TISSUE ENGINEERED CARTILAGE USING CHITOSAN/GELATIN AND NORMAL OR POST-RNA INTERFERENCE-CHONDROCYTES IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANG Zhenghui ¹ , WU Baojun ¹ , Kamal Mustafa. ²

1Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an Shaanxi, 710004, P.R.China;;
2Faculty of Dentistry, University of Bergen, Norway.;

Corresponding author：

Keywords：

Tissue engineered cartilage; RNA interference; Chondrocyte; Aggrecan; Aggrecanase; Rat

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【Abstract】 Objective The seed cells source is a research focus in tissue engineered cartilage. To observe whether the post-RNA interference (RNAi) chondrocytes could be used as the seed cells of tissue engineered cartilage. Methods Chondrocytes were separated from Sprague Dawley rats. The first passage chondrocytes were used and divided into 2 groups: normal chondrocytes (control group) and post-RNAi (experimental group). Normal and post-RNAi chondrocytes were seeded into chitosan/gelatin material and cultured in vitro to prepare tissue engineered cartilage. The contents of Aggrecan and Aggrecanase-1, 2 were measured by HE and Masson staining, scanning electron microscope (SEM), and RT-PCR. Results The histological results: no obvious difference was observed in cell number and extracellular matrix (ECM) between 2 groups at 2 weeks; when compared with control group, the secretion of ECM and the cell number increased in experimental group with time. The RT-PCR results: the expression of Aggrecan mRNA in experimental group was significantly higher than that in control group (P lt; 0.05); but the expressions of Aggrecanase-1, 2 mRNA in experimental group were significantly lower than those in control group (P lt; 0.05). The SEM results: the cell number in experimental group was obviously more than that in control group, and the cells in experimental group were conjugated closely. Conclusion The post-RNAi chondrocytes can be used as the seed cells for tissue engineered cartilage, which can secrete more Aggrecan than normal chondrocytes. But their biological activities need studying further.

Citation： WANG Zhenghui,WU Baojun,Kamal Mustafa.. TISSUE ENGINEERED CARTILAGE USING CHITOSAN/GELATIN AND NORMAL OR POST-RNA INTERFERENCE-CHONDROCYTES IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(1): 106-111. doi: Copy

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3.	using costchondral chondrocytes as a source of primary rat chondrocyte and cryopreservation method. Bone, 2005, 37(4): 530-544.
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6.	, 2009, 27(5): 307-314.
7.	pericellular matrix improves cell-induced cartilage formation. J Cell Biochem, 2010 , 110(1): 260-271.
8.	tumor cell death in vitro and radiation therapy in vivo by use of small interfering RNA targeted to hypoxia-inducible factor-1α. Cancer Res, 2004, 64: 8139-8142.
9.	Efficiencies, kinetics, and non-specific effects of siRNA-mediated gene suppression. Biologicals, 2007, 35(4): 321-328.
10.	passaged chondrocytes in vitro. Tissue Eng Part A, 2009, 15(3): 665-673.
11.	regeneration when subcutaneously implanted in nude mice. J Tissue Eng Regen Med , 2009, 3(7): 493-500.
12.	of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS. Nat Med, 2005, 11(4): 423-428.
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14.	beta. J Rheumatol, 2004 , 31(2): 315-320.
15.	(ADAM-TS5): substrate specificity studies and comparison.
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21.	Gartland A, Mecher J, Mason-Savas A, et al. In vitro chondrocyte differentiation.
22.	Nagase H, Kashiwagi M. Aggrecanases and cartilage matrix degradation.
23.	Vinatier C, Mrugala D, Jorgensen C, et al. Cartilage engineering: a crucial.
24.	Vonk LA, Doulabi BZ, Huang C, et al. Preservation of the chondrocyte’s.
25.	Gründer T, Gaissmaier C, Fritz J, et al. Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads. Osteoarthritis Cartilage, 2004, 12(7): 559-567.
26.	Zhang XW, Takashi K, Wang H, et al. Enhancement of hypoxia-induced.
27.	李卿, 陆峻泓, 刘伟, 等. 软骨细胞的老化对软骨蛋白聚糖代谢的影响. 中国临床康复, 2003, 7(2): 216-217.
28.	Klatt AR, Klinger G, Zech D, et al. RNAi in primary human chondrocytes:.
29.	Ahmed N, Gan L, Nagy A, et al. Cartilage tissue formation using redifferentiated.
30.	王正辉, 杨壮群, 贺西京, 等. 大鼠Aggrecanse-1, 2特异性shRNA慢病毒表达载体的构建及测序. 中国美容医学, 2007, 16(12): 1679-1682.
31.	Kaps C, Frauenschuh S, Endres M, et al. Gene expression profiling of human articular cartilage grafts generated by tissue engineering. Biomaterials,.
32.	Oliveira JT, Santos TC, Martins L, et al. Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage.
33.	Raoul C, Abbas-Terki T, Bensadoun JC, et al. Lentiviral-mediated silencing.
34.	Nishitsuji H, Ikeda T, Miyoshi H, et al. Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary nondividing cells. Microbes Infect, 2004, 6(1): 76-85.
35.	Heilbronn R, Weger S. Viral vectors for gene transfer: current status of gene therapeutics. Handb Exp Pharmacol, 2010, (197): 143-170.
36.	, 27(19): 3617-3630.

