三种方法构建去细胞肌肉生物支架:组织学和生物力学比较
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摘要
背景:目前构建去细胞肌肉生物支架的方法有化学方法、物理冻融法和冷冻法。目的:比较化学方法、物理冻融法和改良化学方法构建去细胞肌肉生物支架组织学和生物力学差异。方法:将6只SD大鼠竖脊肌截成36段,随机分3组(n=12),分别采用化学方法、物理冻融方法、改良化学方法构建去细胞肌肉生物支架。将荧光标记的骨髓间充质干细胞接种于3组支架上进行组织学与生物力学检测。结果与结论:苏木精-伊红染色显示3组支架均可见平行排列结构,化学方法组连续性较物理冻融组、改良化学组差;Masson染色显示3组支架结构主要为胶原纤维,物理冻融组残留一些肌纤维,间隙不均。物理冻融组第7天荧光标记种子细胞计数明显少于化学方法组、改良化学组(P<0.01),第14天物理冻融组少于改良化学组(P<0.05);扫描电镜可见细胞贴附各组支架生长、大量存活;物理冻融组最大载荷高于化学方法组(P<0.05),与改良化学组无明显差别,各组弹性模量无差异;物理冻融组孔隙率低于化学方法组、改良化学组(P<0.05)。表明改良化学方法可以兼顾支架的组织学和生物力学特性,彻底清除肌细胞,较多保留更完整的细胞外基质。
BACKGROUND: The methods of constructing acellular muscle bioscaffolds include chemical method,frozen/thawing method and freezing method.OBJECTIVE: To study the histological and biomechanical differences among the acellular muscle bioscaffolds prepared using chemical method, frozen/thawing method and reformed chemical method.METHODS: Totally 36 segments of the erector spinae from six Sprague-Dawley rats were randomized into three groups by different preparation methods (n=12): chemical method group (group A), frozen/thawing group (group B) and reformed chemical method group (group C). After sterilization and seeding of bone mesenchymal stem cells,the scaffolds from three groups were compared on histology and biomechanics.RESULTS AND CONCLUSION: Hematoxylin-eosin staining showed the internal structure was in parallel rows in the three group, but the continuity of scaffold structure in group A was less than groups B and C. Masson staining confirmed essential component of scaffolds was collagen fibers in the three group and some residual muscle fibers in group B with asymmetry clearance. The number of living seed cells labeled by fluorescence in group B was significantly less than that in groups A and C at 7 days (P < 0.01) and less than that in group C at 14 days (P < 0.05).Lots of cells attaching to each group scaffolds were flourished confirmed by scanning electron microscope. Group B exceeded group A in the maximum load of scaffold (P < 0.05) but not group C. There was no difference among three groups in modulus of elasticity. Porosity of group B was lower than that of groups A and C (P < 0.05). These findings indicate that the reformed chemical method is more ideal, which can balance the histology and biomechanics of acellular muscle bioscaffolds, remove muscle cells more thoroughly and maintain more extracellular matrix.
BACKGROUND: The methods of constructing acellular muscle bioscaffolds include chemical method,frozen/thawing method and freezing method.OBJECTIVE: To study the histological and biomechanical differences among the acellular muscle bioscaffolds prepared using chemical method, frozen/thawing method and reformed chemical method.METHODS: Totally 36 segments of the erector spinae from six Sprague-Dawley rats were randomized into three groups by different preparation methods (n=12): chemical method group (group A), frozen/thawing group (group B) and reformed chemical method group (group C). After sterilization and seeding of bone mesenchymal stem cells,the scaffolds from three groups were compared on histology and biomechanics.RESULTS AND CONCLUSION: Hematoxylin-eosin staining showed the internal structure was in parallel rows in the three group, but the continuity of scaffold structure in group A was less than groups B and C. Masson staining confirmed essential component of scaffolds was collagen fibers in the three group and some residual muscle fibers in group B with asymmetry clearance. The number of living seed cells labeled by fluorescence in group B was significantly less than that in groups A and C at 7 days (P < 0.01) and less than that in group C at 14 days (P < 0.05).Lots of cells attaching to each group scaffolds were flourished confirmed by scanning electron microscope. Group B exceeded group A in the maximum load of scaffold (P < 0.05) but not group C. There was no difference among three groups in modulus of elasticity. Porosity of group B was lower than that of groups A and C (P < 0.05). These findings indicate that the reformed chemical method is more ideal, which can balance the histology and biomechanics of acellular muscle bioscaffolds, remove muscle cells more thoroughly and maintain more extracellular matrix.
