改良型富血小板纤维蛋白与β⁃磷酸三钙复合物诱导成骨作用与机制

Study on the role and mechanism of osteogenesis induced by advanced platelet⁃rich fibrin andβ⁃tricalcium phosphate complex

目的探讨改良型富血小板纤维蛋白(advanced platelet⁃rich fibrin,A⁃PRF)与β⁃磷酸三钙(β⁃tricalcium phosphate,β⁃TCP)构成比例对兔股骨缺损模型骨形成的作用及机制,为临床治疗骨缺损提供新思路。方法24只新西兰大白兔构建骨缺损模型并分为模型组、1∶1复合物组(A⁃PRF∶β⁃TCP=1∶1)、2∶1复合物组(A⁃PRF∶β⁃TCP=2∶1)与4∶1复合物组(A⁃PRF∶β⁃TCP=4∶1),每组6只。复合物组在骨缺损处填充复合材料,模型组不充填材料,8周后安乐法处死动物采标本。采用micro⁃CT测定股骨缺损区新生骨的骨密度(bone mineral densi⁃ty,BMD)、骨体积分数(bone volume fraction,BV/TV)、骨小梁厚度(trabecular thickness,Tb.Th)、骨小梁分离度(trabecular separation/spacing,Tb.Sp)。HE染色观察新骨及新生血管形成情况;扫描电镜观察新生骨组织的形态变化;酶联免疫法检测新生股骨组织中磷酸化丝裂原活化蛋白激酶p38(phospho⁃p38 mitogen activated pro⁃tein kinase,p⁃p38MAPK)、CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer⁃binding protein homologous pro⁃tein,CHOP)、磷酸化含半胱氨酸的天冬氨酸蛋白水解酶3(phospho⁃cysteine aspartic protease⁃3,p⁃Caspase3)含量;实时定量PCR检测新生骨组织中骨保护素(osteoprotegerin,OPG)、骨形态发生蛋白⁃2(bone morphogenetic protein⁃2,BMP⁃2)、核因子κ⁃B配体受体致活剂(receptor activator of nuclear factor kappa⁃B ligand,RANKL)、p38MAPK的mRNA表达量;免疫组织化学法观察新生骨组织中OPG、BMP⁃2、RANKL、p⁃p38MAPK、p⁃Caspase3蛋白表达情况;荧光Tunel染色观察新生股骨组织中细胞凋亡情况。结果模型组股骨缺损区域骨形成缓慢,成骨质量差。与模型组相比,复合物组动物股骨缺损区域成骨情况良好,BMD、BV.TV、Tb.Th、Tb.N升高,Tb.Sp下降,新生股骨组织中见新生毛细血管形成;p⁃p38MAPK、CHOP、p⁃Caspase3蛋白表达降低,OPG、BMP⁃2的mRNA与蛋白表达升高,RANKL与p38MAPK的mRNA表达降低(P<0.05);各组新生骨组织中的凋亡检测表明2:1复合物组样本中细胞凋亡率最低(P<0.05);A⁃PRF∶β⁃TCP=2∶1的配比成骨效果最佳。结论由A⁃PRF与β⁃TCP组成复合物能促进OPG表达,抑制RANKL表达和p38MAPK磷酸化,减少新生骨组织细胞凋亡,促进成骨分化。

Objective To investigate the role and mechanism of bone formation caused by the ratio of advanced platelet⁃rich fibrin(A⁃PRF)andβ⁃tricalcium phosphate(β⁃TCP)in rabbit femur defect model,which provides a new idea for clinical treatment of bone defect.Methods Twenty⁃four New Zealand white rabbits were divided into model group,1∶1 complex group(A⁃PRF∶β⁃TCP=1∶1),2∶1 complex group(A⁃PRF∶β⁃TCP=2∶1)and 4∶1 complex group(A⁃PRF∶β⁃TCP=4∶1),with 6 rabbits in each group.Femoral defect models were constructed in each group.In the com⁃posite group,the bone defect was filled with composite material,while in the model group,no material was filled.After 8 weeks,the animals were euthanized and specimens were collected.Bone mineral density(BMD),bone volume fraction(BV/TV),trabecular thickness(Tb.Th),trabecular separation(Tb.SP)and trabecular number(Tb.N)in femoral defect tis⁃sue were measured by micro⁃CT and photographed.Hematoxylin⁃eosin staining was used to detect the pathological changes of new bone tissue.The morphological changes of the new bone tissue were observed by scanning electron mi⁃croscopy.Determination of phospho⁃mitogen activated protein kinase p38(p⁃p38MAPK),CCAAT/enhancer binding pro⁃tein homologous protein(CHOP)and phospho⁃cysteine aspartic protease⁃3(p⁃Caspase3)in newborn femur by ELISA.The mRNA expressions of osteoprotegerin(OPG),bone morphogenetic protein⁃2(BMP⁃2),receptor activator of nuclear factor kappa⁃B ligand(RANKL)and p38MAPK were detected by real⁃time quantitative PCR.The expression of OPG,BMP⁃2,RANKL,p⁃p38MAPK and p⁃Caspase3 protein in the new bone tissue was observed by immunohistochemistry.Results In the model group,bone formation in the femoral defect area was slow and osteogenic quality was poor.Com⁃pared with the model group,the bone formation and neocapillaries of femoral defect area in the complex group was good,BMD,BV.TV,Tb.Th,Tb.N were increased,and Tb.Sp were decreased,the expressions of p⁃p38MAPK,CHOP and p⁃Caspase3 were decreased,and the mRNA and protein expressions of OPG and BMP⁃2 were increased.The mRNA expression of RANKL and p38MAPK was decreased.Apoptosis in new bone tissue of each group showed the lowest apoptosis rate in samples of the 2∶1 complex group(P<0.05);A⁃PRF:β⁃TCP=2∶1 ratio has the best osteogenic effect.Conclusion The complex composed of A⁃PRF andβ⁃TCP can promote the expression of OPG,inhibit the expression of RANKL and phosphorylation of p38MAPK,reduce the apoptosis of new bone tissue cells,and promote osteogenic dif⁃ferentiation.