脱细胞软骨生物支架材料与大鼠骨髓间充质干细胞共培养及体内埋植后的降解

Co-culture of bone marrow mesenchymal stem cells on acellular cartilage scaffold biomaterials and in vivo biodegradation of acellular cartilage biomaterials

背景：动物体内埋植实验被认为是最常用有效的检测生物相容性的方法。目的：拟观察脱细胞软骨生物支架材料与细胞共培养及体内埋植实验时的生物降解性。时间及地点：观察性实验,于2007-06/08在山西医科大学第二医院骨科实验室完成。材料：SD大鼠,体质量60-80g;6月龄猪由山西畜牧研究所提供。方法：①分离培养大鼠骨髓间充质干细胞,取猪膝关节软骨制备脱细胞关节软骨支架材料,将两者进行复合培养。未经与无细胞胶原基质联合培养的细胞作为正常对照。②将备好的生物支架材料植入大鼠背部皮下,于14,28d取材,对其进行大体及组织染色。主要观察指标：①对复合细胞的无细胞胶原基质进行石蜡切片常规染色,倒置显微镜下观察24,72h细胞与无细胞胶原基质共培养情况。②通过炎性细胞反应及纤维囊形成级别评定标准、材料降解结果评定合格标准分析植入材料在动物体内降解情况。结果：①倒置显微镜下观察与无细胞胶原基质共培养24h细胞多呈长梭形和多角形;72h后与无细胞胶原基质联合培养和未与无细胞胶原基质联合培养的2种细胞形态相似,均呈多角形和圆锥形。苏木精-伊红染色可见细胞嵌入软骨支架材料中,呈圆形和椭圆形,部分细胞可见细胞核,材料呈颗粒状,染色均一。②组织学观察可见试样周围存在少量淋巴细胞和嗜中性粒细胞,致密纤维囊壁将复合细胞的材料包裹,材料被降解为细小颗粒,细胞均匀散在材料中呈椭圆形,可见分裂相。结论：支架材料在与细胞共培养及体内埋植实验中均生长正常,经过4周材料顺利降解为细小颗粒。

BACKGROUND： The method of embedding in animals is considered to be the most effective pathway to detect the biocompatibility. OBJECTIVE： To investigate the biodegradation properties of acellular cartilage scaffold biomaterials in the co-culture with bone marrow mesenchyrnal stem cells （BMSCs） and the in vivo embedding. TIME AND SETTING： An observation was carded out in the laboratory of Orthopedics, the Second Hospital of Shanxi Medical University （Talyuan, Shanxi, China） from June to August in 2007. MATERIALS： SD rats weighing 60-80 g; 6-month pigs were offered by Shanxi Institute of Animal Husbandry. METHODS： The BMSCs were isolated from SD rats and then cultured. Pig knee articular cartilage was harvested to prepare acellular cartilage scaffold material, which was co-cultured with BMSCs. While cells cultured without acellular collagen matrix （ACM） were taken as normal controls. The resultant scaffold material was implanted into the dorsal skin of rats. Gross observation and histology were determined at 14 and 28 days. MAIN OUTCOME MEASURES： The ACM was routinely stained in paraffin sections, the co-culture of BMSCs on ACM was observed at 24 and 72 hours with inverted microscope. By means of inflammatory cells response, fibrous capsule formation and degradation standard, the biodegradation of the ACM was estimated after the ACM was hypodermically embedded in animals. RESULTS： Under inverted microscope, BMSCs co-cultured on the ACM were fusiform-shaped and polygonal at 24 hours, and the morphology was identical with the normal controlled cells at 72 hours, which presented polygon and cop. Hematoxylin-eosin stain revealed that, BMSCs in the ACM were oval and nucleoli appeared in a few cells. The ACM were decomposed into particulates. Histological observation displayed few lymphocytes and polymorphonuclear leucocytes around the ACM, and the ACM were encapsulated by compact fibrous capsule wall and were decomposed into small parts. CONCLUSION： BMSCs on the ACM develop in the course of nature and the ACM embedded hypodermically degrades into particles after four weeks.