Study on the gelatin methacryloyl composite scaffold with exogenous transforming growth factor β<sub>1</sub> to promote the repair of skull defects_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIU Xiangyu ^1,2,3 , WANG Zhaodong ^1,3 , XU Chen ^1,3 , GUAN Jianzhong ^1,3 , WEI Bangguo ^1,2,3 ,  LIU Yajun ^1,3

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;

Corresponding author：

LIU Yajun, Email: lyj19900501@126.com

Keywords：

Exogenous transforming growth factor β₁; gelatin methacryloyl hydrogel; bone marrow mesenchymal stem cells; osteogenic differentiation; bone regeneration; bone defect

DOI：

10.7507/1002-1892.202102008

Video：

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Objective To prepare a bone tissue engineering scaffold for repairing the skull defect of Sprague Dawley (SD) rats by combining exogenous transforming growth factor β₁ (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 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-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 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-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.

Citation： LIU Xiangyu, WANG Zhaodong, XU Chen, GUAN Jianzhong, WEI Bangguo, LIU Yajun. Study on the gelatin methacryloyl composite scaffold with exogenous transforming growth factor β₁ to promote the repair of skull defects. Chinese Journal of Reparative and Reconstructive Surgery, 2021, 35(7): 904-912. doi: 10.7507/1002-1892.202102008 Copy

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2.	Priddy LB, Chaudhuri O, Stevens HY, et al. Oxidized alginate hydrogels for bone morphogenetic protein-2 delivery in long bone defects. Acta Biomater, 2014, 10(10): 4390-4399.
3.	Reichert JC, Epari DR, Wullschleger ME, et al. Establishment of a preclinical ovine model for tibial segmental bone defect repair by applying bone tissue engineering strategies. Tissue Eng Part B Rev, 2010, 16(1): 93-104.
4.	张素珊, 李赟, 于贞成, 等. 甲基丙烯酸酐化明胶水凝胶介导的骨髓间充质干细胞增强肩袖损伤后愈合的研究. 组织工程与重建外科杂志, 2020, 16(4): 270-276.
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6.	Oryan A, Kamali A, Moshiri A, et al. Role of mesenchymal stem cells in bone regenerative medicine: What is the evidence? Cells Tissues Organs, 2017, 204(2): 59-83.
7.	Iijima K, Otsuka H. Cell scaffolds for bone tissue engineering. Bioengineering (Basel), 2020, 7(4): 119. doi: 10.3390/bioengineering7040119.
8.	Li RD, Deng ZL, Hu N, et al. Biphasic effects of TGFβ1 on BMP9-induced osteogenic differentiation of mesenchymal stem cells. BMB Rep, 2012, 45(9): 509-514.
9.	Lin CH, Su JJ, Lee SY, et al. Stiffness modification of photopolymerizable gelatin-methacrylate hydrogels influences endothelial differentiation of human mesenchymal stem cells. J Tissue Eng Regen Med, 2018, 12(10): 2099-2111.
10.	Bayati V, Hashemitabar M, Gazor R, et al. Expression of surface markers and myogenic potential of rat bone marrow- and adipose-derived stem cells: a comparative study. Anat Cell Biol, 2013, 46(2): 113-121.
11.	李文雄, 张晶宇, 杨锋. 中药调控骨髓间充质干细胞成骨分化的研究进展. 中医正骨, 2016, 28(7): 68-71.
12.	王小志, 何惠宇, 杨楠, 等. 基因转染骨髓间充质干细胞复合同种异体骨修复绵羊极限骨缺损. 中国组织工程研究, 2013, 17(47): 8141-8148.
13.	Zhou X, Feng W, Qiu K, et al. BMP-2 derived peptide and dexamethasone incorporated mesoporous silica nanoparticles for enhanced osteogenic differentiation of bone mesenchymal stem cells. ACS Appl Mater Interfaces, 2015, 7(29): 15777-15789.
14.	Shao N, Guo J, Guan Y, et al. Development of organic/inorganic compatible and sustainably bioactive composites for effective bone regeneration. Biomacromolecules, 2018, 19(9): 3637-3648.
15.	Pearson RG, Bhandari R, Quirk RA, et al. Recent advances in tissue engineering. J Long Term Eff Med Implants, 2017, 27(2-4): 199-231.
16.	Bonewald LF, Mundy GR. Role of transforming growth factor-beta in bone remodeling. Clin Orthop Relat Res, 1990, (250): 261-276.
17.	Karsdal MA, Hjorth P, Henriksen K, et al. Transforming growth factor-beta controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression. J Biol Chem, 2003, 278(45): 44975-44987.
18.	Engel ME, McDonnell MA, Law BK, et al. Interdependent SMAD and JNK signaling in transforming growth factor-beta-mediated transcription. J Biol Chem, 1999, 274(52): 37413-37420.
19.	Xu J, Liu J, Gan Y, et al. High-Dose TGF-β1 impairs mesenchymal stem cell-mediated bone regeneration via Bmp2 inhibition. J Bone Miner Res, 2020, 35(1): 167-180.
20.	Salazar VS, Gamer LW, Rosen V. BMP signalling in skeletal development, disease and repair. Nat Rev Endocrinol, 2016, 12(4): 203-221.
21.	张乃丹, 蒋益萍, 薛黎明, 等. 仙茅酚苷类成分促进成骨细胞骨形成和抑制破骨细胞骨吸收. 第二军医大学学报, 2016, 37(5): 562-568.
22.	廖乃顺, 李钻芳, 林如辉, 等. 比较3种形态学方法观察成骨细胞矿化结节的应用价值. 中国组织工程研究, 2014, (33): 5266-5270.

