Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3706
標題: 幾丁聚醣/動物明膠複合物作為軟骨再生支架之研究
Assessment of Chitosan/ Gelatin Complexes as Tissue Engineering Scaffolds for Cartilage Regeneration
作者: 胡淑文
Whu, Shu-Wen
關鍵字: Chitosan;幾丁聚醣;gelatin;cartilage;tissue engineering;dynamic mechanical analysis;TGF-β3;human bone marrow mesenchymal stem cells;dynamic culture;動物明膠;軟骨;組織工程;動態機械分析;TGF-β3;人類骨髓間質幹細胞;動態培養
出版社: 化學工程學系所
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Engineering National Chung Hsing University, Vol.12, No.2, pp.103-108, 2001 4. Freed LE, and Vunjak-Novakovic G, Tissue Engineering of Cartilage, In The Biomedical Engineering Handbook 2nd ed Vol. II, Bronzino JD (ed.), CRC Press, Boca Raton , FL, 2000, Chap.124 chapter 4 1. S. Treppo, H. Koepp, E.C. Quan, A.A. Cole, K.E. Kuettner and A. J. Grodzinsky, Comparison of biomechanical and biochemical properties of cartilage from human knee and ankle pairs, J. Orthop. Res. 18 (2000), 739-48. 2. J.S. Jurvelin, M.D. Buschmann and E.B. Hunziker, Mechanical anisotropy of the human knee articular cartilage in compression, Proc. Inst. Mech. Eng. [H] 217 (2003), 215-9. 3. D.D. Anderson, T.D. Brown, K.H. Yang and E.L. Radin, A dynamic finite element analysis of impulsive loading of the extension-splinted rabbit knee, J. Biomech. Eng. 112 (1990), 119-28. 4. L.J. Bonassar, K.A. Jeffries, E.H. Frank, V.L. Moore, M.W. Lark, E.K. Bayne, J. McDonnell, J. Olszewski, W. Hagmann and K. 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Evaluation of biodegradable polyesters modified by type II collagen and Arg-Gly-Asp as tissue engineering scaffolding materials for cartilage regeneration, Artificial Organs, 30 (2006), 42-55. 13. Y.J. Kim, R.Y. Sah, J.Y. Doong and A.J. Grodzinsky. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal. Biochem. 174 (1988), 168-176. 14. B.O. Enobakhare, D.L. Bader and D.A. Lee. Quantification of sulfated glycosaminoglycans in chondrocyte/alginate culture, by use of 1,9-dimethylmethylene blue, Anal. Biochem. 243 (1996), 189-191. 15. M. Bergman and R. Loxley. Two improved and simplified methods for the spectrophotometric determination of hydroxyproline, Anal. Biochem. 35 (1961), 1961-1965. 16. K. Tomihata, K. Burczak, K. Shiraki and Y. Ikada. Cross-linking and biodegradation of native and denatured collagen, ACS Symposium Series No.540. Appendix A 1. Francis Suh, J. K. and Matthew, H. W. T., Biomaterials, 21, pp.2589-2598, 2000. 2. 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摘要: 
軟骨組織工程中,基於結合軟骨細胞和生醫材料的細胞支架一直不斷在開發。其中最重要的成功關鍵就是找到最佳的材料作成支架。本篇將利用幾丁聚醣-動物明膠聚電性複合材料作為軟骨組織工程支架進行體外和體內相關測試。自戊二醛、雙環氧化物和水溶性氰胺物等三種交聯劑中找到水溶性氰胺物是動物明膠的最佳交聯劑。當幾丁聚醣和動物明膠比例為1:1時複合支架有適當的降解速率與機械穩定性。以水溶性氰胺物交聯後的幾丁聚醣-動物明膠聚電性複合材料有很好的軟骨細胞增殖和細胞間質分泌表現。進行兔子體內實驗後,幾丁聚醣-動物明膠聚電性複合材料仍然確定為適合做為軟骨組織再生支架。
為了獲得更佳的體外培養條件,利用旋轉型生物反應器提供良好的質傳與生理上的翦應力,使得再生軟骨有好的機械強度和壓縮模數。但是動態培養並不是產生類真實組織結構之新生軟骨的必要條件。另外,發現動物明膠能促進人類軟骨細胞增殖,並且幾丁聚醣能維持人類軟骨細胞型態。
為了增加軟骨細胞來源,將人類骨髓間葉幹細胞(hBMSC)植入包括第二型膠原蛋白改質的PLGA/PLLA支架(BCII)與幾丁聚醣/動物明膠混合支架(CG) 的兩種三維支架。結果發現CG支架內的細胞數量較BCII支架為高。支架種類並不影響TGF-β3促進骨髓間葉幹細胞之分化;動態培養並不幫助骨髓間葉幹細胞之分化。
最後,目前沒有任何方法能夠對組織工程軟骨進行非破壞性的品質控管。本研究研發利用動態機械分析儀和流變分析來確認不同部位的軟骨組織 (如關節、肋骨和耳朵)。不同部位的軟骨有不同的storage modulus。當軟骨組織能負載應力高則有較高的storage modulus表現。而關節和肋骨等屬於透明軟骨等組織有相近的loss tan。另ㄧ方面,來自耳朵的彈性軟骨表現出有別於關節和肋骨的loss tan。儘管耳朵軟骨組織有高於關節軟骨的基質含量和細胞數量,機械性質卻遠低於關節軟骨。而組織工程細胞支架經由1和28天的培養後,細胞數量、細胞間質含量和storage modulus會隨時間增加不過仍遠低於真實軟骨組織但是細胞支架的loss tan卻接近真實組織。所以loss tan可以作為組織工程軟骨的品管參數。

