Beagle犬骨髓基质细胞复合A-PCPC裸鼠皮下成骨的实验研究

Experimental study of subcutaneous osteogenesis by bone marrow stromal cells with A-PCPC in Beagle dogs

目的:观察支架材料活性磷酸钙骨水泥(active porous calcium phosphate cement,A-PCPC)及其与Beagle犬骨髓基质细胞(bone marrow stromal cells,BMSCs)复合体在裸鼠体内的成骨性能。方法:抽取Beagle犬骨髓,体外分离培养、成骨诱导、扩增BMSCs并通过细胞化学方法检测其成骨表型。体外构建BMSCs/A-PCPC复合体,并行扫描电镜超微结构观察。将复合体植于裸鼠背部皮下,同期植入单纯A-PCPC作为对照组。术后2、4、8周取材,HE染色,进行组织学及组织形态计量学分析。结果:ALP染色阳性,VonKossa染色可见钙结节形成。扫描电镜可见细胞贴附于材料表面及孔隙表面生长。HE染色显示,2周时,复合体组可见少量编织骨,单纯材料组成骨较少。4周时,复合体组可见编织骨向小梁骨转化,单纯材料组可见大量骨样组织。8周时,复合体组新骨趋于成熟,单纯材料组骨成熟度较差。结论:A-PCPC支架接种BMSCs后显示良好的成骨活性,能够促进未成熟骨矿化,可以作为骨组织工程支架材料。

PURPOSE：To study the osteogenic capability of the construct combined dog＇s bone stromal cells with active porous calcium phosphate cement（A-PCPC） in nude mice in vivo.METHODS：Isolated bone marrow stromal cells（BMSCs） were expanded and osteogenically induced in vitro.Their osteogenic phenotype was evaluated by cytochemistry.The tissue engineering complex was constructed with BMSCs/A-PCPC in vitro.After SEM scanning,the complex of BMSCs/A-PCPC was implanted into the subcutaneous tissue of the nude mice as experimental group,and A-PCPC as control group.The engineered bone was harvested 2,4,8weeks post-implantation and processed for HE staining,then evaluated by histology and histomorphometry.RESULTS：Cytochemistry showed alkaline phosphate activity,Von Kossa staining proved the formation of mineralization nodules.Scanning electron microscopy showed the cells adhered to the inner surface of the A-PCPC.HE staining showed a small group of woven bone formation 2 weeks later in the experimental group,while the formation of bone less in the control group.Woven bone turned into trabecular bone gradually at 4 weeks in the experimental group,while the control group showed a large number of bone-like tissue.Histomorphometry showed more mature bone in the experimental group than the control group at 8 weeks.CONCLUSIONS：The A-PCPC/BMSCs composites show good osteogenetic activity and could promote mineralization of the immature bone.It can be used as the bone tissue engineering scaffolds.