1. Lin B, He Y, Xu Y, et al. Outcome of bone defect reconstruction with clavicle bone cement prosthesis after tumor resection: a case series study. BMC Musculoskelet Disord, 2014, 15: 183. doi: 10.1186/1471-2474-15-183.
2. Priddy LB, Chaudhuri O, Stevens HY, et al. Oxidized alginate hydrogels for bone morphogenetic protein-2 delivery in long bone defects. Acta Biomater, 2014, 10(10): 4390-4399.
3. Reichert JC, Epari DR, Wullschleger ME, et al. Establishment of a preclinical ovine model for tibial segmental bone defect repair by applying bone tissue engineering strategies. Tissue Eng Part B Rev, 2010, 16(1): 93-104.
4. 张素珊, 李赟, 于贞成, 等. 甲基丙烯酸酐化明胶水凝胶介导的骨髓间充质干细胞增强肩袖损伤后愈合的研究. 组织工程与重建外科杂志, 2020, 16(4): 270-276.
5. Shi Z, Xu Y, Mulatibieke R, et al. Nano-silicate-reinforced and SDF-1α-loaded gelatin-methacryloyl hydrogel for bone tissue engineering. Int J Nanomedicine, 2020, 15: 9337-9353.
6. Oryan A, Kamali A, Moshiri A, et al. Role of mesenchymal stem cells in bone regenerative medicine: What is the evidence? Cells Tissues Organs, 2017, 204(2): 59-83.
7. Iijima K, Otsuka H. Cell scaffolds for bone tissue engineering. Bioengineering (Basel), 2020, 7(4): 119. doi: 10.3390/bioengineering7040119.
8. Li RD, Deng ZL, Hu N, et al. Biphasic effects of TGFβ1 on BMP9-induced osteogenic differentiation of mesenchymal stem cells. BMB Rep, 2012, 45(9): 509-514.
9. Lin CH, Su JJ, Lee SY, et al. Stiffness modification of photopolymerizable gelatin-methacrylate hydrogels influences endothelial differentiation of human mesenchymal stem cells. J Tissue Eng Regen Med, 2018, 12(10): 2099-2111.
10. Bayati V, Hashemitabar M, Gazor R, et al. Expression of surface markers and myogenic potential of rat bone marrow- and adipose-derived stem cells: a comparative study. Anat Cell Biol, 2013, 46(2): 113-121.
11. 李文雄, 张晶宇, 杨锋. 中药调控骨髓间充质干细胞成骨分化的研究进展. 中医正骨, 2016, 28(7): 68-71.
12. 王小志, 何惠宇, 杨楠, 等. 基因转染骨髓间充质干细胞复合同种异体骨修复绵羊极限骨缺损. 中国组织工程研究, 2013, 17(47): 8141-8148.
13. Zhou X, Feng W, Qiu K, et al. BMP-2 derived peptide and dexamethasone incorporated mesoporous silica nanoparticles for enhanced osteogenic differentiation of bone mesenchymal stem cells. ACS Appl Mater Interfaces, 2015, 7(29): 15777-15789.
14. Shao N, Guo J, Guan Y, et al. Development of organic/inorganic compatible and sustainably bioactive composites for effective bone regeneration. Biomacromolecules, 2018, 19(9): 3637-3648.
15. Pearson RG, Bhandari R, Quirk RA, et al. Recent advances in tissue engineering. J Long Term Eff Med Implants, 2017, 27(2-4): 199-231.
16. Bonewald LF, Mundy GR. Role of transforming growth factor-beta in bone remodeling. Clin Orthop Relat Res, 1990, (250): 261-276.
17. Karsdal MA, Hjorth P, Henriksen K, et al. Transforming growth factor-beta controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression. J Biol Chem, 2003, 278(45): 44975-44987.
18. Engel ME, McDonnell MA, Law BK, et al. Interdependent SMAD and JNK signaling in transforming growth factor-beta-mediated transcription. J Biol Chem, 1999, 274(52): 37413-37420.
19. Xu J, Liu J, Gan Y, et al. High-Dose TGF-β1 impairs mesenchymal stem cell-mediated bone regeneration via Bmp2 inhibition. J Bone Miner Res, 2020, 35(1): 167-180.
20. Salazar VS, Gamer LW, Rosen V. BMP signalling in skeletal development, disease and repair. Nat Rev Endocrinol, 2016, 12(4): 203-221.
21. 张乃丹, 蒋益萍, 薛黎明, 等. 仙茅酚苷类成分促进成骨细胞骨形成和抑制破骨细胞骨吸收. 第二军医大学学报, 2016, 37(5): 562-568.
22. 廖乃顺, 李钻芳, 林如辉, 等. 比较3种形态学方法观察成骨细胞矿化结节的应用价值. 中国组织工程研究, 2014, (33): 5266-5270.

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Study on the gelatin methacryloyl composite scaffold with exogenous transforming growth factor β₁ to promote the repair of skull defects

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Study on the gelatin methacryloyl composite scaffold with exogenous transforming growth factor β₁ to promote the repair of skull defects