猪源性异种骨支架材料的生物相容性

Biocompatibility of heterogeneous bone derived from pigs as scaffold materials

目的:异种骨来源广泛,价格低廉,经过适当处理既能保留骨诱导作用,并作为骨支架使其具有骨传导作用。检测猪源性骨支架材料与人源性骨支架材料的生物相容性有无明显差异,为猪源性骨支架材料取代人源性骨支架材料提供实验依据。方法:实验于2006-08/2007-03在南方医科大学人体解剖学系实验室(广东省组织构建与检测重点实验室)完成。①实验材料:人源性、猪源性骨支架材料(为课题组前期制备);脂肪源间充质细胞(前期培养)。②实验方法及评估:体外细胞毒性实验:提取人源性、猪源性骨支架材料浸提液,用此浸提液培养脂肪源间充质细胞,并用无浸提液的DMEM培养基做对照,镜下观察细胞生长状况,流式细胞术检测细胞生长周期;人源性、猪源性骨支架材料皮下植入试验:于8只新西兰大白兔皮下,脊柱左侧植入异种骨支架材料,右侧植入同种异体骨支架材料。分别于4,8,12,16周将植入材料连同周围组织取出,进行组织切片观察及电镜观察。对动物处置符合动物伦理学标准。结果:①体外细胞毒性实验结果:在同一时间点,浸提液组、正常培养组细胞生长状况均良好,未见明显细胞抑制;流式细胞术检测细胞周期,3组细胞G1、G2、S期细胞百分率差异无统计学意义(P>0.05)。②皮下植入实验结果:组织切片光镜及电镜观察,4周两种植入材料周围均见结缔组织包绕,有大量巨噬细胞、浆细胞、中性粒细胞、单核细胞浸润,材料表面光滑,与结缔组织界限清楚;8周时炎症反应减轻,可见部分材料开始降解,材料表面稍显粗糙,有少量结缔组织长入材料内部;12周炎症反应基本消失、材料降解明显、异位成骨趋势明显,材料表面更加粗糙,周围有大量结缔组织长入材料;16周均无炎症反应,两种材料大部分降解,并且猪源性骨支架材料降解更为迅速,均有新骨形成,材料表面粗糙程度较12周时为甚,骨支架材料基本消失。结论:猪源性异种骨支架材料有良好的生物相容性,与人源性异种骨支架材料无明显差别。

AIM： Heterogeneous bone, which is widespread and cheap, can be applied as scaffold materials due to its capacities of bone induction and bone conduction. This study was designed to provide experimental evidences for using heterogeneous bone scaffold material to replace homogeneous bone scaffold material by detecting the difference in biocompatibility between homogeneous bone scaffold derived from human and heterogeneous bone scaffold from pigs. METHODS： The experiment was performed at the Department of Human Anatomy, Southern Medical University （Guangdong Province Key Laboratory of Tissue Construction and Detection） from August 2006 to March 2007. ① Participants/materials： Human-derived homogeneous bone scaffold material, pig-derived heterogeneous bone scaffold material, and adipose-derived mesenchymal stem cells （ADMSCs）. ②Methods and evaluations： Experiment of cytotoxicity in vitror. Scaffold materials extracted from human-derived and pig-derived bone were used to culture ADMSCs in leaching liquor. While those in DMEM without leaching liquor were served as controls. Then the growth of cells was observed through inverted microscope. And the cell circle was detected by flow cytometry. Experiment of subcutaneous implantation in viva. Two kinds of bone scaffold materials were implanted in eight New Zealand rabbits subcutaneously, one kind for either side. At 4, 8, 12, 16 weeks after implantation, the materials were dislodged with surrounding tissues, then the obtained tissue sections were observed under light microscope and electron microscopy. All the experimental procedures were consistent with the ethnical standard for animals. RESULTS： ①In vitro cytotoxicity experiment： Cells cultured in normal nutritive medium and extract leaching liquor were both good, and no significant inhibition was detected; There was no significant difference in cell cycle among three groups of G1, G2 and S phases. ②In vivo subcutaneous implantation experiment： Under the light microscope and electron microscope at the fourth week, there were connective tissues, and a lot of macrophages, plasma cells, neutrophils, monocyte infiltrating around the two kinds of implants, the surfaces of which were smooth, and boundaries with connective tissue were clear; at the eighth week, inflammation was relieved. The materials were starting to degrade and surfaces were slightly rough, with small amounts of connective tissues ingrew into the materials. At the twelfth week, inflammation disappeared, material degradation became obvious, and ectopic bone was formed apparently. Surface became rougher, and a large number of surrounding connective tissues ingrew into the materials; at the sixteenth week, no inflammatory reaction was found, two kinds of materials degraded mostly, and pig-derived bone scaffold degraded more rapidly. With new bone formation, surfaces were rougher than that at the twelfth week, and there was not integrity implants. CONCLUSION： Xenotransplantation of pig-derived bone scaffolds and human-derived bone scaffolds both have good biocompatibility, and there are no significant differences between two kinds of xenogeneic bone scaffolds.