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 with α-MEM medium [containing 10% fetal bovine serum (FBS) and 1% double antibody] containing 2%, 5%, and 10%CGFe, respectively; group D was cultured with α-MEM medium (containing 10%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 activity was proportional to the osteogenic induction time and CGFe concentration ( P<0.05). Western blot analysis of osteogenic induction culture for 14 days showed that the relative expression of OPN protein in groups A, B, and C was significantly higher than that in group D, and the higher the CGFe concentration, the higher the relative expression of OPN protein ( P<0.05). In vivo experiment: Micro-CT observation showed that the new bone formation and material degradation of the experimental group were better than those of the control group at 4, 8, and 12 weeks after operation. Quantitative detection showed that the volume of new bone volume, new bone volume fraction, trabeculae number, and trabecular thickness of the experimental group were significantly higher than those of the control group at each time point, the residual material volume, residual material volume fraction, and trabecular separation were significantly lower than those of the control group, all showing significant differences ( P<0.05).
Conclusion: CGF can effectively promote the adhesion, proliferation, and osteogenic differentiation of BMSCs on MC materials, and 10%CGFe has the most significant effect. The combined application of CGF and MC material can significantly promote bone formation in vivo.
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。CGF 和 MC 材料联合应用可显著促进体内成骨。.
Keywords: Concentrated growth factor; bone marrow mesenchymal stem cells; mineralized collagen; osteogenic differentiation.