胰蛋白酶法和Triton X-100法脱猪软骨细胞的比较

Trypsin versus Triton X-100 for decellularization of porcine articular cartilage

背景:作为修复软骨缺损的一种材料,脱细胞软骨基质已受到越来越多研究者的关注,但是制备脱细胞软骨基质的两种主要方法其脱细胞效果孰优孰劣尚不清楚。目的:对比胰蛋白酶法和Triton X-100法脱猪关节软骨细胞的效果。设计、时间及地点:对比观察,于2008-03/07在山西医科大学第二医院骨科中心实验室完成。材料:新鲜猪膝、髋关节软骨市场上购得。方法:选取新鲜猪关节软骨片分别置入2.5g/L胰酶溶液和2.5g/LTriton X-100溶液中脱细胞处理,并以未经处理的新鲜软骨片作对照。主要观察指标:①大体观察脱细胞后关节软骨的外观形态。②免疫组织化学染色及扫描电镜下观察两种脱细胞方式对细胞外基质胶原的影响。③每组随机选取10片软骨片进行加载、卸载试验和拉断试验,测定断裂强度和断裂拉伸率。结果:①经过两种方法脱细胞后,软骨脱细胞基质在外观上与未脱细胞软骨差别甚微,只是颜色较软骨略白,触摸较未脱细胞基质柔软。②免疫组织化学染色及扫描电镜可见,胰酶法和Triton X-100法均彻底脱去了关节软骨上的细胞成分,胰酶法观察到胶原排列有序,纹理清晰,可见胰酶在脱细胞的同时对细胞外基质没有明显的破坏;Triton X-100法观察到胶原仍比较多,但纹理结构有些紊乱,可能是Triton X-100法脱细胞对细胞外基质进行了破坏。③新鲜关节软骨组、胰酶组和Triton X-100组断裂强度和断裂拉伸率相比,差异均无显著性意义。结论:两种方法均彻底脱去了关节软骨上的细胞成分,脱胰酶法脱猪关节软骨细胞的方法作用时间较短,胶原保存好,且成本较低。Triton X-100法脱细胞步骤较多,时间较长,对胶原结构有轻微的破坏,但对软骨的力学性能没有显著影响。

BACKGROUND： As a material to repair osteochondral defect, the acellular cartilage matrix has attracted more and more researchers＇ attention, however, there is no comparison between two ways of decellularization. OBJECTIVE： To test trypsin and Triton X-100 methods for their potential of cell removal. DESIGN, TIME AND SETTING： Controlled observation was performed at the Central Laboratory of Orthopaedics in the Second Hospital of Shanxi Medical University between March and July in 2008. MATERIALS： Fresh knee and hip articular cartilages of pig were purchased from the market. METHODS： Porcine articular cartilages were treated with either 0.25% trypsin or 0.25% Triton X-100 respectively for decellularization, while fresh untreated articular cartilage served as control. MAIN OUTCOME MEASURES： ①3eneral observation on the morphology of acellular articular cartilage. ②lmmunohistochemistry and scanning electron microscopy were used to determine the effect of two decelluladzation methods on the extracellular matrix collagen. ③Ten articular cartilage leaflets in each group were measured for breaking strength and percentage elongation through loading, unloading and breaking tests. RESULTS：①By use of trypsin and Triton X-100 methods, the acellular matrix had no significance difference in the morphology compared with untreated cartilage, only presenting slightly white color and feeling less soft. ②lmmunohistochemistry and scanning electron microscopy results showed that, trypsin and Triton X-100 methods achieved complete decellularization. Trypsin had no significant influence in extracellular matdx and cartilage leaflets microstructure; Triton X-100 caused some tiny structural alterations, such as more collagen and disordered structure. It is possibly that Triton X-100 damaged extracellular matrix. ③No significant difference was identified between untreated and trypsin, Triton X-100 groups in breaking strength and percentage elongation. CONCLUSION： Trypsin and Triton X-100 all achieve complete decellularization, but trypsin uses short time and low costs with well preservation, while Triton X-100 with long procedure and slight damage to collagen structure. They all have no significance influence to the mechanics characteristics of articular cartilage.