生物反应器中不同载荷培养促进传代软骨细胞的软骨生成效应_《中国修复重建外科杂志》

作者：

王宁 , 陈继营 , 张国强 , 柴伟

解放军总医院骨科（北京，100853）;

关键词：

软骨组织工程传代软骨细胞生物支架生物反应器载荷培养软骨缺损修复牛

DOI：

10.7507/1002-1892.20130174

视频：

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目的探讨第3代软骨细胞在生物反应器中经载荷培养的软骨生成效应，为自体软骨移植临床应用提供实验依据。方法取3～4月龄西门塔尔小牛新鲜膝关节软骨，采用酶消化法分离培养软骨细胞。将第3代软骨细胞与多孔聚氨酯支架复合制备细胞-支架复合体。实验分为5组，分别为空载培养2周组（A组）、直接载荷培养2周组（B组）、空载培养4周组（C组）、直接载荷培养4周组（D组）、空载培养2周后载荷培养2周组（E组）。空载培养时将细胞-支架复合体置于培养箱中孵育；载荷培养是在生物反应器中模拟体内关节微环境，对细胞-支架复合体进行垂直加压和界面旋转的复合载荷运动。各组于各时间点取材，行糖胺聚糖（glycosaminoglycan，GAG）/DNA定量分析，实时定量PCR检测Ⅰ型胶原、Ⅱ型胶原、蛋白聚糖、软骨寡聚基质蛋白（cartilage oligomeric matrix protein，COMP）和表层蛋白（superficial zone protein，SZP）mRNA表达，并行甲苯胺蓝组织学观察和免疫组织化学染色观察。结果各组细胞-支架复合体分泌的DNA含量、GAG含量以及GAG/DNA比率比较差异均无统计学意义（P gt; 0.05）。载荷培养后，大量GAG从支架释放至培养液中，且随载荷时间增加GAG释放相应增加（P lt; 0.05）。Ⅰ型胶原mRNA相对表达量各组间比较差异均无统计学意义（P gt; 0.05）。经不同载荷培养后，B组Ⅱ型胶原mRNA相对表达量显著高于A组（P lt; 0.01），D、E组显著高于C组（P lt; 0.01）；D、E组蛋白聚糖mRNA相对表达量显著高于C组（P lt; 0.01），E组显著高于D组（P lt; 0.01）；B组COMP mRNA相对表达量显著高于A组（P lt; 0.01），E组显著高于C组（P lt; 0.01）；E组SZP mRNA相对表达量显著高于C、D组（P lt; 0.05）。甲苯胺蓝染色和免疫组织化学染色示，载荷培养能增强软骨细胞合成分泌GAG；各组Ⅰ型胶原和Ⅱ型胶原免疫组织化学染色无变化，但D、E组表现较强的蛋白聚糖免疫染色增强作用。结论不同载荷均能促进以传代软骨细胞为基础的软骨再生，空载培养后载荷培养可能是最佳的软骨再生培养模式，但载荷诱导第3代软骨细胞的软骨生成效应弱化。

引用本文： 王宁,陈继营,张国强,柴伟. 生物反应器中不同载荷培养促进传代软骨细胞的软骨生成效应. 中国修复重建外科杂志, 2013, 27(7): 786-792. doi: 10.7507/1002-1892.20130174 复制

