犬成肌细胞复合聚乳酸聚乙酸共聚物支架在裸鼠体内异位成软骨的实验研究_《中国修复重建外科杂志》

作者：

顾羊林 ¹ , 王予彬 ¹ , 施克勤 ¹ , 杨玉生 ¹ , 陈鹏 ¹ , 朱文辉 ² , 朱国兴 ¹

1南京医科大学附属无锡第二医院骨科（江苏无锡，214002）;;
2同济大学附属东方医院运动创伤关节外科;

关键词：

组织工程软骨成肌细胞聚乳酸聚乙酸共聚物犬裸鼠

DOI：

10.7507/1002-1892.20130166

视频：

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目的探讨与聚乳酸聚乙酸共聚物[poly（lactide-co-glycolide），PLGA]支架复合后的成肌细胞体外经软骨来源形成蛋白2（cartilage-derived morphogenetic protein 2，CDMP-2）联合TGF-β1诱导后，能否在裸鼠体内异位构建组织工程软骨。方法取1岁龄雄性比格犬股直肌肌肉分离培养成肌细胞，取第4代成肌细胞接种于PLGA支架，加入含CDMP-2与TGF-β1的软骨诱导液体外培养2周。实验分为4组：A组为经软骨诱导的成肌细胞-PLGA复合物组，B组为未经软骨诱导的成肌细胞-PLGA复合物组，C组为经软骨诱导的单纯PLGA支架组，D组为单纯PLGA支架组。于24只裸鼠背部皮下两侧制备4个皮下腔隙，分别植入4组材料。术后8、12周处死裸鼠，取材行大体观察、HE及甲苯胺蓝染色、Ⅰ型胶原及Ⅱ型胶原免疫组织化学染色观察，RT-PCR检测Ⅰ型胶原、Ⅱ型胶原、蛋白聚糖（Aggrecan）、Sox9 mRNA表达，阿利新蓝染色检测糖胺聚糖（glycosaminoglycans，GAG）含量，生物力学试验检测标本压缩弹性模量。结果A组标本术后8周呈类软骨样，乳白色，表面光洁，有弹性；12周时外观和弹性与正常软骨相似。B组术后8周仅残余少许组织，12周已完全降解；C、D组标本术后8周均降解消失。HE染色示A组8、12周时有成熟软骨陷窝形成；B组8周时组织内未见软骨陷窝形成。甲苯胺蓝染色示A组8、12周时组织内新生软骨细胞形成，细胞椭圆，排列整齐，细胞陷窝形成，细胞质和细胞外基质均蓝染阳性；B组8周时组织内未见蓝染的细胞外基质。Ⅰ、Ⅱ型胶原免疫组织化学染色示A组8、12周时均呈阳性表达；B组8周时呈阴性表达。RT-PCR检测示术后8、12周A组均可见Ⅰ型胶原、Ⅱ型胶原、Aggrecan和Sox9 mRNA表达，B组均呈阴性。术后12周A组压缩弹性模量为（90.79 ± 1.78） MPa，GAG含量为（10.20 ± 1.07）μg/mL与正常半月板相比，差异有统计学意义（P lt; 0.05）。结论体外CDMP-2联合TGF-β1软骨诱导的比格犬成肌细胞复合PLGA支架后具备在裸鼠体内异位构建组织工程软骨的能力。

引用本文： 顾羊林,王予彬,施克勤,杨玉生,陈鹏,朱文辉,朱国兴. 犬成肌细胞复合聚乳酸聚乙酸共聚物支架在裸鼠体内异位成软骨的实验研究. 中国修复重建外科杂志, 2013, 27(6): 749-754. doi: 10.7507/1002-1892.20130166 复制

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9.	Freymann U, Endres M, Neumann K, et al. Expanded human meniscus-derived cells in 3-D polymer-hyaluronan scaffolds for meniscus repair. Acta Biomater, 2012, 8(2): 677-685.
10.	Koning M, Harmsen MC, van Luyn MJ, et al. Current opportunities and challenges in skeletal muscle tissue engineering. J Tissue Eng Regen Med, 2009, 3(6): 407-415.
11.	Liu Y, Li C, Wang HY, et al. Synthesis of Thermo- and pH-Sensitive Polyion Complex Micelles for Fluorescent Imaging. Chemistry, 2012, 18(8): 2297-2304.
12.	Asadi H, Rostamizadeh K, Salari D, et al. Preparation and characterization of tri-block poly (lactide)-poly (ethylene glycol)-poly (lactide) nanogels for controlled release of naltrexone. Int J Pharm, 2011, 416(1): 356-364.
13.	Singh D, Nayak V, Kumar A. Proliferation of myoblast skeletal cells on three-dimensional supermacroporous cryogels. Int J Biol Sci, 2010, 6(4): 371-381.
14.	Steinert AF, Ghivizzani SC, Rethwilm A, et al. Major biological obstacles for persistent cell-based regeneration of articular cartilage. Arthritis Res Ther, 2007, 9(3): 213.
15.	Tian H, Yang S, Xu L, et al. Chondrogenic differentiation of mouse bone marrow mesenchymal stem cells induced by cartilage-derived morphogenetic protein-2 in vitro. J Huazhong Univ Sci Technolog Med Sci, 2007, 27(4): 429-432.
16.	Bobick BE, Chen FH, Le AM, et al. Regulation of the chondrogenic phenotype in culture. Birth Defects Res C Embryo Today, 2009, 87(4): 351-371.
17.	Garg T, Singh O, Arora S, et al. Scaffold: a novel carrier for cell and drug delivery. Crit Rev Ther Drug Carrier Syst, 2012, 29(1): 1-63.
18.	Subhash HM, Xie H, Smith JW, et al. Optical detection of indocyanine green encapsulated biocompatible poly (lactic-co-glycolic) acid nanoparticles with photothermal optical coherence tomography. Opt Lett, 2012, 37(5): 981-983.
19.	Espandar L, Bunnell B, Wang GY, et al. Adipose-derived stem cells on hyaluronic Acid-derived scaffold: a new horizon in bioengineered cornea. Arch Ophthalmol, 2012, 130(2): 202-208.
20.	Cui L, Wu Y, Cen L, et al. Repair of articular cartilage defect in non-weight bearing areas using adipose derived stem cells loaded polyglycolic acid mesh. Biomaterials, 2009, 30(14): 2683-2693.

