Effect of concentrated growth factor combined with mineralized collagen material on the adhesion, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells and the osteogenic effect <i>in vivo</i>_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Yue , LIU Keda , YAN Ming ,  WANG Wei

Comprehensive Department, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang Liaoning, 110002, P.R.China;

Corresponding?author：

WANG Wei, Email: wwang75@cmu.edu.cn

Keywords：

Concentrated growth factor; mineralized collagen; bone marrow mesenchymal stem cells; osteogenic differentiation

DOI：

10.7507/1002-1892.202009070

Video：

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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.

Citation： ZHANG Yue, LIU Keda, YAN Ming, WANG Wei. Effect of concentrated growth factor combined with mineralized collagen material on the adhesion, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells and the osteogenic effect in vivo. Chinese Journal of Reparative and Reconstructive Surgery, 2021, 35(3): 295-302. doi: 10.7507/1002-1892.202009070 Copy

1.	Sun XC, Wang H, Li JH, et al. Repair of alveolar cleft bone defects by bone collagen particles combined with human umbilical cord mesenchymal stem cells in rabbit. Biomed Eng Online, 2020, 19(1): 62.
2.	Chivu M, Tantar C, Hutu E, et al. Evaluation of bone healing of artificial defects in laboratory animals after covering with alloplastic material. Revista de Chimie, 2018, 69(7): 1728-1732.
3.	Zhao YQ, Tang RK. Improvement of organisms by biomimetic mineralization: A material incorporation strategy for biological modification. Acta Biomater, 2020. doi: 10.1016/j.actbio.2020.06.038.
4.	Cui FZ, Li Y, Ge J. Self-assembly of mineralized collagen composites. Materials Science and Engineering, 2007, 57(1-6): 1-27.
5.	Xu SJ, Qiu ZY, Wu JJ, et al. Osteogenic differentiation gene expression profiling of hMSCs on hydroxyapatite and mineralized collagen. Tissue Eng Part A, 2016, 22(1-2): 170-181.
6.	姜力銘, 宋戈, 夏商, 等. 轉化生長因子 β1 對人牙髓干細胞成骨分化作用研究. 中國實用口腔科雜志, 2018, 11(9): 530-533.
7.	Qiao J, An N, Ouyang X. Quantification of growth factors in different platelet concentrates. Platelets, 2017, 28(8): 774-778.
8.	Qin J, Wang L, Sun Y, et al. Concentrated growth factor increases Schwann cell proliferation and neurotrophic factor secretion and promotes functional nerve recovery in vivo. Int J Mol Med, 2016, 37(2): 493-500.
9.	孫玉環, 何冬梅, 楊馳, 等. 自體濃縮生長因子膜對山羊髁突軟骨缺損的修復作用. 中國口腔頜面外科雜志, 2014, 12(3): 205-209.
10.	Chen X, Chen Y, Hou Y, et al. Modulation of proliferation and differentiation of gingiva-derived mesenchymal stem cells by concentrated growth factors: Potential implications in tissue engineering for dental regeneration and repair. Int J Mol Med, 2019, 44(1): 37-46.
11.	Fang D, Long Z, Hou J. Clinical Application of concentrated growth factor fibrin combined with bone repair materials in jaw defects. J Oral Maxillofac Surg, 2020, 78(6): 882-892.
12.	?ankaya ZT, ünsal B, Gürbüz S, et al. Efficiency of concentrated growth factor in the surgical treatment of multiple adjacent papillary losses: A randomized, controlled, examiner-blinded clinical trial using CAD/CAM. Int J Periodontics Restorative Dent, 2020, 40(2): e73-e83.
