犬成肌細胞復合聚乳酸聚乙酸共聚物支架在裸鼠體內異位成軟骨的實驗研究_《中國修復重建外科雜志》

作者：

顧羊林 ¹ , 王予彬 ¹ , 施克勤 ¹ , 楊玉生 ¹ , 陳鵬 ¹ , 朱文輝 ² , 朱國興 ¹

1南京醫科大學附屬無錫第二醫院骨科（江蘇無錫，214002）;;
2同濟大學附屬東方醫院運動創傷關節外科;

關鍵詞：

組織工程軟骨成肌細胞聚乳酸聚乙酸共聚物犬裸鼠

DOI：

10.7507/1002-1892.20130166

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目的探討與聚乳酸聚乙酸共聚物[poly（lactide-co-glycolide），PLGA]支架復合后的成肌細胞體外經軟骨來源形成蛋白2（cartilage-derived morphogenetic protein 2，CDMP-2）聯合TGF-β1誘導后，能否在裸鼠體內異位構建組織工程軟骨。方法取1歲齡雄性比格犬股直肌肌肉分離培養成肌細胞，取第4代成肌細胞接種于PLGA支架，加入含CDMP-2與TGF-β1的軟骨誘導液體外培養2周。實驗分為4組：A組為經軟骨誘導的成肌細胞-PLGA復合物組，B組為未經軟骨誘導的成肌細胞-PLGA復合物組，C組為經軟骨誘導的單純PLGA支架組，D組為單純PLGA支架組。于24只裸鼠背部皮下兩側制備4個皮下腔隙，分別植入4組材料。術后8、12周處死裸鼠，取材行大體觀察、HE及甲苯胺藍染色、Ⅰ型膠原及Ⅱ型膠原免疫組織化學染色觀察，RT-PCR檢測Ⅰ型膠原、Ⅱ型膠原、蛋白聚糖（Aggrecan）、Sox9 mRNA表達，阿利新藍染色檢測糖胺聚糖（glycosaminoglycans，GAG）含量，生物力學試驗檢測標本壓縮彈性模量。結果A組標本術后8周呈類軟骨樣，乳白色，表面光潔，有彈性；12周時外觀和彈性與正常軟骨相似。B組術后8周僅殘余少許組織，12周已完全降解；C、D組標本術后8周均降解消失。HE染色示A組8、12周時有成熟軟骨陷窩形成；B組8周時組織內未見軟骨陷窩形成。甲苯胺藍染色示A組8、12周時組織內新生軟骨細胞形成，細胞橢圓，排列整齊，細胞陷窩形成，細胞質和細胞外基質均藍染陽性；B組8周時組織內未見藍染的細胞外基質。Ⅰ、Ⅱ型膠原免疫組織化學染色示A組8、12周時均呈陽性表達；B組8周時呈陰性表達。RT-PCR檢測示術后8、12周A組均可見Ⅰ型膠原、Ⅱ型膠原、Aggrecan和Sox9 mRNA表達，B組均呈陰性。術后12周A組壓縮彈性模量為（90.79 ± 1.78） MPa，GAG含量為（10.20 ± 1.07）μg/mL與正常半月板相比，差異有統計學意義（P lt; 0.05）。結論體外CDMP-2聯合TGF-β1軟骨誘導的比格犬成肌細胞復合PLGA支架后具備在裸鼠體內異位構建組織工程軟骨的能力。

引用本文： 顧羊林,王予彬,施克勤,楊玉生,陳鵬,朱文輝,朱國興. 犬成肌細胞復合聚乳酸聚乙酸共聚物支架在裸鼠體內異位成軟骨的實驗研究. 中國修復重建外科雜志, 2013, 27(6): 749-754. doi: 10.7507/1002-1892.20130166 復制

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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.
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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. 顧羊林, 朱文輝, 王予彬, 等. 犬成肌細胞體外向纖維軟骨細胞表型分化的實驗研究. 同濟大學學報: 醫學版, 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.

《中國修復重建外科雜志》

犬成肌細胞復合聚乳酸聚乙酸共聚物支架在裸鼠體內異位成軟骨的實驗研究

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《中國修復重建外科雜志》

犬成肌細胞復合聚乳酸聚乙酸共聚物支架在裸鼠體內異位成軟骨的實驗研究

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