1. Kalff S, Meachem S, Preston C. Incidence of medial meniscal tears after arthroscopic assisted tibial plateau leveling osteotomy. Vet Surg, 2011, 40(8): 952-956.
2. Gu YL, Wang YB. Treatment of meniscal injury: a current concept review. Chin J Traumatol, 2010, 13(6): 370-376.
3. Eli N, Oragui E, Khan W. Advances in meniscal tissue engineering. Ortop Traumatol Rehabil, 2011, 13(4): 319-326.
4. 顾羊林, 朱文辉, 王予彬, 等. 犬成肌细胞体外向纤维软骨细胞表型分化的实验研究. 同济大学学报: 医学版, 2012, 33(2): 1-6.
5. Gu Y, Wang Y, Dai H, et al. Chondrogenic differentiation of canine myoblasts induced by cartilage-derived morphogenetic protein-2 and transforming growth factor-β1 in vitro. Mol Med Rep, 2012, 5(3): 767-772.
6. Asakura A, Komaki M, Rudnicki M. Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic and adipogenic differentiation. Differentiation, 2001, 68(4-5): 245-253.
7. Matsushita T, Matsui N, Fujioka H, et al. Expression of transcription factor Sox9 in rat L6 myoblastic cells. Connect Tissue Res, 2004, 45(3): 164-173.
8. Tuli R, Seghatoleslami MR, Tuli S, et al. p38 MAP kinase regulation of AP-2 binding in TGF-beta1-stimulated chondrogenesis of human trabecular bone-derived cells. Ann NY Acad Sci, 2002, 961: 172-177.
9. Freymann U, Endres M, Neumann K, et al. Expanded human meniscus-derived cells in 3-D polymer-hyaluronan scaffolds for meniscus repair. Acta Biomater, 2012, 8(2): 677-685.
10. Koning M, Harmsen MC, van Luyn MJ, et al. Current opportunities and challenges in skeletal muscle tissue engineering. J Tissue Eng Regen Med, 2009, 3(6): 407-415.
11. Liu Y, Li C, Wang HY, et al. Synthesis of Thermo- and pH-Sensitive Polyion Complex Micelles for Fluorescent Imaging. Chemistry, 2012, 18(8): 2297-2304.
12. Asadi H, Rostamizadeh K, Salari D, et al. Preparation and characterization of tri-block poly (lactide)-poly (ethylene glycol)-poly (lactide) nanogels for controlled release of naltrexone. Int J Pharm, 2011, 416(1): 356-364.
13. Singh D, Nayak V, Kumar A. Proliferation of myoblast skeletal cells on three-dimensional supermacroporous cryogels. Int J Biol Sci, 2010, 6(4): 371-381.
14. Steinert AF, Ghivizzani SC, Rethwilm A, et al. Major biological obstacles for persistent cell-based regeneration of articular cartilage. Arthritis Res Ther, 2007, 9(3): 213.
15. Tian H, Yang S, Xu L, et al. Chondrogenic differentiation of mouse bone marrow mesenchymal stem cells induced by cartilage-derived morphogenetic protein-2 in vitro. J Huazhong Univ Sci Technolog Med Sci, 2007, 27(4): 429-432.
16. Bobick BE, Chen FH, Le AM, et al. Regulation of the chondrogenic phenotype in culture. Birth Defects Res C Embryo Today, 2009, 87(4): 351-371.
17. Garg T, Singh O, Arora S, et al. Scaffold: a novel carrier for cell and drug delivery. Crit Rev Ther Drug Carrier Syst, 2012, 29(1): 1-63.
18. Subhash HM, Xie H, Smith JW, et al. Optical detection of indocyanine green encapsulated biocompatible poly (lactic-co-glycolic) acid nanoparticles with photothermal optical coherence tomography. Opt Lett, 2012, 37(5): 981-983.
19. Espandar L, Bunnell B, Wang GY, et al. Adipose-derived stem cells on hyaluronic Acid-derived scaffold: a new horizon in bioengineered cornea. Arch Ophthalmol, 2012, 130(2): 202-208.
20. Cui L, Wu Y, Cen L, et al. Repair of articular cartilage defect in non-weight bearing areas using adipose derived stem cells loaded polyglycolic acid mesh. Biomaterials, 2009, 30(14): 2683-2693.

《中国修复重建外科杂志》

犬成肌细胞复合聚乳酸聚乙酸共聚物支架在裸鼠体内异位成软骨的实验研究

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《中国修复重建外科杂志》

犬成肌细胞复合聚乳酸聚乙酸共聚物支架在裸鼠体内异位成软骨的实验研究

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