13.	Honda H, Tamai N, Naka N, et al. Bone tissue engineering with bone marrow-derived stromal cells integrated with concentrated growth factor in Rattus norvegicus calvaria defect model. J Artif Organs, 2013, 16(3): 305-315.
14.	陳琦, 李石巖, 禹鑫等. CGF 復合絲素蛋白-羥基磷灰石支架對兔下頜骨缺損的修復作用. 河北醫學, 2020, 26(4): 698-702.
15.	Qiu ZY, Cui Y, Tao CS, et al. Mineralized collagen: rationale, current status, and clinical applications. Materials (Basel), 2015, 8(8): 4733-4750.
16.	Dohan Ehrenfest DM, Doglioli P, de Peppo GM, et al. Choukroun’s platelet-rich fibrin (PRF) stimulates in vitro proliferation and differentiation of human oral bone mesenchymal stem cell in a dose-dependent way. Arch Oral Biol, 2010, 55(3): 185-194.
17.	Hong S, Chen W, Jiang B. A Comparative evaluation of concentrated growth factor and platelet-rich fibrin on the proliferation, migration, and differentiation of human stem cells of the apical papilla. J Endod, 2018, 44(6): 977-983.
18.	Sohn DS, Heo JU, Kwak DH, et al. Bone regeneration in the maxillary sinus using an autologous fibrin-rich block with concentrated growth factors alone. Implant Dent, 2011, 20(5): 389-395.
19.	Qiao J, An N. Effect of concentrated growth factors on function and Wnt3a expression of human periodontal ligament cells in vitro. Platelets, 2017, 28(3): 281-286.
20.	Yu X, Ren H, Shang Q, et al. Effect of bone marrow mesenchymal stem cell combined with concentrate growth factor (CGF) on postmenopausal bone defects. Spine J, 2019, 19(9): S166.
21.	Pirpir C, Yilmaz O, Candirli C, et al. Evaluation of effectiveness of concentrated growth factor on osseointegration. Int J Implant Dent, 2017, 3(1): 7.
22.	宦俊, 竇磊, 嚴崎方, 等. 濃縮生長因子促人臍靜脈血管內皮細胞成血管化作用研究. 華西口腔醫學雜志, 2018, 36(3): 247-251.
23.	Chen X, Wang J, Yu L, et al. Concentrated growth factor promotes bone marrow-derived stromal cells osteogenesis: Optimal concentration and cell proliferation time. J Biomater Tissue Eng, 2017, 7(7): 595-604.
24.	Jun H, Lei D, Qi FY, et al. Effects of concentrated growth factors on the angiogenic properties of dental pulp cells and endothelial cells: An in vitro study. Braz Oral Res, 2018, 32: e48.
25.	Masuki H, Okudera T, Watanebe T, et al. Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF). Int J Implant Dent, 2016, 2(1): 19.
26.	Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoieticorgans. Exp Hematol, 1976, 4(5): 267-274.
27.	Liu Y, Ming L, Luo H, et al. Integration of a calcined bovine bone and BMSC-sheet 3D scaffold and the promotion of bone regeneration in large defects. Biomaterials, 2013, 34(38): 9998-10006.
28.	Tang J, Yu H, Wang Y, et al. microRNA-199a counteracts glucocorticoid inhibition of bone marrow mesenchymal stem cell osteogenic differentiation through regulation of Klotho expression in vitro. Cell Biol Int, 2020, 44(12): 2532-2540.
29.	Pei B, Wang W, Dunne N, et al. Applications of carbon nanotubes in bone tissue regeneration and engineering: Superiority, concerns, current advancements, and prospects. Nanomaterials, 2019, 9(10): 1501.
30.	Li XJ, Yang HX, Zhang ZJ, et al. Platelet-rich fibrin exudate promotes the proliferation and osteogenic differentiation of human periodontal ligament cells in vitro. Mol Med Rep, 2018, 18: 4477-4485.
31.	Li X, Yang H, Zhang Z, et al. Concentrated growth factor exudate enhances the proliferation of human periodontal ligament cells in the presence of TNF-α. Molecular medicine reports, 2019, 19(2): 943-950.

