成骨细胞特异性钙黏蛋白涂布脱钙骨基质材料对BMSCs黏附及成骨分化能力的影响  

EFFECTS OF DEMINERALIZED BONE MATRIX MODIFIED WITH TYPE II CADHERIN ECTODOMAIN ON ADHESION AND OSTEOGENIC DIFFERENTIATION OF BMSCs

作　　者：向强[1] 邓聪颖[2] 张远[1] 张超[1] 周跃[1] 

机构地区：[1]第三军医大学新桥医院骨科,重庆400037 [2]解放军第324医院神经外科

出　　处：《中国修复重建外科杂志》2009年第5期602-606,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30700892);重庆市自然科学基金资助项目(2007BB5058)~~

摘　　要：目的探讨成骨细胞特异性钙黏蛋白(cadherin ectodomainⅡ,Cad-Ⅱ)涂布于同种异体脱钙骨基质材料(freeze-dried demineralized bone matrix,FDBM)对兔BMSCs黏附、增殖及成骨分化能力的影响。方法取4周龄日本大耳白兔10只,体重0.61～0.88kg,雌雄不限,常规分离培养BMSCs。取第2代BMSCs(细胞密度1×106个/mL)分别与经Cad-Ⅱ修饰的FDBM(实验组)和未经Cad-Ⅱ修饰的FDBM(对照组)复合,行MTT检测细胞增殖能力,细胞黏附实验检测细胞上架率和上架数,倒置相差显微镜、扫描电镜及HE染色观察细胞生长情况。另取第2代BMSCs(细胞密度5×105个/mL)分别与经Cad-Ⅱ修饰的FDBM(实验组)和未经Cad-Ⅱ修饰的FDBM(对照组)复合培养,行ALP活性检测和骨钙素免疫组织化学染色观察细胞成骨分化情况。结果MTT检测发现BMSCs在经Cad-Ⅱ修饰的支架材料上能正常增殖,但与对照组比较差异无统计学意义(P>0.05)。实验组细胞上架率为87.41%±5.19%,明显高于对照组35.56%±1.75%(P<0.01);对照组每片材料细胞上架数平均为2.6×104个,实验组平均高达5.0×105个,明显多于对照组(P<0.05)。倒置相差显微镜、扫描电镜及HE染色观察均显示实验组支架上黏附细胞数量明显多于对照组。成骨诱导培养7d,实验组和对照组细胞均可表达骨钙素,具有成骨细胞表型;培养14d,实验组和对照组ALP活性分别为29.33±1.53和18.31±1.32,骨钙素免疫组织化学染色阳性率分别为83%±7%和56%±7%,两组比较差异均有统计学意义(P<0.01);与同组7、21d比较差异有统计学意义(P<0.01),7d与21d组间及组内比较差异均无统计学意义(P>0.05)。结论Cad-Ⅱ修饰的FDBM对BMSCs增殖无明显促进作用,但能提高细胞黏附性,并促进BMSCs向成骨细胞分化。Objective To evaluate the adhesion, proliferation and osteogenic differentiation of rabbit BMSCs after cultured on freeze-dried demineralized bone matrix（FDBM） modified with type II cadherin ectodomain（Cad-II）.Methods BMSCs isolated from 10 Japanese white rabbits（male and female, 4-week-old, 0.61-0.88 kg） were cultured.The second generation of BMSCs（cell density 1 × 10^6/mL） were seeded onto the Cad-II modi ed allogenic FDBM（experimental group） and only FDBM（control group） respectively, and then cocultured in vitro.The densities of seeded cells, the adhesion rate and their ALP activity were measured.The complex was observed through inverted phase contrast microscope and scanning electron microscope to evaluate the interaction between cells and FDBM.Another group of second generation of BMSCs（cell density 5 × 10^5/mL） were seeded onto the Cad-II modified FDBM（experimental group） and only FDBM（control group） respectively, and then cocultured in vitro too.The ALP activity and osteocalcin immunohistochemical was measured.Results There was no signi cant di erence in cell proliferation between experimental group and control group.The adhesion rate of cells in the experimental group was 87.41% ± 5.19%, higher than that in the the control group 35.56% ± 1.75%（P 〈 0.01）;the densities of seeded cells reached 5.0 × 10^5, showing signi cant di erence compared with the control group（2.6 × 10^4, P 〈 0.05）.Inverted phase contrast microscope showed that in the experimental group, more cultured BMSCs pasted in the hole and edge of the sca old than that in the control group.HE staining showed the densities of seeded cells in the experimental group was higher than that in the control group.Scanning electron microscope showed that in the experimental group, a lot of cultured BMSCs adhered, spreaded in the sca old, in the control group only a few BMSCs unevenly distributed in the sca old.After 7 days of culture, the cultured BMSCs on modi ed FDBM expressed higher ALP activity;afte

关 键 词：组织工程骨 钙黏蛋白 脱钙骨基质 BMSCS 黏附 成骨分化 兔 

分 类 号：R318.08[医药卫生—生物医学工程]