载外源性TGF-β1甲基丙烯酰化明胶复合支架促进颅骨缺损修复实验研究

Study on the gelatin methacryloyl composite scaffold with exogenous transforming growth factorβto promote the repair of skull defects

目的通过外源性TGF-β1加载修饰甲基丙烯酰化明胶(gelatin methacryloyl,GelMA)水凝胶,制备可修复SD大鼠颅骨缺损的骨组织工程支架。方法首先制备含浓度为0、150、300、600、900、1200 ng/mL外源性TGF-β1的GelMA水凝胶复合支架(分别设为A、B、C、D、E、F组),运用细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测培养1、4、7 d各组支架对SD大鼠BMSCs增殖活性的影响;通过ALP染色、茜素红染色、骨钙素(osteocalcin,OCN)免疫荧光染色及Western blot检测探究对BMSCs成骨分化的影响,筛选最适浓度的TGF-β1/GelMA水凝胶支架。取36只8周龄SD大鼠制备5 mm直径颅骨骨缺损模型后随机均分为3组,分别为对照组、GelMA组和GelMA+TGF-β1组(采用最适浓度TGF-β1/GelMA水凝胶支架),术后4、8周分别处死大鼠行micro-CT、HE染色和OCN免疫组织化学染色观察颅骨缺损修复效果。结果CCK-8法检测示各组TGF-β1/GelMA水凝胶支架对BMSCs增殖均有促进作用,D组作用最强,细胞活性显著高于其余各组(P<0.05)。ALP染色、茜素红染色、OCN免疫荧光染色和Western blot检测结果表明,D组BMSCs的ALP阳性面积百分比、茜素红阳性面积百分比、ALP和OCN蛋白相对表达量均显著高于其余各组(P<0.05),成骨作用最强。因此体外实验筛选出TGF-β1/GelMA水凝胶支架最适浓度为600 ng/mL。大鼠颅骨缺损修复实验micro-CT、HE染色和OCN免疫组织化学染色示,术后4、8周,TGF-β1+GelMA组新生骨质最多,新生骨组织和骨体积/组织体积比值均显著高于GelMA组和对照组(P<0.05)。结论浓度为600 ng/mL的TGF-β1/GelMA水凝胶支架具有促BMSCs成骨分化作用,能够显著促进颅骨缺损部位骨再生,可作为生物活性材料应用于骨组织再生。

Objective To prepare a bone tissue engineering scaffold for repairing the skull defect of Sprague Dawley(SD)rats by combining exogenous transforming growth factorβ1(TGF-β1)with gelatin methacryloyl(GelMA)hydrogel.Methods Firstly,GelMA hydrogel composite scaffolds containing exogenous TGF-β1 at concentrations of 0,150,300,600,900,and 1200 ng/mL(set to groups A,B,C,D,E,and F,respectively)were prepared.Cell counting kit 8(CCK-8)method was used to detect the effect of composite scaffold on the proliferation of bone marrow mesenchymal stem cells(BMSCs)in SD rats.ALP staining,alizarin red staining,osteocalcin(OCN)immunofluorescence staining,and Western blot were used to explore the effect of scaffolds on osteogenic differentiation of BMSCs,and the optimal concentration of TGF-β1/GelMA scaffold was selected.Thirty-six 8-week-old SD rats were taken to prepare a 5 mm diameter skull bone defect model and randomly divided into 3 groups,namely the control group,the GelMA group,and the GelMA+TGF-β1 group(using the optimal concentration of TGF-β1/GelMA scaffold).The rats were sacrificed at 4 and8 weeks after operation,and micro-CT,HE staining,and OCN immunohistochemistry staining were performed to observe the repair effect of skull defects.Results The CCK-8 method showed that the TGF-β1/GelMA scaffolds in each group had a promoting effect on the proliferation of BMSCs.Group D had the strongest effect,and the cell activity was significantly higher than that of the other groups(P<0.05).The results of ALP staining,alizarin red staining,OCN immunofluorescence staining,and Western blot showed that the percentage of ALP positive area,the percentage of alizarin red positive area,and the relative expressions of ALP and OCN proteins in group D were significantly higher than those of the other groups(P<0.05),the osteogenesis effect in group D was the strongest.Therefore,in vitro experiments screened out the optimal concentration of TGF-β1/GelMA scaffold to be 600 ng/mL.Micro-CT,HE staining,and OCN immunohistochemistry staining of rat skull defect repair experiments showed that the new bone tissue and bone volume/tissue volume ratio in the TGF-β1+GelMA group were significantly higher than those in the GelMA group and control group at 4 and 8 weeks after operation(P<0.05).Conclusion The TGF-β1/GelMA scaffold with a concentration of 600 ng/mL can significantly promote the osteogenic differentiation of BMSCs,can significantly promote bone regeneration at the skull defect,and can be used as a bioactive material for bone tissue regeneration.