1. Sun XC, Wang H, Li JH, et al. Repair of alveolar cleft bone defects by bone collagen particles combined with human umbilical cord mesenchymal stem cells in rabbit. Biomed Eng Online, 2020, 19(1): 62.
2. Chivu M, Tantar C, Hutu E, et al. Evaluation of bone healing of artificial defects in laboratory animals after covering with alloplastic material. Revista de Chimie, 2018, 69(7): 1728-1732.
3. Zhao YQ, Tang RK. Improvement of organisms by biomimetic mineralization: A material incorporation strategy for biological modification. Acta Biomater, 2020. doi: 10.1016/j.actbio.2020.06.038.
4. Cui FZ, Li Y, Ge J. Self-assembly of mineralized collagen composites. Materials Science and Engineering, 2007, 57(1-6): 1-27.
5. Xu SJ, Qiu ZY, Wu JJ, et al. Osteogenic differentiation gene expression profiling of hMSCs on hydroxyapatite and mineralized collagen. Tissue Eng Part A, 2016, 22(1-2): 170-181.
6. 姜力銘, 宋戈, 夏商, 等. 轉化生長因子 β1 對人牙髓干細胞成骨分化作用研究. 中國實用口腔科雜志, 2018, 11(9): 530-533.
7. Qiao J, An N, Ouyang X. Quantification of growth factors in different platelet concentrates. Platelets, 2017, 28(8): 774-778.
8. Qin J, Wang L, Sun Y, et al. Concentrated growth factor increases Schwann cell proliferation and neurotrophic factor secretion and promotes functional nerve recovery in vivo. Int J Mol Med, 2016, 37(2): 493-500.
9. 孫玉環, 何冬梅, 楊馳, 等. 自體濃縮生長因子膜對山羊髁突軟骨缺損的修復作用. 中國口腔頜面外科雜志, 2014, 12(3): 205-209.
10. Chen X, Chen Y, Hou Y, et al. Modulation of proliferation and differentiation of gingiva-derived mesenchymal stem cells by concentrated growth factors: Potential implications in tissue engineering for dental regeneration and repair. Int J Mol Med, 2019, 44(1): 37-46.
11. Fang D, Long Z, Hou J. Clinical Application of concentrated growth factor fibrin combined with bone repair materials in jaw defects. J Oral Maxillofac Surg, 2020, 78(6): 882-892.
12. ?ankaya ZT, ünsal B, Gürbüz S, et al. Efficiency of concentrated growth factor in the surgical treatment of multiple adjacent papillary losses: A randomized, controlled, examiner-blinded clinical trial using CAD/CAM. Int J Periodontics Restorative Dent, 2020, 40(2): e73-e83.
13. Honda H, Tamai N, Naka N, et al. Bone tissue engineering with bone marrow-derived stromal cells integrated with concentrated growth factor in Rattus norvegicus calvaria defect model. J Artif Organs, 2013, 16(3): 305-315.
14. 陳琦, 李石巖, 禹鑫等. CGF 復合絲素蛋白-羥基磷灰石支架對兔下頜骨缺損的修復作用. 河北醫學, 2020, 26(4): 698-702.
15. Qiu ZY, Cui Y, Tao CS, et al. Mineralized collagen: rationale, current status, and clinical applications. Materials (Basel), 2015, 8(8): 4733-4750.
16. Dohan Ehrenfest DM, Doglioli P, de Peppo GM, et al. Choukroun’s platelet-rich fibrin (PRF) stimulates in vitro proliferation and differentiation of human oral bone mesenchymal stem cell in a dose-dependent way. Arch Oral Biol, 2010, 55(3): 185-194.
17. Hong S, Chen W, Jiang B. A Comparative evaluation of concentrated growth factor and platelet-rich fibrin on the proliferation, migration, and differentiation of human stem cells of the apical papilla. J Endod, 2018, 44(6): 977-983.
18. Sohn DS, Heo JU, Kwak DH, et al. Bone regeneration in the maxillary sinus using an autologous fibrin-rich block with concentrated growth factors alone. Implant Dent, 2011, 20(5): 389-395.
19. Qiao J, An N. Effect of concentrated growth factors on function and Wnt3a expression of human periodontal ligament cells in vitro. Platelets, 2017, 28(3): 281-286.
20. Yu X, Ren H, Shang Q, et al. Effect of bone marrow mesenchymal stem cell combined with concentrate growth factor (CGF) on postmenopausal bone defects. Spine J, 2019, 19(9): S166.
21. Pirpir C, Yilmaz O, Candirli C, et al. Evaluation of effectiveness of concentrated growth factor on osseointegration. Int J Implant Dent, 2017, 3(1): 7.
22. 宦俊, 竇磊, 嚴崎方, 等. 濃縮生長因子促人臍靜脈血管內皮細胞成血管化作用研究. 華西口腔醫學雜志, 2018, 36(3): 247-251.
23. Chen X, Wang J, Yu L, et al. Concentrated growth factor promotes bone marrow-derived stromal cells osteogenesis: Optimal concentration and cell proliferation time. J Biomater Tissue Eng, 2017, 7(7): 595-604.
24. Jun H, Lei D, Qi FY, et al. Effects of concentrated growth factors on the angiogenic properties of dental pulp cells and endothelial cells: An in vitro study. Braz Oral Res, 2018, 32: e48.
25. Masuki H, Okudera T, Watanebe T, et al. Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF). Int J Implant Dent, 2016, 2(1): 19.
26. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoieticorgans. Exp Hematol, 1976, 4(5): 267-274.
27. Liu Y, Ming L, Luo H, et al. Integration of a calcined bovine bone and BMSC-sheet 3D scaffold and the promotion of bone regeneration in large defects. Biomaterials, 2013, 34(38): 9998-10006.
28. Tang J, Yu H, Wang Y, et al. microRNA-199a counteracts glucocorticoid inhibition of bone marrow mesenchymal stem cell osteogenic differentiation through regulation of Klotho expression in vitro. Cell Biol Int, 2020, 44(12): 2532-2540.
29. Pei B, Wang W, Dunne N, et al. Applications of carbon nanotubes in bone tissue regeneration and engineering: Superiority, concerns, current advancements, and prospects. Nanomaterials, 2019, 9(10): 1501.
30. Li XJ, Yang HX, Zhang ZJ, et al. Platelet-rich fibrin exudate promotes the proliferation and osteogenic differentiation of human periodontal ligament cells in vitro. Mol Med Rep, 2018, 18: 4477-4485.
31. Li X, Yang H, Zhang Z, et al. Concentrated growth factor exudate enhances the proliferation of human periodontal ligament cells in the presence of TNF-α. Molecular medicine reports, 2019, 19(2): 943-950.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of concentrated growth factor combined with mineralized collagen material on the adhesion, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells and the osteogenic effect in vivo

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