引文
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[10]Liu J,Xu GY.Riyong Huaxue Gongye.2003;33(1):29-32.刘静,徐桂英.表面活性剂与蛋白质相互作用的研究进展[J].日用化学工业,2003,33(1):29-32.
[11]Nguyen H,Morgan DA,Forwood MR.Sterilization of allograft bone:is25kGy the gold standard for gamma irradiation?J Cell Tissue Bank.2007;8(2):81-91.
[12]Balsly CR,Cotter AT,Williams LA,et al.Effect of low dose and moderate dose gamma irradiation on the mechanical properties of bone and soft tissue allografts.J Cell Tissue Bank.2008;9(4):289-298.
[2]Zhang XY.Jilin Daxue.2007.张秀英.肌基膜管移植大鼠脊髓半切损伤模型血管生成情况的研究[D].吉林大学,2007.
[3]Mligiliche N,Kitada M,Ide C.Grafting of detergent-denatured skeletal muscles provides effective conduits for extension of regenerating axons in the rat sciatic nerve.Arch Histol Cytol.2001;64(1):29-36.
[4]Arai T,Kanje M,Lundborg G,et al.Axonal outgrowth in muscle grafts made acellular by chemical extraction.Restor Neurol Neurosci.2000;17(4):165-174.
[5]Xu XZ,Sun L,Hu YY.Zhonghua Wuli Yixue yu Kangfu Zazhi.2000;22(2):2.徐新智,孙磊,胡蕴玉.肌基膜管桥接周围神经缺损的电生理研究[J].中华物理医学与康复杂志,2000,22(2):2.
[6]Keilhoff G,Pratsch F,Wolf G,et al.Bridging extra large defects of peripheral nerves:possibilities and limitations of alternative biological grafts from acellular muscle and Schwann cells.Tissue Eng.2005;11(7-8):1004-1014.
[7]Zhang HX,Xu SD,Wu X,et al.Zhonghua Guke Zazhi.1995;15(1):32-35.张红旭,胥少汀,吴霞,等.肌基膜管结合神经生长因子修复脊髓缺损的实验研究[J].中华骨科杂志,1995,15(1):32-35.
[8]Carlson EC,Carlson BM.A method for preparing skeletal muscle fiber basal laminae.Anat Rec.1991;230(3):325-331.
[9]9Wang DB,Wen YM,Lan X,et al.Zhongugo Jiaoxing Waike Zazhi.2010;18(10):836-839.汪大彬,文益民,蓝旭,等.壳聚糖-藻酸盐多通道支架材料细胞相容性研究[J].中国矫形外科杂志,2010,18(10):836-839.
[10]Liu J,Xu GY.Riyong Huaxue Gongye.2003;33(1):29-32.刘静,徐桂英.表面活性剂与蛋白质相互作用的研究进展[J].日用化学工业,2003,33(1):29-32.
[11]Nguyen H,Morgan DA,Forwood MR.Sterilization of allograft bone:is25kGy the gold standard for gamma irradiation?J Cell Tissue Bank.2007;8(2):81-91.
[12]Balsly CR,Cotter AT,Williams LA,et al.Effect of low dose and moderate dose gamma irradiation on the mechanical properties of bone and soft tissue allografts.J Cell Tissue Bank.2008;9(4):289-298.