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Assessment of Chitosan/ Gelatin Complexes as Tissue Engineering Scaffolds for Cartilage Regeneration
|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||引用:||Chapter 1 1. Hunter, W. Of the structure and diseases of articulating cartilages, Philosophical Transactions 470:514-21, 1743. 2. Buckwalter, J.A., and Mankin, H.J. Articular cartilage repair and transplantation. Arthrit Rheum 41 (8):1331-42, 1998. 3. Buckwalter, J.A., and Mankin, H.J. Articular cartilage. Part I: tissue design ad chondrocyte-matrix interactions, J Bone Joint Surg 79a:600, 1997. 4. Freeman, M.A.R. Adult articular cartilage, Oxford: Alden Press, 1973. 5. Mitchell, N., and Shepard, N. The resurfacing of adult articular cartilage by multiple perforation through the subchondral bone, J Bone Jt Surg 58a(2): 230-3, 1976. 6. Francis Suh, J.K., and Matthew, H.W.T. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. 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The effect of two different bioreactors on the neocartilage formation in type II collagen modified polyester scaffolds seeded with chondrocytes, Artificial Organs, 29 (2005), 467-74. 12. S. Hsu, S.H. Chang, H. Yen, S.W. Whu, C. Tsai and D.C. Chen. 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. Hye-Won, Yasuhiko Tabata and Yoshito Ikada, Biomaterials, 20, pp. 1339-1344, 1999. 3. Ross Tubo and Francois Binettem, Methods in Molecular Medicine, Vol. 18: Tissue Engineering Methods and Protocols pp.205-215, 1999. 4. Shan-hui Hsu, Chi-Ching Kuo, Shu Wen Whu, Chen-Huan Lin,and Ching-Lin Tsai. The effect of ultrasound stimulat1h3ion versus bioreactors on neocartilage formation in tissue engineering scaffolds seeded with human chondrocytes in vitro. Biomol Eng, Oct;23(5):259-64, 2006 5. Muller-Rath R, Gavénis K, Andereya S, Mumme T, Schmidt-Rohlfing B, Schneider U. A novel rat tail collagen type-I gel for the cultivation of human articular chondrocytes in low cell density. Int J Artif Organs Dec;30(12):1057-67, 2007. 6. Lin YJ, Yen CN, Hu YC, Wu YC, Liao CJ, Chu IM. Chondrocytes culture in three-dimensional porous alginate scaffolds enhanced cell proliferation, matrix synthesis and gene expression. J Biomed Mater Res A. Feb 6, 2008. 7. Yen CN, Lin YR, Chang MD, Tien CW, Wu YC, Liao CJ, Hu YC. Use of porous alginate sponges for substantial chondrocyte expansion and matrix production: effects of seeding density. Biotechnol Prog. Mar-Apr;24(2):452-7, 2008.||摘要:||
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.
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