1. with aggrecanase-1 (ADAM-TS4). Matrix Biol, 2002, 21(6): 499-511.
2. chondrocytes in vivo and in vitro, is downregulated by interleukin.
3. using costchondral chondrocytes as a source of primary rat chondrocyte and cryopreservation method. Bone, 2005, 37(4): 530-544.
4. Arithritis Res Ther, 2003, 5(2): 94-103.
5. combination of cells, biomaterials and biofactors. Trends Biotechnol.
6. , 2009, 27(5): 307-314.
7. pericellular matrix improves cell-induced cartilage formation. J Cell Biochem, 2010 , 110(1): 260-271.
8. tumor cell death in vitro and radiation therapy in vivo by use of small interfering RNA targeted to hypoxia-inducible factor-1α. Cancer Res, 2004, 64: 8139-8142.
9. Efficiencies, kinetics, and non-specific effects of siRNA-mediated gene suppression. Biologicals, 2007, 35(4): 321-328.
10. passaged chondrocytes in vitro. Tissue Eng Part A, 2009, 15(3): 665-673.
11. regeneration when subcutaneously implanted in nude mice. J Tissue Eng Regen Med , 2009, 3(7): 493-500.
12. of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS. Nat Med, 2005, 11(4): 423-428.
13. Tortorella MD, Liu RQ, Burn T, et al. Characterization of human aggrecanase-.
14. beta. J Rheumatol, 2004 , 31(2): 315-320.
15. (ADAM-TS5): substrate specificity studies and comparison.
16. Wachsmuth L, Bau B, Fan Z, et al. ADAMTS-1, a gene product of articular.
17. Miller JA, Liu RQ, Davis GL, et al. A microplate assay specific for the enzyme aggrecanase. Anal Biochem, 2003, 314(2): 260-265.
18. Wang Z, Yang ZQ, He XJ, et al. Effects of RNAi-mediated inhibition of aggrecanase-1 and aggrecanase-2 on rat costochondral chondrocytes in vitro. Acta Pharmacol Sin, 2008, 29(10): 1215-1226.
19. Wang Z, Yang ZQ, He XJ, et al. Lentivirus-mediated knockdown of aggrecanase-1 and -2 promotes chondrocyte-engineered cartilage formation in vitro. Biotechnol Bioeng, 2010, 107(4): 730-736.
20. 王正辉, 李国光, 杨壮群, 等. 大鼠肋软骨细胞体外培养及老化对基质代谢的影响. 组织工程与重建外科, 2007, 5(6): 312-316.
21. Gartland A, Mecher J, Mason-Savas A, et al. In vitro chondrocyte differentiation.
22. Nagase H, Kashiwagi M. Aggrecanases and cartilage matrix degradation.
23. Vinatier C, Mrugala D, Jorgensen C, et al. Cartilage engineering: a crucial.
24. Vonk LA, Doulabi BZ, Huang C, et al. Preservation of the chondrocyte’s.
25. Gründer T, Gaissmaier C, Fritz J, et al. Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads. Osteoarthritis Cartilage, 2004, 12(7): 559-567.
26. Zhang XW, Takashi K, Wang H, et al. Enhancement of hypoxia-induced.
27. 李卿, 陆峻泓, 刘伟, 等. 软骨细胞的老化对软骨蛋白聚糖代谢的影响. 中国临床康复, 2003, 7(2): 216-217.
28. Klatt AR, Klinger G, Zech D, et al. RNAi in primary human chondrocytes:.
29. Ahmed N, Gan L, Nagy A, et al. Cartilage tissue formation using redifferentiated.
30. 王正辉, 杨壮群, 贺西京, 等. 大鼠Aggrecanse-1, 2特异性shRNA慢病毒表达载体的构建及测序. 中国美容医学, 2007, 16(12): 1679-1682.
31. Kaps C, Frauenschuh S, Endres M, et al. Gene expression profiling of human articular cartilage grafts generated by tissue engineering. Biomaterials,.
32. Oliveira JT, Santos TC, Martins L, et al. Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage.
33. Raoul C, Abbas-Terki T, Bensadoun JC, et al. Lentiviral-mediated silencing.
34. Nishitsuji H, Ikeda T, Miyoshi H, et al. Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary nondividing cells. Microbes Infect, 2004, 6(1): 76-85.
35. Heilbronn R, Weger S. Viral vectors for gene transfer: current status of gene therapeutics. Handb Exp Pharmacol, 2010, (197): 143-170.
36. , 27(19): 3617-3630.

Chinese Journal of Reparative and Reconstructive Surgery

TISSUE ENGINEERED CARTILAGE USING CHITOSAN/GELATIN AND NORMAL OR POST-RNA INTERFERENCE-CHONDROCYTES IN VITRO

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