EXPERIMENTAL STUDY ON COLLAGEN HYDROGEL SCAFFOLDS FOR CARTILAGE TISSUE ENGINEERING_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LI Kuifeng , GUO Likun , FAN Yujiang , ZHANG Xingdong.

National Engineering Research Center for Biomaterials, Sichuan University, Chengdu Sichuan, 610065, P.R.China. Corresponding author: ZHANG Xingdong, E-mail: zhangxd@scu.edu.cn;

Corresponding author：

Keywords：

Cartilage tissue engineering; Collagen type I; Collagen hydrogel; Chondrocytes; Scaffold material

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Objective To investigate the effect of collagen type I concentration on the physical and chemical properties of the collagen hydrogel, and to analyze the effect of different concentrations of collagen type I hydrogel on the phenotype and gene expression of the chondrocytes in vitro. Methods Three kinds of collagen hydrogels with concentrations of 12, 8, and 6 mg/ mL (C12, C8, and C6) were prepared, respectively. The micro-structure, compressive modulus, and swelling ratio of the hydrogels were measured and analyzed. The chondrocytes at 2nd passage were cocultured with three kinds of collagen hydrogels in vitro, respectively. After 1-day culture, the samples were stained with fluorescein diacetate (FDA) / propidium iodide (PI) and the cell activity was observed under confocal laser microscope. After 14-day culture, HE staining and toluidine blue staining were carried out to observe the histological morphology, and mRNA expressions of chondrocytes related genes (collagen type II, Aggrecan, collagen type I, collagen type X, Sox9) were determined by real-time fluorescent quantitative PCR. Results With the increase of collagen type I concentration from 6 to 12 mg/mL, the physical and chemical properties of the collagen hydrogels changed significantly: the fiber network became dense; the swelling ratios of C6, C8, and C12 were 0.260 ± 0.055, 0.358 ± 0.072, and 0.539 ± 0.033 at 192 hours, respectively, showing significant differences among 3 groups (P lt; 0.05); and the compression modulus were (4.86 ± 0.96), (7.09 ± 2.33), and (11.08 ± 3.18) kPa, respectively, showing significant differences among 3 groups (P lt; 0.05). After stained with FDA/PI, most cells were stained green, and few were stained red. The histological observation results showed that the chondrocytes in C12 hydrogels aggregated obviously with b heterochromia, chondrocytes in C8 hydrogels aggregated partly with obvious heterochromia, and chondrcytes in C6 hydrogels uniformly distributed with weak heterochromia. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of collagen type II and Aggrecan were at the same level in C12, C8, and C6; the expressions of collagen type I, Sox9, and collagen type X were up-regulated with the increase of collagen type I hydrogels concentration, and the expressions were the highest at 12 mg/mL and were the lowest at 6 mg/mL, showing significant differences among 3 groups (P lt; 0.05). Conclusion Increasing the concentration of collagen hydrogels leads to better mechanical properties and higher shrink-resistance, but it may induce the up-regulation of cartilage fibrosis and hypertrophy related gene expression.

Citation： LI Kuifeng,GUO Likun,FAN Yujiang,ZHANG Xingdong.. EXPERIMENTAL STUDY ON COLLAGEN HYDROGEL SCAFFOLDS FOR CARTILAGE TISSUE ENGINEERING. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(11): 1356-1361. doi: Copy

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.

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.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON COLLAGEN HYDROGEL SCAFFOLDS FOR CARTILAGE TISSUE ENGINEERING

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