BMSCs來源細胞外基質支架對微骨折骨髓刺激術后血凝塊體外軟骨化分化的作用研究_《中國修復重建外科雜志》

作者：

魏波 , 金成哲 , 徐燕 , 唐成 , 胡文浩 , 王黎明

南京醫科大學附屬南京醫院（南京市第一醫院）骨科（南京，210006）;

關鍵詞：

BMSCs 細胞外基質支架微骨折骨髓刺激術血凝塊軟骨化分化兔

DOI：

10.7507/1002-1892.20130107

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目的探討BMSCs來源細胞外基質（extracellular matrix，ECM）支架結合微骨折骨髓刺激術后血凝塊在體外軟骨化分化的可行性及有效性。方法選用5～6月齡新西蘭大白兔40只，隨機取20只兔骨髓分離、培養BMSCs并行多向分化鑒定；利用凍干技術制備BMSCs來源的ECM支架，通過掃描電鏡觀察其微結構。于兔股骨滑車處作直徑6 mm軟骨缺損，垂直缺損面作5個直徑1 mm、深3 mm的微骨折孔洞。將未穿刺的20只動物隨機分為2組（n=10）：A組取微骨折術后滲出的血凝塊直接體外培養；B組在A組基礎上用TGF-β3誘導其成軟骨分化。將抽取骨髓的20只動物隨機分為2組（n=10）：C組用制備的ECM支架植入軟骨缺損處，待ECM支架與滲出的骨髓血充分融合后取出體外直接培養；D組在C組基礎上給于TGF-β3誘導其成軟骨分化。體外培養1、2、4、8周時分別行大體、組織學、免疫組織化學及生化成分分析觀察，檢測其分化效果。結果分離、培養的細胞經多向分化鑒定，呈成骨細胞、軟骨細胞、脂肪細胞的表型特征。掃描電鏡觀察示ECM支架呈三維多孔狀結構。在培養過程中，A組組織塊逐漸降解消失；C組組織塊逐漸形成質地柔軟的疏松樣結構；B、D組組織塊逐漸形成表面平滑的新生軟骨樣組織。體外培養4、8周，C、D組組織塊面積均大于B組，差異有統計學意義（P lt; 0.05）；D組大于C組，差異亦有統計學意義（P lt; 0.05）。A、C組HE、番紅O及免疫組織化學染色示組織內僅有少量細胞分布，未見糖胺多糖（glycosaminoglycan，GAG）及Ⅱ型膠原的積累；B、D組可見組織內出現許多軟骨陷窩樣結構，且GAG及Ⅱ型膠原分布逐漸增多。生化成分分析示，培養過程中，B、D組GAG及總膠原含量逐漸增加，其中D組增加更明顯，培養至4、8周，D組與B組比較差異有統計學意義（P lt; 0.05）。結論BMSCs來源的ECM支架結合微骨折骨髓刺激術后血凝塊在體外給予TGF-β3誘導培養時具有良好的軟骨化分化潛能。

引用本文： 魏波,金成哲,徐燕,唐成,胡文浩,王黎明. BMSCs來源細胞外基質支架對微骨折骨髓刺激術后血凝塊體外軟骨化分化的作用研究. 中國修復重建外科雜志, 2013, 27(4): 464-474. doi: 10.7507/1002-1892.20130107 復制