1.	Brittberg M, Lindahl A, Nilsson A, et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med, 1994, 331(14): 889-895.
2.	Marlovits S, Zeller P, Singer P, et al. Cartilage repair: generations of autologous chondrocyte transplantation. Eur J Radiol, 2006, 57(1): 24-31.
3.	Peterson L, Vasiliadis HS, Brittberg M, et al. Autologous chondrocyte implantation: a long-term follow-up. Am J Sports Med, 2010, 38(6): 1117-1124.
4.	Niemeyer P, Pestka JM, Salzmann GM, et al. Influence of cell quality on clinical outcome after autologous chondrocyte implantation. Am J Sports Med, 2012, 40(3): 556-561.
5.	Brun P, Dickinson SC, Zavan B, et al. Characteristics of repair tissue in second-look and third-look biopsies from patients treated with engineered cartilage: relationship to symptomatology and time after implantation. Arthritis Res Ther, 2008, 10(6): R132.
6.	Kon E, Filardo G, Di Matteo B, et al. Matrix assisted autologous chondrocyte transplantation for cartilage treatment: A systematic review. Bone Joint Res, 2013, 2(2): 18-25.
7.	Brix MO, Stelzeneder D, Trattnig S, et al. Cartilage repair of the knee with Hyalograft C: ® magnetic resonance imaging assessment of the glycosaminoglycan content at midterm. Int Orthop, 2013, 37(1): 39-43.
8.	Grad S, Gogolewski S, Alini M, et al. Effects of simple and complex motion patterns on gene expres sion of chondrocytes seeded in 3-D scaffolds. Tissue Eng, 2006, 12(11): 3171-3179.
9.	Grad S, Eglin D, Alini M, et al. Physical stimulation of chondrogenic cells in vitro: a review. Clin Orthop Relat Res, 2011, 469(10): 2764-2772.
10.	Crawford DC, Heveran CM, Cannon WD, et al. An autologous cartilage tissue implant NeoCart for treatment of grade III chondral injury to the distal femur: prospective clinical safety trial at 2 years. Am J Sports Med, 2009, 37(7): 1334-1343.
11.	Lee CR, Grad S, Gorna K, et al. Fibrin-polyurethane composites for articular cartilage tissue engineering: a preliminary analysis. Tissue Eng, 2005, 11(9-10): 1562-1573.
12.	Wimmer MA, Grad S, Kaup T, et al. Tribology approach to the engineering and study of articular cartilage. Tissue Eng, 2004, 10(9-10): 1436-1445.
13.	Wimmer MA, Alini M, Grad S. The effect of sliding velocity on chondrocytes activity in 3D scaffolds. J Biomech, 2009, 42(4): 424-449.
14.	Salzmann GM, Nuernberger B, Schmitz P, et al. Physicobiochemical synergism through gene therapy and functional tissue engineering for in vitro chondrogenesis. Tissue Eng Part A, 2009, 15(9): 2513-2524.
15.	Wysocka A, Mann K, Bursig H, et al. Chondrocyte suspension in fibrin glue. Cell Tissue Bank, 2010, 11(2): 209-215.
16.	Verdonk R, Verdonk P, Huysse W, et al. Tissue ingrowth after implantation of a novel, biodegradable polyurethane scaffold for treatment of partial meniscal lesions. Am J Sports Med, 2011, 39(4): 774-782.
17.	Oldershaw RA. Cell sources for the regeneration of articular cartilage: the past, the horizon and the future. Int J Exp Pathol, 2012, 93(6): 389-400.
18.	Das RH, Jahr H, Verhaar JA, et al. In vitro expansion affects the response of chondrocytes to mechanical stimulation. Osteoarthritis Cartilage, 2008, 16(3): 385-391.
19.	Mauck RL, Byers BA, Yuan X, et al. Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading. Biomech Model Mechanobiol, 2007, 6(1): 113-125.
20.	Xie J, Han Z, Kim SH, et al. Mechanical loading-dependence of mRNA expressions of extracellular matrices of chondrocytes inoculated into elastomeric microporous poly (L-lactide-co-epsilon-caprolactone) scaffold. Tissue Eng, 2007, 13(1): 29-40.
21.	Fehrenbacher A, Steck E, Roth W, et al. Long-term mechanical loading of chondrocyte-chitosan biocomposites in vitro enhanced their proteoglycan and collagen content. Biorheology, 2006, 43(6): 709-720.
22.	Hung BP, Hutton DL, Grayson WL. Mechanical control of tissue-engineered bone. Stem Cell Res Ther, 2013, 4(1): 10.

