Xiangyu Liu1,2,3, Zhaodong Wang1,3, Chen Xu1,3, Jianzhong Guan1,3, Bangguo Wei1,2,3, Yajun Liu1,3. 1. Department of Orthopedics, the First Affiliated Hospital of Bengbu Medical College, Bengbu Anhui, 233000, P.R.China. 2. Bengbu Medical College, Bengbu Anhui, 233000, P.R.China. 3. Key Laboratory of Anhui Province for Tissue Transplantation, Bengbu Anhui, 233000, P.R.China.
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.
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.
Entities:
Keywords:
Exogenous transforming growth factor β1; bone defect; bone marrow mesenchymal stem cells; bone regeneration; gelatin methacryloyl hydrogel; osteogenic differentiation
Authors: Johannes C Reichert; Devakara R Epari; Martin E Wullschleger; Siamak Saifzadeh; Roland Steck; Jasmin Lienau; Scott Sommerville; Ian C Dickinson; Michael A Schütz; Georg N Duda; Dietmar W Hutmacher Journal: Tissue Eng Part B Rev Date: 2010-02 Impact factor: 6.389