1.	Buckwalter JA, Mankin HJ. Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation. Instr Course Lect, 1998, 47: 487-504.
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3.	Steadman JR, Rodkey WG, Rodrigo JJ, et al. Microfracture: surgical technique and rehabilitation to treat chondral defects. Clin Orthop Relat Res, 2001, (391 Suppl): S362-369.
4.	Kaul G, Cucchiarini M, Remberger K, et al. Failed cartilage repair for early osteoarthritis defects: a biochemical, histological and immunohistochemical analysis of the repair tissue after treatment with marrow-stimulation techniques. Knee Surg Sports Traumatol Arthrosc, 2012, 20(11): 2315-2324.
5.	Breinan HA, Martin SD, Hsu HP, et al. Healing of canine articular cartilage defects treated with microfracture, a type-II collagen matrix, or cultured autologous chondrocytes. J Orthop Res, 2000, 18(5): 781-789.
6.	Milano G, Sanna Passino E, Deriu L, et al. The effect of platelet rich plasma combined with microfractures on the treatment of chondral defects: an experimental study in a sheepmodel. Osteoarthritis Cartilage, 2010, 18(7): 971-980.
7.	Zantop T, Petersen W. Arthroscopic implantation of a matrix to cover large chondral defect during microfracture. Arthroscopy, 2009, 25(11): 1354-1360.
8.	Lu H, Hoshiba T, Kawazoe N, et al. Autologous extracellular matrix scaffolds for tissue engineering. Biomaterials, 2011, 32(10): 2489-2499.
9.	Jin CZ, Choi BH, Park SR, et al. Cartilage engineering using cell-derived extracellular matrix scaffold in vitro. J Biomed Mater Res A, 2010, 92(4): 1567-1577.
10.	Kang SW, Lee SJ, Kim JS, et al. Effect of a scaffold fabricated thermally from acetylated PLGA on the formation of engineered cartilage. Macromol Biosci, 2011, 11(2): 267-274.
11.	莫湘濤, 鄧力, 李秀群, 等. 軟骨細胞-海藻酸鈉水凝膠-SIS復合體修復全層關節軟骨缺損的實驗研究. 中國修復重建外科雜志, 2009, 23(8): 974-979.
12.	Christensen BB, Foldager CB, Hansen OM, et al. A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit?model compared to a collagen type I/III scaffold: in vitro and in vivo studies. Knee Surg Sports Traumatol Arthrosc, 2012, 20(6): 1192-1204.
13.	Kirn-Safran C, Farach-Carson MC, Carson DD. Multifunctionality of extracellular and cell surface heparan sulfate proteoglycans. Cell Mol Life Sci, 2009, 66(21): 3421-3434.
14.	Jin RL, Park SR, Chio BH, et al. Scaffold-free cartilage system using passaged porcine chondrocytes and basic fibroblast growth factor. Tissue Eng Part A, 2009, 15(8): 1887-1895.
15.	Jin CZ, Park SR, Choi BH, et al. In vivo cartilage tissue engineering using a cell-derived extracellular matrix scaffold. Artif Organs, 2007, 31(3): 183-192.
16.	Jin CZ, Cho JH, Choi BH, et al. The maturity of tissue-engineered cartilage in vitro affects the repairability for osteochondral defect. Tissue Eng Part A, 2011, 17(23-24): 3057-3065.
17.	Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411): 143-147.
18.	Lu H, Hoshiba T, Kawazoe N, et al. Cultured cell-derived extracellular matrix scaffolds for tissue engineering. Biomaterials, 2011, 32(36): 9658-9666.
19.	Discher DE, Mooney DJ, Zandstra PW. Growth factors, matrices, and forces combine and control stem cells. Science, 2009, 324(5935): 1673-1677.
20.	Fortier LA, Potter HG, Rickey EJ, et al. Concentrated bone marrow aspirate improves full-thickness cartilage repair compared with microfracture in the equine model. J Bone Joint Surg (Am), 2010, 92(10) : 1927-1937.
21.	Yang HS, La WG, Bhang SH, et al. Hyaline cartilage regeneration by combined therapy of microfracture and long-term bone morphogenetic protein-2 delivery. Tissue Eng Part A, 2011, 17(13-14): 1809-1818.
22.	Kuo AC, Rodrigo JJ, Reddi AH, et al. Microfracture and bone morphogenetic protein 7 (BMP-7) synergistically stimulate articular cartilage repair. Osteoarthritis Cartilage, 2006, 14(11): 1126-1135.
23.	Hoemann CD, Sun J, McKee MD, et al. Rabbit hyaline cartilage repair after marrow stimulation depends on the surgical approach and a chitosan-GP stabilised in situ blood clot. Trans Orthop Res Soc, 2005, 30: 1372.
24.	Thorpe SD, Buckley CT, Vinardell T. The response of bone marrow-derived mesenchymal stem cells to dynamic compression following TGF-β3 induced chondrogenic differentiation. Ann Biomed Eng, 2010, 38(9): 2896-2909.