1. Brittberg M, Lindahl A, Nilsson A, et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med, 1994, 331(14): 889-895.
2. Marlovits S, Zeller P, Singer P, et al. Cartilage repair: generations of autologous chondrocyte transplantation. Eur J Radiol, 2006, 57(1): 24-31.
3. Peterson L, Vasiliadis HS, Brittberg M, et al. Autologous chondrocyte implantation: a long-term follow-up. Am J Sports Med, 2010, 38(6): 1117-1124.
4. Niemeyer P, Pestka JM, Salzmann GM, et al. Influence of cell quality on clinical outcome after autologous chondrocyte implantation. Am J Sports Med, 2012, 40(3): 556-561.
5. Brun P, Dickinson SC, Zavan B, et al. Characteristics of repair tissue in second-look and third-look biopsies from patients treated with engineered cartilage: relationship to symptomatology and time after implantation. Arthritis Res Ther, 2008, 10(6): R132.
6. Kon E, Filardo G, Di Matteo B, et al. Matrix assisted autologous chondrocyte transplantation for cartilage treatment: A systematic review. Bone Joint Res, 2013, 2(2): 18-25.
7. Brix MO, Stelzeneder D, Trattnig S, et al. Cartilage repair of the knee with Hyalograft C: ® magnetic resonance imaging assessment of the glycosaminoglycan content at midterm. Int Orthop, 2013, 37(1): 39-43.
8. Grad S, Gogolewski S, Alini M, et al. Effects of simple and complex motion patterns on gene expres sion of chondrocytes seeded in 3-D scaffolds. Tissue Eng, 2006, 12(11): 3171-3179.
9. Grad S, Eglin D, Alini M, et al. Physical stimulation of chondrogenic cells in vitro: a review. Clin Orthop Relat Res, 2011, 469(10): 2764-2772.
10. Crawford DC, Heveran CM, Cannon WD, et al. An autologous cartilage tissue implant NeoCart for treatment of grade III chondral injury to the distal femur: prospective clinical safety trial at 2 years. Am J Sports Med, 2009, 37(7): 1334-1343.
11. Lee CR, Grad S, Gorna K, et al. Fibrin-polyurethane composites for articular cartilage tissue engineering: a preliminary analysis. Tissue Eng, 2005, 11(9-10): 1562-1573.
12. Wimmer MA, Grad S, Kaup T, et al. Tribology approach to the engineering and study of articular cartilage. Tissue Eng, 2004, 10(9-10): 1436-1445.
13. Wimmer MA, Alini M, Grad S. The effect of sliding velocity on chondrocytes activity in 3D scaffolds. J Biomech, 2009, 42(4): 424-449.
14. Salzmann GM, Nuernberger B, Schmitz P, et al. Physicobiochemical synergism through gene therapy and functional tissue engineering for in vitro chondrogenesis. Tissue Eng Part A, 2009, 15(9): 2513-2524.
15. Wysocka A, Mann K, Bursig H, et al. Chondrocyte suspension in fibrin glue. Cell Tissue Bank, 2010, 11(2): 209-215.
16. Verdonk R, Verdonk P, Huysse W, et al. Tissue ingrowth after implantation of a novel, biodegradable polyurethane scaffold for treatment of partial meniscal lesions. Am J Sports Med, 2011, 39(4): 774-782.
17. Oldershaw RA. Cell sources for the regeneration of articular cartilage: the past, the horizon and the future. Int J Exp Pathol, 2012, 93(6): 389-400.
18. Das RH, Jahr H, Verhaar JA, et al. In vitro expansion affects the response of chondrocytes to mechanical stimulation. Osteoarthritis Cartilage, 2008, 16(3): 385-391.
19. Mauck RL, Byers BA, Yuan X, et al. Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading. Biomech Model Mechanobiol, 2007, 6(1): 113-125.
20. Xie J, Han Z, Kim SH, et al. Mechanical loading-dependence of mRNA expressions of extracellular matrices of chondrocytes inoculated into elastomeric microporous poly (L-lactide-co-epsilon-caprolactone) scaffold. Tissue Eng, 2007, 13(1): 29-40.
21. Fehrenbacher A, Steck E, Roth W, et al. Long-term mechanical loading of chondrocyte-chitosan biocomposites in vitro enhanced their proteoglycan and collagen content. Biorheology, 2006, 43(6): 709-720.
22. Hung BP, Hutton DL, Grayson WL. Mechanical control of tissue-engineered bone. Stem Cell Res Ther, 2013, 4(1): 10.

《中国修复重建外科杂志》

生物反应器中不同载荷培养促进传代软骨细胞的软骨生成效应

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《中国修复重建外科杂志》

生物反应器中不同载荷培养促进传代软骨细胞的软骨生成效应

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