生物反應器中不同載荷培養促進傳代軟骨細胞的軟骨生成效應_《中國修復重建外科雜志》

作者：

王寧 , 陳繼營 , 張國強 , 柴偉

解放軍總醫院骨科（北京，100853）;

關鍵詞：

軟骨組織工程傳代軟骨細胞生物支架生物反應器載荷培養軟骨缺損修復牛

DOI：

10.7507/1002-1892.20130174

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目的探討第3代軟骨細胞在生物反應器中經載荷培養的軟骨生成效應，為自體軟骨移植臨床應用提供實驗依據。方法取3～4月齡西門塔爾小牛新鮮膝關節軟骨，采用酶消化法分離培養軟骨細胞。將第3代軟骨細胞與多孔聚氨酯支架復合制備細胞-支架復合體。實驗分為5組，分別為空載培養2周組（A組）、直接載荷培養2周組（B組）、空載培養4周組（C組）、直接載荷培養4周組（D組）、空載培養2周后載荷培養2周組（E組）。空載培養時將細胞-支架復合體置于培養箱中孵育；載荷培養是在生物反應器中模擬體內關節微環境，對細胞-支架復合體進行垂直加壓和界面旋轉的復合載荷運動。各組于各時間點取材，行糖胺聚糖（glycosaminoglycan，GAG）/DNA定量分析，實時定量PCR檢測Ⅰ型膠原、Ⅱ型膠原、蛋白聚糖、軟骨寡聚基質蛋白（cartilage oligomeric matrix protein，COMP）和表層蛋白（superficial zone protein，SZP）mRNA表達，并行甲苯胺藍組織學觀察和免疫組織化學染色觀察。結果各組細胞-支架復合體分泌的DNA含量、GAG含量以及GAG/DNA比率比較差異均無統計學意義（P gt; 0.05）。載荷培養后，大量GAG從支架釋放至培養液中，且隨載荷時間增加GAG釋放相應增加（P lt; 0.05）。Ⅰ型膠原mRNA相對表達量各組間比較差異均無統計學意義（P gt; 0.05）。經不同載荷培養后，B組Ⅱ型膠原mRNA相對表達量顯著高于A組（P lt; 0.01），D、E組顯著高于C組（P lt; 0.01）；D、E組蛋白聚糖mRNA相對表達量顯著高于C組（P lt; 0.01），E組顯著高于D組（P lt; 0.01）；B組COMP mRNA相對表達量顯著高于A組（P lt; 0.01），E組顯著高于C組（P lt; 0.01）；E組SZP mRNA相對表達量顯著高于C、D組（P lt; 0.05）。甲苯胺藍染色和免疫組織化學染色示，載荷培養能增強軟骨細胞合成分泌GAG；各組Ⅰ型膠原和Ⅱ型膠原免疫組織化學染色無變化，但D、E組表現較強的蛋白聚糖免疫染色增強作用。結論不同載荷均能促進以傳代軟骨細胞為基礎的軟骨再生，空載培養后載荷培養可能是最佳的軟骨再生培養模式，但載荷誘導第3代軟骨細胞的軟骨生成效應弱化。

引用本文： 王寧,陳繼營,張國強,柴偉. 生物反應器中不同載荷培養促進傳代軟骨細胞的軟骨生成效應. 中國修復重建外科雜志, 2013, 27(7): 786-792. doi: 10.7507/1002-1892.20130174 復制

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.

《中國修復重建外科雜志》

生物反應器中不同載荷培養促進傳代軟骨細胞的軟骨生成效應

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《中國修復重建外科雜志》

生物反應器中不同載荷培養促進傳代軟骨細胞的軟骨生成效應

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