本研究利用膠原蛋白的降解物—明膠，作為組織工程體外培養軟骨的支架，明膠屬於天然高分子，具有良好的生物相容性以及降解性等優點，且降解後的分子不容易造成生物體內的發炎或是免疫反應，這些特性使明膠適於軟骨細胞的貼附、增生以及分泌ECM，但由於明膠體的機械性質較弱，所以我們採用由茜草提煉的天然交聯劑(Genipin)來提高其機械性質，延緩它在培養液或體內的降解速率，使之更適合軟骨細胞的培養。
由本實驗室之前的研究可知明膠相當適合做為培養軟骨的材料，故我們在此利用改變交聯反應的溫度以及調整冷凍乾燥的程序，製造出四種不同孔徑範圍的明膠支架，並植入乳鼠的軟骨細胞觀察其在不同孔徑的孔洞下的生長情形，探討孔洞的大小是否會對軟骨的增生和分化產生影響。
實驗並針對各孔洞大小的支架進行壓力測試，觀察細胞形成組織後支架機械性質的增減。此外，利用二次冷凍乾燥將各組別的孔洞改良得更具均勻性以及聯通性，觀察培養後的細胞成長情形。未來可以引入醣胺素(GAG)、生長因子(growth-factor)等方式來培養，期望藉由這些因子增加軟骨細胞的ECM分泌、增生的數量與表型的維持，使明膠支架能對培養軟骨組織有更佳的效果。

The research utilizes gelatin—a degradated product of collagen as scaffolds for cartilage tissue engineering in vitro. Gelatin is a kind of natural polymers, it has advantage of very good biocompatibility and biodegradation property; besides, it’s degradation molecular would hardly induce inflammation and immunization reaction inside bodies. These benefits make it suitable for the adherence, proliferation and synthesizing ECM of cartilage cells. But due to its weak mechanical properties, we use natural cross-linking reagent---genipin which was extracted from Gardeniae to make gelatin more stable, and elongate its degradation time in the medium or even in the body, to make it more suitable for chondrocyte culturing.
From our lab’s previous study, we know that gelatin is an excellent material for cartilage culture. Hence I further altered the temperature of cross-linking reactions and adjusted the processes of lyophilization, to produce scaffolds with four ranges of different pore size.
To investigate whether different pore sizes influence proliferation and differentiation of cartilage cells, articular chondrocytes of Wistar rats within 7 days of birth were transplanted into the scaffolds. DNA assay, GAG assay, H&E staining, Safranin-O staining and RT-PCR were used to analyze the behavior of chondrocytes.
We found that cell’s metabolism could be affected by the configuration of the scaffold. The secretion of ECM of cells in the scaffolds with largest inner pores has the most significant amount over the other groups. We also found that cells in the smallest pores of scaffolds often show a de-differentiation form, because of lacking space in scaffolds. The phenotype of the cells will be maintained easily through the progressive increment of the pore size in scaffolds. In conclusion, cells prefer a pore size between 250μm to 500μm to produce ECM and proliferate, and the size of the space is the key factor for cell’s metabolism.
On the other hand, the mechanical tests showed that our scaffolds have a good resilience. After 30 days culture, the scafflds’ anti-compression capability have made a great progress in the group with the largest inner pores. These facts have proven that gelatin scaffold is a very suitable material for cartilage tissue engineering.

1. 洪娟瑜 Osteoarthritis 課程講義 2006年7月
2. 宋信文 人工器官與組織工程 課程用書 2004年 9月
3. D. Nesic, R. Whiteside, M. Brittberg, D. Wendt,I. Martin, P. Mainil-Varlet, Cartilage tissue engineering for degenerative joint disease, Advanced Drug Delivery Reviews 58 (2006) 300–322
4. J. S. Temeno, A. G. Mikos, Review: tissue engineering for regeneration of articular cartilage Biomaterials 21 (2000) 431–440
5. T. Aigner, A. Sachse, P.M. Gebhard, H.I. Roach, Osteoarthritis: Pathobiology—targets and ways for therapeutic intervention Advanced Drug Delivery Reviews 58 (2006) 128–149
6. 李偉德 明膠作為關節軟骨組織工程支架對軟骨細胞生長的影響 清華大學化工系 碩士論文 民國94年
7. T. Bhardwaj, R. M. Pilliar, M. D. Grynpas, R. A. Kandel1, Effect of material geometry on cartilagenous tissue formation in vitro, material geometry and cartilagenous tissue, 30 March (2001)
8. D. J. GriVon,M.R. Sedighi, D. V. SchaeVer,J. A. Eurell, A. L. Johnson, Chitosan scaffolds: Interconnective pore size and cartilage engineering Acta Biomaterialia 2 (2006) 313–320
9. 李夢仰 退化性關節炎之概況與治療 藥學雜誌78期(1) P119-129
10. J. J. Gartland, M.D. Fundamentals of Orthopaedics 基礎骨科學 賴祐平譯 藝軒出版社 2001年9月
11. P. Dieppe, Subchondral bone should be the main target for the treatment of pain and disease progression in osteoarthritis, Osteoarthritis and Cartilage7 (1999), 325–326
12. N. A. Segal, J. A. Buckwalter, A. Amendola, Other surgical techniques for osteoarthritis Best Practice & Research Clinical Rheumatology Vol. 20, No. 1, (2006) 155–176
13. R.G. LeBaron, K.A. Athanasiou, Ex vivo synthesis of articular cartilage Biomaterials 21 (2000) 2575–2587
14. L. C. Dijkgraaf, G.M. Lambert, G. Boering, S.B. Robert, Normal Cartilage Structure, Biochemistry,and Metabolism: A Review of the Literature J Oral Maxillofac Surg 53: (1995),924–929
15. J. A. Buckwalter, H.J. Mankin, Articular cartilage: tissue design and Chondrocyte matrix interactions. AAOS Inst Course Lect ;47(1998):477–486.
