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Title: [Study on the gelatin methacryloyl composite scaffold with exogenous transforming growth factor β ₁ to promote the repair of skull defects].
Author: Liu X, Wang Z, Xu C, Guan J, Wei B, Liu Y.
Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2021 Jul 15; 35(7):904-912. PubMed ID: 34308601.
Abstract:
OBJECTIVE: To prepare a bone tissue engineering scaffold for repairing the skull defect of Sprague Dawley (SD) rats by combining exogenous transforming growth factor β ₁ (TGF-β ₁) with gelatin methacryloyl (GelMA) hydrogel. METHODS: Firstly, GelMA hydrogel composite scaffolds containing exogenous TGF-β ₁ at concentrations of 0, 150, 300, 600, 900, and 1 200 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-β ₁/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-β ₁ group (using the optimal concentration of TGF-β ₁/GelMA scaffold). The rats were sacrificed at 4 and 8 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-β ₁/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 vitroexperiments screened out the optimal concentration of TGF-β ₁/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-β ₁+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-β ₁/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. 目的: 通过外源性 TGF-β ₁ 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、1 200 ng/mL 外源性 TGF-β ₁ 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-β ₁/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-β ₁ 组（采用最适浓度 TGF-β ₁/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-β ₁/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-β ₁/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-β ₁+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-β ₁/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。.

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