膠原水凝膠支架用于軟骨組織工程的實驗研究_《中國修復重建外科雜志》

作者：

李奎鋒 , 郭立坤 , 樊渝江 , 張興棟

四川大學國家生物醫學材料工程技術研究中心（成都，610065）;

關鍵詞：

軟骨組織工程 Ⅰ型膠原膠原水凝膠軟骨細胞支架材料

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目的探討Ⅰ型膠原濃度對膠原水凝膠理化性能的影響，以及在體外組織工程模型中不同濃度的Ⅰ型膠原水凝膠對軟骨細胞表型和基因表達的影響。方法制備Ⅰ型膠原濃度為12、8、6 mg/mL的3種膠原水凝膠，記為C12、C8、C6。掃描電鏡觀察形貌，并通過溶脹性能和抗壓性能測試表征其理化性能。取2只新生7日齡新西蘭大白兔的關節軟骨，酶消化法分離、培養軟骨細胞。取第2代軟骨細胞與3種膠原水凝膠復合體外培養（C12組、C8組及C6組），1 d后采用二乙酸熒光素（fluorescein diacetate，FDA）/碘化丙啶（propidium iodide，PI）染色，激光共聚焦顯微鏡觀察細胞活性，體外培養14 d后行HE和甲苯胺藍染色觀察組織學形態，實時熒光定量PCR測定軟骨細胞相關基因，即Ⅱ型膠原、蛋白聚糖（Aggrecan）、Ⅰ型膠原、Ⅹ型膠原、Sox9 mRNA表達。結果隨著Ⅰ型膠原濃度從6 mg/mL提高到12 mg/ mL，膠原水凝膠的理化性質出現明顯變化，掃描電鏡下纖維網絡變得致密； 192 h時C6、C8、C12溶脹率依次增大，分別為0.260 ± 0.055、0.358 ± 0.072、0.539 ± 0.033，比較差異均有統計學意義（P lt; 0.05）；壓縮模量逐漸增加，分別為（4.86 ± 0.96）、（7.09 ± 2.33）、（11.08 ± 3.18）kPa，比較差異均有統計學意義（P lt; 0.05）。體外培養1 d，3組凝膠中軟骨細胞經FDA/PI染色后，絕大部分被染成綠色，僅有少數被染成紅色。14 d后組織學觀察示：C12組軟骨細胞在組織中聚集明顯，并出現明顯的甲苯胺藍異染為紫紅色的現象；C8組中也有部分增殖聚集的區域，細胞周圍有較明顯的甲苯胺藍異染現象；C6組細胞分布較為均勻，也有較明顯的甲苯胺藍異染現象。實時熒光定量PCR檢測顯示，3組Ⅱ型膠原mRNA和Aggrecan mRNA表達水平相近；Ⅰ型膠原、Sox9和X型膠原mRNA表達水平C12組最高，C6組最低，C8組介于二者之間，組間差異均有統計學意義（P lt; 0.05）。結論提高Ⅰ型膠原濃度至12 mg/mL后，膠原水凝膠具有更好的理化性質，但軟骨細胞纖維化和肥大相關基因表達也有所上調。

引用本文： 李奎鋒,郭立坤,樊渝江,張興棟. 膠原水凝膠支架用于軟骨組織工程的實驗研究. 中國修復重建外科雜志, 2012, 26(11): 1356-1361. doi: 復制

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2.
3.	Haleem AM, Chu CR. Advances in tissue engineering techniques for articular cartilage repair. Oper Tech Orthop, 2010, 20(2): 76-89.
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6.	Eyre DR, Weis MA, Wu JJ. Advances in collagen cross-link analysis. Methods, 2008, 45(1): 65-74.
7.	Riesle J, Hollander A, Langer R, et al. Collagen in tissue-engineered cartilage: types, structure, and crosslinks. J Cell Biochem, 1998, 71(3): 313-327.
8.	Lee CH, Singla A, Lee Y. Biomedical applications of collagen. Int J Pharm, 2001, 221(1-2): 1-22.
9.	Balakrishnan B, Banerjee R. Biopolymer-based hydrogels for cartilage tissue engineering. Chem Rev, 2011, 111(8): 4453-4474.
10.	Schindler OS. Current concepts of articular cartilage repair. Acta Orthop Belg, 2011, 77(6): 709-726.
11.	Zheng L, Sun J, Chen XN, et al. In vivo cartilage engineering with collagen hydrogel and allogenous chondrocytes after diffusion chamber implantation in immunocompetent host. Tissue Eng Part A, 2009, 15(8): 2145-2153.
12.	Fan J, Gong Y, Ren L, et al. In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels. Acta Biomater, 2010, 6(3): 1178-1185.
13.	Jin R, Moreira Teixeira LS, Krouwels A, et al. Synthesis and characterization of hyaluronic acid-poly (ethylene glycol) hydrogels via Michael addition: An injectable biomaterial for cartilage repair. Acta Biomater, 2010, 6(6): 1968-1977.
14.	Tang Y, Sun J, Fan H, et al. An improved complex gel of modified gellan gum and carboxymethyl chitosan for chondrocytes encapsulation. Carbohydrate Polymers, 2011, 88(1): 46-53.
15.	Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell, 1982, 30(1): 215-224.
16.	Schinagl RM, Gurskis D, Chen AC, et al. Depth-dependent confined compression modulus of full-thickness bovine articular cartilage. J Orthop Res, 1997, 15(4): 499-506.
17.	Park H, Guo X, Temenoff JS, et al. Effect of swelling ratio of injectable hydrogel composites on chondrogenic differentiation of encapsulated rabbit marrow mesenchymal stem cells in vitro. Biomacromolecules, 2009, 10(3): 541-546.
18.	Cancedda R, Dozin B, Giannoni P, et al. Tissue engineering and cell therapy of cartilage and bone. Matrix Biol, 2003, 22(1): 81-91.
19.	Keeney M, Lai JH, Yang F. Recent progress in cartilage tissue engineering. Curr Opin Biotechnol, 2011, 225(5): 734-740.
20.	Schmitz N, Laverty S, Kraus V, et al. Basic methods in histopathology of joint tissues. Osteoarthritis Cartilage, 2010, 18 Suppl 3: S113-116.
21.	Lien SM, Ko LY, Huang TJ. Effect of pore size on ECM secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering. Acta Biomater, 2009, 5(2): 670-679.
22.	Shen G. The role of type X collagen in facilitating and regulating endochondral ossification of articular cartilage. Orthod Craniofac Res, 2005, 8(1): 11-17.

