成骨细胞复合原位成型磷酸钙骨水泥的细胞学研究  

Cytological study on osteoblasts and in-situ setting calcium phosphate cements

作　　者：张洲[1] 乔朋艳[1] 肖军军[2] 董利民[3] 谢秋菲[1] 徐韬[4] 

机构地区：[1]北京大学口腔医学院·口腔医院修复科,北京100081 [2]北京大学基础医学院细胞生物学系,北京100191 [3]清华大学核能与新能源技术研究院,精细陶瓷北京市重点实验,北京100084 [4]北京大学口腔医学院·口腔医院预防科,北京100081

出　　处：《北京大学学报（医学版）》2011年第1期67-72,共6页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(30772447);北京大学医学部引进人才计划(bmu2009139)资助~~

摘　　要：目的:通过体外细胞培养实验,观察分析壳聚糖微球/磷酸钙骨水泥、β-磷酸三钙(β-tricalcium phosphate,β-TCP)/磷酸钙骨水泥、含钾磷酸钙骨水泥浆料固化过程对成骨细胞的影响以及固化后10 d细胞的生物活性变化。方法:首先以兔的骨髓为组织来源,采用密度梯度分离法和贴壁分离法分离培养原代兔骨髓基质细胞(rabbitmarrow stromal cell,rMSC),通过流式细胞分析和分选得到成分比较单一的兔骨髓基质细胞,经过体外诱导得到兔成骨细胞,用茜素红染色法验证其成骨功能。将rMSC分别与上述3种骨水泥固化块及其浆料复合培养,空白对照组是直接在24孔板上接种细胞,每组4个样本。在复合培养的第1、4、7、10天,通过酸性磷酸酶(acid phosphataseassay,APA)和碱性磷酸酶(alkaline phosphatase,ALP)活性测定,检测细胞的增殖和分化能力;吖啶橙(acridine or-ange,AO)染色后荧光显微镜观察,对细胞进行计数并分析。环境扫描电子显微镜观察细胞在材料表面的生长、黏附状况。各组结果进行双因素方差分析,用LSD法进行组间比较。结果:3种骨水泥浆料在固化过程中均对rMSC有较大影响,细胞的增殖活性(复合培养第10天APA活性浆料组吸光度平均值分别为0.049,0.050,0.049;固化块组分别为0.898,0.867,0.909;P<0.001)和分化能力(复合培养第10天ALP活性浆料组平均值分别为0.775,0.782,0.798 U/g protein;固化块组分别为49.288,49.631,49.744 U/g protein;P<0.001)均明显低于固化块组,细胞数目也明显减少(复合培养第10天细胞数目每视野平均为3.7,3.7,3.7个;固化块组分别为91.1,89.7,93.7个,P<0.001),且这种影响是不可恢复的;rMSC在3种骨水泥的固化块上能较好地黏附,细胞数目、增殖和分化能力基本不受影响。结论:可注射性磷酸钙骨水泥浆料固化过程对成骨细胞有较大的影响,用成骨细胞复合浆料前需对细胞采取保护措施。Objective： To evaluate biocompatibility of three kinds of self-developed injectable calcium phosphate cements（CPCs）： chitosan microspheres/CPC,β-tricalcium phosphate（β-TCP）/CPC,and K＋/CPC and the viability of the osteogenic cells cultured with CPC pastes and discs for 10 days.Methods： The rabbit marrow stromal cells（rMSCs）,isolated from rabbit bone marrow with density gradient centrifugation and flow cytometer,were cultured,expended and induced into osteoblasts.Alizarin red staining was used to determine the function of ossification.Then,rMSCs were incubated randomly on both the pastes and solidified discs of the 3 kinds of CPCs.The cells cultured on a 24-well plate were as blank control.Each group had 4 samples.The proliferation and differentiation of each group were observed using acid phosphatase assay（APA） and by testing the activity of alkaline phosphatase（ALP） at day 1,4,7,and 10.After stained by acridine orange（AO）,the cells were observed,counted and analyzed with an epifluorescence microscopy.The morphology of the cells on CPCs was observed with scanning electron microscope（SEM）.The data was subjected to two-way ANOVA followed by LSD test to compare between groups.Results： The process of solidification of the three kinds of CPC pastes has the toxic effect on cells,which is irreversible.The proliferation（the average absortion of pastes are 0.049,0.050,0.049;the discs are 0.898,0.867,0.909;P0.001）,function（the average ALP activity of pastes after ten days are 0.775,0.782,0.798 U/g protein;the discs are 49.288,49.631,49.744 U/g protein;P0.001） and number of cells（the average number of cells of pastes after ten days are 3.7,3.7,3.7;the discs are 91.1,89.7,93.7;P0.001） directly exposed to CPC paste significantly decreased compared with those contacting with the discs.By contrast,cells on the three kinds of discs showed better viability,proliferation,and ossification and cell numbers increased obviously with culture days.Conclusion： The process of solidifica

关 键 词：成骨细胞 磷酸钙类 骨代用品 

分 类 号：R68[医药卫生—骨科学]