16. 簡千翔 明膠與陶瓷複合式支架培養關節軟骨細胞的研究 清華大學化工系 碩士論文 民國95年
17. 黃彥富 鲨魚軟骨素與物理刺激對體外軟骨再生組織的影響 中興大學化工系 碩士論文 民國93年
18. J. E. Aubin, F. Liu, L. Malaval, A. K. Gupta, Osteoblast and chondroblast deffieriention Bone Vol. 17, NO. 2, (1995) : 77S-88S
19. 蔡偉博 Tissue Engineering–Articular cartilage repair 化工資訊6,(2002) p.41–45
20. A. Barbero, S. Grogan , D. Scha¨ fer, M. Heberer,P. Mainil-Varlet, I. Martin, Age related changes in human articular chondrocyte yield, proliferation and post-expansion chondrogenic capacity OsteoArthritis and Cartilage (2004) 12, 476–484
21. M. Huber, S. Trattnig, F. Lintner, Anatomy, Biochemistry, and Physiology of Articular Cartilage investigative radiology Vol. 35, No. 10, (2000) 573–580
22. D. Lajeunesse, G. Hilal, J. P. Pelletier, M. P. Johanne, Subchondral bone morphological and biochemical alterations in Osteoarthritis, Osteoarthritis and Cartilage (1999) 7, 321–322
23. D. R. Eyre, J. J. Wu, P. E. Woods, The cartilage collagens: Structural and metabolic studies, J. Rheumatol (1991) 18,49–51
24. C. B. Knudson, W. Knudson, Cartilage proteoglycans, cell & Developmental Biology, Vol. 12, (2001):pp.69–78
25. J. K. Francis Suh, Howard W.T. Matthew, Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review,Biomaterials 21 (2000) 2589–2598
26. P. M. van der Kraan, P. Buma, T. van Kuppevelt, W. B. van den Berg, Interaction of chondrocytes, extracellular matrix and growth factors:relevance for articular cartilage tissue engineering Osteoarthritis and Cartilage (2002) 10, 631–637
27. W. Knudson, B. Casey, Y. Nishida, W. Eer, K. E. Kuettner, C. H. Knudson, Hyaluronan oliosaccharides perturb cartilage matrix homeostasis and induce chondrocytic chondrolysis. Arthritis Rheum 43 (2000):1165–74
28. J. K. Heath growth factors, Oxford University Press, Printed by Interprint Ltd, Malta (1993)
29. I. McKay, I. Leigh, Growth Factors A Practical Approach, Oxford University Press(1993)
30. R. C. Olney, J. Wang, J. E. Sylvester, E. B. Mougeya, Growth factor regulation of human growth plate chondrocyte proliferation in vitro Biochemical and Biophysical Research Communications 317 (2004) 1171–1182
31. E. B. Hunziker, Growth-factor-induced healing of partial-thickness defects in adult articular cartilage, OsteoArthritis and Cartilage (2001) 9, 22–32
32. H. L. Moses,R. Serra, Regulation of differentiation by TGF-β,Current Opinion in Genetics & Development 6,(1996):581–586
33. H. Mizuta , A. Sanyal , T. Fukumoto, J. S. Fitzsimmons ,N. Matsui , M. E. Bolander , M. J. Oursler , S. W. O’Driscoll, The spatiotemporal expression of TGF-b1 and its receptors during periosteal chondrogenesis in vitro, Journal of Orthopaedic Research 20 (2002) 562–574
34. G. Schmidmaier, B.Wildemann, D. Ostapowicz, F. Kandziora,R. Stange, N. P. Haas, M.l Raschke, Long-term effects of local growth factor (IGF-I and TGF-b1) treatment on fracture healing A safety study for using growth factors, Journal of Orthopaedic Research 22 (2004) 514–519
35. M. M. Stevens, R. P. Marini, I. Martin ,R. Langer , V. P. Shastri, FGF-2 enhances TGF-b1-induced periosteal chondrogenesis, Journal of Orthopaedic Research 22 (2004) 1114–1119
36. A. Fukudaa, K. Katoa, M. Hasegawaa, H. Hirataa, A. Sudoa, K. Okazakib, K. Tsutab, Y. Shikinamib, A. Uchidaa, Enhanced repair of large osteochondral defects using a combination of artificial cartilage and basic fibroblast growth factor , Biomaterials 26 (2005) 4301–4308
37. H. Akaogia, T. Akimotob, S. Miyakia, T. Ushidad, N. Ochiaia, T. Tateishib, J. Tanaka, Basic fibroblast growth factor supports in vitro chondrogenesis of bone marrow-derived mesenchymal stem cells from patients with osteoarthritis, Materials Science and Engineering 24 (2004) 403–406
38. J. I. Shida, S. jingushi, Basic fibroblast growth factor regulate expression of growth factors in rat epiphyseal chondrocyte, Journal of Orthopaedic Research 19 (2001) 259–264
39. M. C. Honorati, L. Cattini, A. Facchini, IL-17, IL-1b and TNF-a stimulate VEGF production by dedifferentiated chondrocytes, OsteoArthritis and Cartilage (2004) 12, 683–691