1. Buckwalter JA, Mankin HJ. Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation. Instr Course Lect, 1998, 47: 487-504.
2. Lützner J, Kasten P, Günther KP, et al. Surgical options for patients with osteoarthritis of the knee. Nat Rev Rheumatol, 2009, 5(6): 309-316.
3. Steadman JR, Rodkey WG, Rodrigo JJ, et al. Microfracture: surgical technique and rehabilitation to treat chondral defects. Clin Orthop Relat Res, 2001, (391 Suppl): S362-369.
4. Kaul G, Cucchiarini M, Remberger K, et al. Failed cartilage repair for early osteoarthritis defects: a biochemical, histological and immunohistochemical analysis of the repair tissue after treatment with marrow-stimulation techniques. Knee Surg Sports Traumatol Arthrosc, 2012, 20(11): 2315-2324.
5. Breinan HA, Martin SD, Hsu HP, et al. Healing of canine articular cartilage defects treated with microfracture, a type-II collagen matrix, or cultured autologous chondrocytes. J Orthop Res, 2000, 18(5): 781-789.
6. Milano G, Sanna Passino E, Deriu L, et al. The effect of platelet rich plasma combined with microfractures on the treatment of chondral defects: an experimental study in a sheepmodel. Osteoarthritis Cartilage, 2010, 18(7): 971-980.
7. Zantop T, Petersen W. Arthroscopic implantation of a matrix to cover large chondral defect during microfracture. Arthroscopy, 2009, 25(11): 1354-1360.
8. Lu H, Hoshiba T, Kawazoe N, et al. Autologous extracellular matrix scaffolds for tissue engineering. Biomaterials, 2011, 32(10): 2489-2499.
9. Jin CZ, Choi BH, Park SR, et al. Cartilage engineering using cell-derived extracellular matrix scaffold in vitro. J Biomed Mater Res A, 2010, 92(4): 1567-1577.
10. Kang SW, Lee SJ, Kim JS, et al. Effect of a scaffold fabricated thermally from acetylated PLGA on the formation of engineered cartilage. Macromol Biosci, 2011, 11(2): 267-274.
11. 莫湘濤, 鄧力, 李秀群, 等. 軟骨細胞-海藻酸鈉水凝膠-SIS復合體修復全層關節軟骨缺損的實驗研究. 中國修復重建外科雜志, 2009, 23(8): 974-979.
12. Christensen BB, Foldager CB, Hansen OM, et al. A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit?model compared to a collagen type I/III scaffold: in vitro and in vivo studies. Knee Surg Sports Traumatol Arthrosc, 2012, 20(6): 1192-1204.
13. Kirn-Safran C, Farach-Carson MC, Carson DD. Multifunctionality of extracellular and cell surface heparan sulfate proteoglycans. Cell Mol Life Sci, 2009, 66(21): 3421-3434.
14. Jin RL, Park SR, Chio BH, et al. Scaffold-free cartilage system using passaged porcine chondrocytes and basic fibroblast growth factor. Tissue Eng Part A, 2009, 15(8): 1887-1895.
15. Jin CZ, Park SR, Choi BH, et al. In vivo cartilage tissue engineering using a cell-derived extracellular matrix scaffold. Artif Organs, 2007, 31(3): 183-192.
16. Jin CZ, Cho JH, Choi BH, et al. The maturity of tissue-engineered cartilage in vitro affects the repairability for osteochondral defect. Tissue Eng Part A, 2011, 17(23-24): 3057-3065.
17. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411): 143-147.
18. Lu H, Hoshiba T, Kawazoe N, et al. Cultured cell-derived extracellular matrix scaffolds for tissue engineering. Biomaterials, 2011, 32(36): 9658-9666.
19. Discher DE, Mooney DJ, Zandstra PW. Growth factors, matrices, and forces combine and control stem cells. Science, 2009, 324(5935): 1673-1677.
20. Fortier LA, Potter HG, Rickey EJ, et al. Concentrated bone marrow aspirate improves full-thickness cartilage repair compared with microfracture in the equine model. J Bone Joint Surg (Am), 2010, 92(10) : 1927-1937.
21. Yang HS, La WG, Bhang SH, et al. Hyaline cartilage regeneration by combined therapy of microfracture and long-term bone morphogenetic protein-2 delivery. Tissue Eng Part A, 2011, 17(13-14): 1809-1818.
22. Kuo AC, Rodrigo JJ, Reddi AH, et al. Microfracture and bone morphogenetic protein 7 (BMP-7) synergistically stimulate articular cartilage repair. Osteoarthritis Cartilage, 2006, 14(11): 1126-1135.
23. Hoemann CD, Sun J, McKee MD, et al. Rabbit hyaline cartilage repair after marrow stimulation depends on the surgical approach and a chitosan-GP stabilised in situ blood clot. Trans Orthop Res Soc, 2005, 30: 1372.
24. Thorpe SD, Buckley CT, Vinardell T. The response of bone marrow-derived mesenchymal stem cells to dynamic compression following TGF-β3 induced chondrogenic differentiation. Ann Biomed Eng, 2010, 38(9): 2896-2909.

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BMSCs來源細胞外基質支架對微骨折骨髓刺激術后血凝塊體外軟骨化分化的作用研究

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《中國修復重建外科雜志》

BMSCs來源細胞外基質支架對微骨折骨髓刺激術后血凝塊體外軟骨化分化的作用研究

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