2 Tissue engineering principle 1 1.3 Orthopedic disorder 2 1.3.1 Osteogenesis 2 1.3.2 Osteoarthritis 2 1.4 Mesenchyme and mesenchymal cells 3 1.5 Biomaterials 4 1.4.1 Chitosan 4 1.4.2 Calcium phosphate 4 1.4.3 Hyaluronic acid (HA) 5 1.4.4 Collagen 5 1.6 Growth factors 6 1.5.1 Connective tissue growth factor (CTGF) 6 1.5.2 Transforming growth factor-β1 (TGF-β1) 6 1.5.3 Vascular endothelial growth factor (VEGF) 7 1.7 Hypothesis 7 1.8 Experiment design 8 Chapter 2 Materials and Methods 9 2.1 Materials 9 2.2 Experimental apparatus 11 2.3 Solution preparation 11 2.3.1 Phosphate buffered saline (PBS) 11 2.3.2 MTT reagent 12 2.3.3 Trypsin 12 2.3.4 Paraformaldehyde solution 12 2.4 Preparation of chitosan/calcium phosphate membrane scaffolds 12 2.5 Isolation of human mesenchymal cells 13 2.5.1 Human mesenchymal stem cells (hMSCs) 13 2.5.2 Human synovial cells and cartilage cells 14 2.6 Seeding and culture of hMSC on the chitosan/CaP membranes 14 2.7 Cell culture toward osteogenic differentiation 15 2.8 Flow cytometry 15 2.9 Scanning electron microscopy (SEM) 16 2.10 Electron dispersion spectroscopy (EDS) 16 2.11 Thin film X-ray diffraction (TF-XRD) 16 2.12 Cell viability assay (MTT) 17 2.13 Cellular metabolic activity assay (Alamar Blue) 17 2.14 Alkaline phosphatase (ALP) assay 18 2.15 Immunofluorescence stain 18 2.16 Preparation of hyaluronic acid 18 2.17 Real-time quantitative polymerase chain reaction (PCR) 19 2.18 Statistical analysis 19 Chapter 3 Results 21 3.1 PART I : 21 Effect of adding micro- and nano-crystalline calcium phosphate apatite to chitosan membrane scaffolds on the proliferation and osteogenic differentiation of human mesenchymal stem cells in vitro 21 3.1.1 Scanning electron microscopy (SEM) 21 3.1.2 Electron dispersion spectroscopy (EDS) 21 3.1.3 Thin film X-ray diffraction (TF-XRD) 22 3.1.2 Cell morphology 22 3.1.3 Flow cytometry analysis 22 3.1.4 Cell proliferation 23 3.1.5 ALP assay 23 3.1.6 Immunofluorescence staining 24 3.2 PART II – 1 : 25 Hyaluronic acid modulates gene expression of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) in human fibroblast-like synovial cells from advanced stage osteoarthritis in vitro 25 3.2.1 Effect of HA and hylan on human synovial cells morphology 25 3.2.2 Immunofluorescence staining of F-actin of human synovial cells with HA and Hylan stimulation 25 3.2.3 Effect of HA and Hylan on human synovial cells viability 26 3.2.4 Gene expression of human synovial cells with HA and Hylan stimulation 26 3.3 PART II–2 : 28 Hyaluronic acid stimulates the gene expressions of OA-related growth factors in human cartilage cells in vitro 28 3.3.1 Cellular morphology 28 3.3.2 Gene expression of human cartilage cells with hyaluronic acids stimulation 28 Chapter 4 Discussions 30 4.1 PART I: 30 Effect of adding micro- and nano-crystalline calcium phosphate apatite to chitosan membrane scaffolds on the proliferation and osteogenic differentiation of human mesenchymal stem cells in vitro 30 4.2 PART II-1: 34 Hyaluronic acid modulates gene expression of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) in human fibroblast-like synovial cells from advanced stage osteoarthritis in vitro 34 4.3 PART II-2 : 37 Hyaluronic acid stimulates the gene expressions of OA-related growth factors in human cartilage cells in vitro 37 Chapter 5 Conclusion and Perspectives 40 Reference 41 List of Tables 48 List of Figures 50 Curriculum Vitae 75 List of Publication 76 List of Conference 78 | |
dc.language.iso | en | |
dc.title | 人類間葉細胞在生醫材料上的行為調控和基因型表現 | zh_TW |
dc.title | Modulation of Cellular Behaviors and Phenotypic Responses of Human Mesenchymal Cells on Biomaterials | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 劉華昌,侯勝茂 | |
dc.contributor.oralexamcommittee | 鄭廖平,孫一明,林宏殷 | |
dc.subject.keyword | 間葉幹細胞,滑囊細胞,軟骨細胞,奈米磷酸鈣,玻尿酸,生長因子,生醫材料,基因表現, | zh_TW |
dc.subject.keyword | mesenchymal stem cells,synovial cells,cartilage cells,nano-scale calcium phosphate,hyaluronic acid,growth factor,biomaterials,phenotype response, | en |
dc.relation.page | 90 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-03 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
顯示於系所單位: | 醫學工程學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-99-1.pdf 目前未授權公開取用 | 2.45 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。