40. W. Hunter, The structure and diseases of articulating cartilages. Philos Trans R Soc Lond (1743);42:514–21.
41. E. B. Hunziker, E. Kapfinger, Removal of proteoglycans from the surface of defects in articular cartilage transiently enhances coverage by repair cells. J Bone Jt Surg (1998);80-B:144–50.
42. 洪士傑 組織工程於軟骨的應用與發展 生物產業 Bioindustry Vol. 12 No.4 (2001) pp.261–265
43. 徐善慧, 陳俊宇 巧奪天工的人類智慧—組織工程 科學發展 (2002),8 356期 pp.5-9
44. R. Shalak, Fox CF, Preface. In: Tissue Engineering. Shalak R, Fox CF, eds. Alan R.Liss, New York. (1988) pp. 26-29.
45. E. Sachlos, J.T. Czernuszka, Making Tissue Engineering Scaffolds Work. Reriew on the application of solid freeform fabrication technology to the production of tissue Engineering scaffolds, European cells and materials Vol.5 (2003) pp.29-40
46. Chun-Hsu Yao, Bai-Shuan Liu, Chen-Jung Chang, Shan-Hui Hsub, Yueh-Sheng Chen, Preparation of networks of gelatin and genipinas degradable biomaterials, Materials Chemistry and Physics 83 (2004) 204–208
47. A. Bigia, G. Cojazzib, S. Panzavolta, N. Roveri, K. Rubini, Stabilization of gelatin films by crosslinking with genipin, Biomaterials 23 (2002) 4827–4832
48. WEN-HSIANG CHANG, YEN CHANG, PO-HONG LAI, HSING-WEN SUNG, A genipin-crosslinked gelatin membrane as wound-dressing material: in vitro and in vivo studies, J. Biomater. Sci. Polymer Edn, Vol. 14, No. 5 (2003), pp. 481–495
49. P. X. Ma, Scaffolds for tissue fabrication materialstoday (2004), 5 pp.30–40
50. Hsing-Wen Sung, Don-Mou Huang, Wen-Hsiang Chang, Rong-Nan Huang, Jer-Chen Hsu1,Evaluation of gelatin hydrogel crosslinked with various crosslinking agents as bioadhesives: In vitro study John Wiley & Sons, Inc. 1999 pp.520–530
51. Hye-Won Kang, Yasuhiko Tabata, Yoshito Ikada, Fabrication of porous gelatin scaffolds for tissue engineering Biomaterials 20 (1999) 1339–1344
52. Chih-Hung Chang, Hwa-Chang Liu, Chien-Cheng Lin, Cheng-Hung Chou, Feng-Huei Lin, Gelatin–chondroitin–hyaluronan tri-copolymer scaffold for cartilage tissue engineering Biomaterials 24 (2003) 4853–4858
53. M. Pei, J. Seidel, G. Vunjak-Novakovic, L.E. Freed ,Growth factors for sequential cellular de- and re-differentiation in tissue engineering Biochemical and Biophysical Research Communications 294 (2002) 149–154
54. Chih-Hung Chang, Feng-Huei Lin, Chien-Cheng Lin, Cheng-Hung Chou, Hwa-Chang Liu Cartilage Tissue Engineering on the Surface of a Novel Gelatin–Calcium-Phosphate Biphasic Scaffold in a double-Chamber Bioreactor Published online 2 July 2004 in Wiley InterScience
55. S. Van Vlierberghe, V. Cnudde, B. Masschaele, P. Dubruel, I. De Paepe, P.J.S. Jacobs, L. Van Hoorebeke, R. Unger, C.J. Kirkpatrick and E.H. Schacht, Porous gelatin cryogels as cell delivery tool in tissue engineering , Journal of Controlled Release, Volume 116, Issue 2, 28 November 2006, Pages e95-e98
56. C. M. Thomas, C. J. Fuller,C. E. Whittles,and M. Sharif, Chondrocyte death by apoptosis is associated with cartilage matrix degradation,OsteoArthritis and Cartilage (2007) 15, 27-34
57. A. L. Clark, L. D. Barclay, J. R. Matyasb, W. Herzog, In situ chondrocyte deformation with physiological compression of the feline patellofemoral joint, Journal of Biomechanics 36 (2003) 553–568