1. 4 Lee CR, Grodzinsky AJ, Spector M. The effects of cross-linking of collagen-glycosaminoglycan scaffolds on compressive stiffness, chondrocyte-mediated contraction, proliferation and biosynthesis. Biomaterials, 2001, 22(23): 3145-3154. 5 Zheng L, Fan H, Sun J, et al. Chondrogenic differentiation of mesenchymal stem cells induced by collagen-based hydrogel: an in vivo study. J Biomed Mater Res A, 2010, 93(2): 783-792.
2.
3. Haleem AM, Chu CR. Advances in tissue engineering techniques for articular cartilage repair. Oper Tech Orthop, 2010, 20(2): 76-89.
4. Zhang L, Li K, Xiao W, et al. Preparation of collagen-chondroitin sulfate-hyaluronic acid hybrid hydrogel scaffolds and cell compatibility in vitro. Carbohydrate Polymers, 2010, 84(1): 118-125.
5. van Susante JLC, Pieper J, Buma P, et al. Linkage of chondroitin-sulfate to type I collagen scaffolds stimulates the bioactivity of seeded chondrocytes in vitro. Biomaterials, 2001, 22(17): 2359-2369.
6. Eyre DR, Weis MA, Wu JJ. Advances in collagen cross-link analysis. Methods, 2008, 45(1): 65-74.
7. Riesle J, Hollander A, Langer R, et al. Collagen in tissue-engineered cartilage: types, structure, and crosslinks. J Cell Biochem, 1998, 71(3): 313-327.
8. Lee CH, Singla A, Lee Y. Biomedical applications of collagen. Int J Pharm, 2001, 221(1-2): 1-22.
9. Balakrishnan B, Banerjee R. Biopolymer-based hydrogels for cartilage tissue engineering. Chem Rev, 2011, 111(8): 4453-4474.
10. Schindler OS. Current concepts of articular cartilage repair. Acta Orthop Belg, 2011, 77(6): 709-726.
11. Zheng L, Sun J, Chen XN, et al. In vivo cartilage engineering with collagen hydrogel and allogenous chondrocytes after diffusion chamber implantation in immunocompetent host. Tissue Eng Part A, 2009, 15(8): 2145-2153.
12. Fan J, Gong Y, Ren L, et al. In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels. Acta Biomater, 2010, 6(3): 1178-1185.
13. Jin R, Moreira Teixeira LS, Krouwels A, et al. Synthesis and characterization of hyaluronic acid-poly (ethylene glycol) hydrogels via Michael addition: An injectable biomaterial for cartilage repair. Acta Biomater, 2010, 6(6): 1968-1977.
14. Tang Y, Sun J, Fan H, et al. An improved complex gel of modified gellan gum and carboxymethyl chitosan for chondrocytes encapsulation. Carbohydrate Polymers, 2011, 88(1): 46-53.
15. Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell, 1982, 30(1): 215-224.
16. Schinagl RM, Gurskis D, Chen AC, et al. Depth-dependent confined compression modulus of full-thickness bovine articular cartilage. J Orthop Res, 1997, 15(4): 499-506.
17. Park H, Guo X, Temenoff JS, et al. Effect of swelling ratio of injectable hydrogel composites on chondrogenic differentiation of encapsulated rabbit marrow mesenchymal stem cells in vitro. Biomacromolecules, 2009, 10(3): 541-546.
18. Cancedda R, Dozin B, Giannoni P, et al. Tissue engineering and cell therapy of cartilage and bone. Matrix Biol, 2003, 22(1): 81-91.
19. Keeney M, Lai JH, Yang F. Recent progress in cartilage tissue engineering. Curr Opin Biotechnol, 2011, 225(5): 734-740.
20. Schmitz N, Laverty S, Kraus V, et al. Basic methods in histopathology of joint tissues. Osteoarthritis Cartilage, 2010, 18 Suppl 3: S113-116.
21. Lien SM, Ko LY, Huang TJ. Effect of pore size on ECM secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering. Acta Biomater, 2009, 5(2): 670-679.
22. Shen G. The role of type X collagen in facilitating and regulating endochondral ossification of articular cartilage. Orthod Craniofac Res, 2005, 8(1): 11-17.

《中國修復重建外科雜志》

膠原水凝膠支架用于軟骨組織工程的實驗研究

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《中國修復重建外科雜志》

膠原水凝膠支架用于軟骨組織工程的實驗研究

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

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