浓缩生长因子联合矿化胶原材料对BMSCs黏附、增殖和成骨分化的影响及体内成骨效应  

Effect of concentrated growth factor combined with mineralized collagen material on the adhesion,proliferation,and osteogenic differentiation of bone marrow mesenchymal stem cells and the osteogenic effect in vivo

作　　者：张月 刘克达 闫明 王蔚[1] ZHANG Yue;LIU Keda;YAN Ming;WANG Wei(Comprehensive Department,School and Hospital of Stomatology,China Medical University,Liaoning Provincial Key Laboratory of Oral Diseases,Shenyang Liaoning,110002,P.R.China)

机构地区：[1]中国医科大学口腔医学院·附属口腔医院综合科辽宁省口腔疾病重点实验室,沈阳110002

出　　处：《中国修复重建外科杂志》2021年第3期295-302,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81970980);沈阳市中青年科技创新人才支持计划项目(RC200060);辽宁省重点研究计划指导计划项目(2018225078);辽宁省自然科学基金指导项目(2019-ZD-0749);沈阳市重大科技创新研发计划(19-112-4-027)。

摘　　要：目的探讨浓缩生长因子(concentrated growth factor,CGF)联合矿化胶原(mineralized collagen,MC)材料对BMSCs黏附、增殖和分化的影响及其体内成骨效应,为CGF和MC材料在骨缺损修复中的联合应用提供理论依据。方法取健康志愿者静脉血制成CGF,然后制备CGF提取液(CGF extracts,CGFe)。体外实验:取人BMSCs(human BMSCs,hBMSCs)分为4组,A、B、C组分别用含2%、5%、10%CGFe的α-MEM培养基(含10%FBS和1%双抗)培养;D组用不含CGFe的α-MEM培养基(含10%FBS和1%双抗)培养。扫描电镜观察CGFe对细胞黏附的影响,细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测CGFe对细胞增殖的影响,成骨诱导后行ALP活性检测及Western blot检测骨桥蛋白(osteopontin,OPN)表达。体内实验:取18只新西兰大耳兔,左、右侧下颌骨分别制备圆形骨缺损模型;分别植入自体静脉血制备的CGF凝胶+MC材料(体积比1∶1,实验组)和单纯MC材料(对照组)。术后4、8、12周分别处死6只兔取材,行Micro-CT扫描观察体内新骨形成及材料降解情况。结果体外实验:扫描电镜观察示A、B、C组细胞在MC材料上铺展情况优于D组,伪足较多;CCK-8法检测示不同浓度CGFe均能促进MC材料上细胞增殖,培养2、3、5、7 d细胞吸光度(A)值C组>B组>A组>D组(P<0.05);ALP活性检测示其活性与成骨诱导时间和CGFe浓度成正比(P<0.05);成骨诱导培养14 d Western blot检测示A、B、C组OPN蛋白相对表达量显著高于D组,且CGFe浓度越高,OPN蛋白相对表达量越高(P<0.05)。体内实验:Micro-CT观察示术后4、8、12周实验组新骨形成、材料降解均优于对照组。定量检测示,各时间点实验组新生骨体积、新生骨体积百分比、骨小梁数、骨小梁厚度均显著高于对照组,材料剩余体积、材料剩余体积百分比、骨小梁分离均显著低于对照组,差异有统计学意义(P<0.05)。结论CGF能有效促进MC材料上BMSCs的黏附、增殖及成骨分化,其中10%CGFe效果最显著�Objective To explore the effects of concentrated growth factor(CGF)combined with mineralized collagen(MC)materials on the adhesion,proliferation,and differentiation of bone marrow mesenchymal stem cells(BMSCs)and their osteogenic effects in vivo,and to provide a theoretical basis for the combined application of CGF and MC materials in bone defect regeneration and repair.Methods CGF was prepared from venous blood of healthy volunteers,and then CGF extracts(CGFe)were prepared.In vitro experiment:human BMSCs(hBMSCs)were divided into 4 groups.Groups A,B,and C were cultured withα-MEM medium[containing 10%fetal bovine serum(FBS)and 1%double antibody]containing 2%,5%,and 10%CGFe,respectively;group D was cultured withα-MEM medium(containing10%FBS and 1%double antibody)without CGFe.Scanning electron microscopy was used to observe the effect of CGFe on cell adhesion.Cell counting kit 8(CCK-8)was used to detect the effect of CGFe on cell proliferation.After osteogenic induction,alkaline phosphatase(ALP)activity was detected and Western blot was performed to detect osteopontin(OPN)expression.In vivo experiment:Eighteen New Zealand big-eared rabbits were used to prepare circular bone defect models on the left and right mandibles,and implant CGF gel(prepared from autologous venous blood)+MC material(volume ratio 1∶1,experimental group)and simple MC material(control group),respectively.At 4,8,and 12 weeks after operation,6 rabbits were sacrificed respectively to obtain materials,and Micro-CT scanning was performed to observe the formation of new bone and material degradation in vivo.Results In vitro experiments:Scanning electron microscopy showed that the cells of groups A,B,and C spread better on MC materials than group D,with more pseudopodia.CCK-8 method showed that different concentrations of CGFe could promote cell proliferation,and the absorbance(A)value of cells cultured for 2,3,5,and 7 days was in the order of group C>group B>group A>group D,the differences were significant(P<0.05).ALP activity test showed that its

关 键 词：浓缩生长因子 矿化胶原 BMSCS 成骨分化 

分 类 号：R318.08[医药卫生—生物医学工程]