Constructs based on chondrocytes and biomaterial scaffolds were developed for cartilage tissue engineering. One of the keys for success is to select suitable materials for fabrication of the scaffolds. In this study, chitosan-gelatin polyelectrolyte complexes were evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for gelatin was selected among glutaradehyde, bisepoxy and water-soluble carbodiimide (WSC), based upon the growth of chondrocytes on the crosslinked gelatin. WSC was found to be the most ideal crosslinker for the system. The complex scaffolds with chitosan/gelatin ratio equal to one possessed the proper degradation profile and mechanical stability. Chondrocytes proliferated well and secreted extracellular matrix in chitosan-gelatin complex scaffolds crosslinked by WSC. Rabbit implantation confirmed the suitability of using chitosan-gelatin complex scaffolds for cartilage tissue engineering.
For better culture condition, it was hypothesized that good mass transfer and the physiological shear provided by the rotating-type bioreactor were important for the neocartilage formed in the scaffolds to exhibit satisfactory mechanical strength and compression modulus; However, the dynamic culture condition was not prerequisite for the constructs to develop a histological resemblance to the real tissues. Then gelatin was observed to promote the human chondocytes proliferation; while chitosan was observed to maintain the human chondocytes morphology.
For more cell source, human bone marrow mesenchymal stem cells (hBMSC) were seeded into two scaffolds, including blended polymers of PLGA50/50 and PLLA modified by type II collagen (BCII) and chitosan-gelatin complexes (CG). Cell numbers in CG scaffolds were higher than those in BCII scaffolds. The materials of the scaffolds had no effect on TGF-β3 induced hBMSC transformation into differentiated cells. The dynamic culture system promoted cell proliferation, but not cell differentiation.
Finally, there is no current method for non-destructive quality control of tissue-engineered cartilage. This study explored a way to utilize a dynamic mechanical analyzer and rheological analysis to assess the cartilage tissues from different anatomic locations (e.g. arthrosis, costa and ear). Cartilage from different locations showed different storage modulus. Higher storage modulus was observed in positions that offered a greater loading force. Hyaline cartilage, either from arthrosis or costa, had similar values in loss tan. On the other hand, elastic cartilage (from ear) showed a distinct value of loss tan from that of arthrosis or costa. In spite of the much higher matrix content and cell number for ear cartilage vs. arthrosis cartilage, the mechanical properties of ear cartilage were much lower than those of arthrosis cartilage. Tissue-engineered constructs were cultured for 1 and 28 days, where the cell number, matrix content and storage modulus all increased with the culture time, but were still much lower than the values in the real cartilage. The values of loss tan of all constructs, however, approached those of real cartilage. It thus appeared that values of loss tan may serve as one of the major performance indice for tissue-engineered cartilage.
URI: http://hdl.handle.net/11455/3706
其他識別: U0005-2508200815195500
Appears in Collections:化學工程學系所

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