Content of bone morphogenetic protein 2 in demineralized bone matrix prepared from different long bones and study of the osteogenic properties <i>in vitro</i>_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHAO Yongjie ¹ , YIN Gang ¹ , DU Rui ¹ , WANG Limin ² , DENG Mingming ¹ , GUAN Guofeng ¹ , SUN Guangchao ¹ ,  LIU Ying ¹

1. Department of Foot and Ankle Surgery, Binzhou Medical University Hospital, Binzhou Shandong, 256603, P. R. China;
2. Beijing Weidafeng Medical Biomaterials Co., Ltd, Beijing, 102200, P. R. China;

Corresponding author：

LIU Ying, Email: ly_510@163.com

Keywords：

Demineralized bone matrix; bone morphogenetic protein 2; cell proliferation; osteogenic differentiation

DOI：

10.7507/1002-1892.202303132

Video：

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Objective To measure the concentration of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) prepared from different long bones and to evaluate the osteoinductivity of different DBM on MC3T3-E1 cells. Methods Different bones from the same cadaver donor were used as the initial materials for making DBM, which were divided into ulna group (uDBM), humerus group (hDBM), tibia group (tDBM), and femur group (fDBM) according to the origins, and boiled DBM (cDBM) was taken as the control group. The proteins of DBM were extracted by guanidine hydrochloride, and the concentrations of BMP-2 were determined by ELISA assay. Then the DBM were co-cultured with MC3T3-E1 cells, the proliferation of MC3T3-E1 cells was observed by cell counting kit 8 (CCK-8) assay. The osteogenic differentiation ability of MC3T3-E1 cells was qualitatively observed by alizarin red, alkaline phosphatase (ALP), and Van Gieson staining, and the osteogenic differentiation ability of MC3T3-E1 cells was quantitatively analyzed by ALP content. Linear regression was used to analyze the effect of BMP-2 concentration in DBM on ALP synthesis. Results There were significant differences in the concentration of BMP-2 among the DBM groups (P<0.05). The concentrations of BMP-2 in the lower limb long bone were higher than those in the upper limb long bone, and the concentration of BMP-2 in the fDBM group was about 35.5 times that in the uDBM group. CCK-8 assay showed that the cells in each group continued to proliferate within 5 days of co-culture, and the absorbance (A) values at different time points were in the order of cDBM group<uDBM group<hDBM group<tDBM group<fDBM group. After co-culture for 14 days, the expressions of ALP, calcified nodules, and collagen fibers in each group were consistent with the distribution of BMP-2 concentration in DBM. The order of ALP content from low to high was cDBM group<uDBM group<hDBM group<tDBM group<fDBM group, and the differences between the groups were significant (P<0.05). Linear regression analysis showed that =0.361+0.017x, the effect of BMP-2 concentration in DBM on cellular ALP content was significant (t=3.552, P=0.005); for every 1 ng/g increase in BMP-2 concentration, ALP content would increase by 0.017 [95%CI (0.006, 0.027)] U/100 mL. Conclusion The concentration of natural BMP-2 in different long bones varies greatly, and the lower limb long bone is higher than the upper limb long bone. The harvested location of bone material was an important factor affecting the osteoinductivity of DBM.

Citation： ZHAO Yongjie, YIN Gang, DU Rui, WANG Limin, DENG Mingming, GUAN Guofeng, SUN Guangchao, LIU Ying. Content of bone morphogenetic protein 2 in demineralized bone matrix prepared from different long bones and study of the osteogenic properties in vitro. Chinese Journal of Reparative and Reconstructive Surgery, 2023, 37(8): 945-951. doi: 10.7507/1002-1892.202303132 Copy

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2.	Bhamb N, Kanim LEA, Drapeau S, et al. Comparative efficacy of commonly available human bone graft substitutes as tested for posterolateral fusion in an athymic rat model. Int J Spine Surg, 2019, 13(5): 437-458.
3.	Urist MR. Bone: formation by autoinduction. Science, 1965, 150(3698): 893-899.
4.	Oliveira Pinho F, Pinto Joazeiro P, Santos AR. Evaluation of the growth and differentiation of human fetal osteoblasts (hFOB) cells on demineralized bone matrix (DBM). Organogenesis, 2021, 17(3-4): 136-149.
5.	Ku JK, Kim IH, Um IW, et al. Effect of gamma irradiation on the osteoinductivity of demineralized dentin matrix for allografts: A preliminary study. J Funct Biomater, 2022, 13(1): 14.
6.	李淼, 白玉龙, 潘小亮, 等. 脱钙骨基质中BMP-2含量与其体内/外成骨活性的相关性研究. 中国修复重建外科杂志, 2021, 35(5): 620-626.
7.	李矛, 白玉龙, 李淼, 等. 两种去抗原异种骨性能评价及修复大鼠颅骨缺损实验研究. 中国修复重建外科杂志, 2021, 35(10): 1303-1310.
8.	Bae HW, Zhao L, Kanim LE, et al. Intervariability and intravariability of bone morphogenetic proteins in commercially available demineralized bone matrix products. Spine (Phila Pa 1976), 2006, 31(12): 1299-1306.
9.	Bae H, Zhao L, Zhu D, et al. Variability across ten production lots of a single demineralized bone matrix product. J Bone Joint Surg (Am), 2010, 92(2): 427-435.
10.	Zhang M, Powers RM, Wolfinbarger L. A quantitative assessment of osteoinductivity of human demineralized bone matrix. J Periodontol, 1997, 68(11): 1076-1084.
11.	Murray SS, Brochmann EJ, Harker JO, et al. A statistical model to allow the phasing out of the animal testing of demineralised bone matrix products. Altern Lab Anim, 2007, 35(4): 405-409.
12.	Hu ZM, Peel SA, Clokie CM. Osteoinductivity assay of the variability of repeated extractions of bone morphogenetic proteins from bovine bone at different times. Chin J Traumatol, 2004, 7(5): 301-307.
13.	Ellis SL, Grassinger J, Jones A, et al. The relationship between bone, hemopoietic stem cells, and vasculature. Blood, 2011, 118(6): 1516-1524.
14.	Siclari VA, Zhu J, Akiyama K, et al. Mesenchymal progenitors residing close to the bone surface are functionally distinct from those in the central bone marrow. Bone, 2013, 53(2): 575-586.
15.	Han B, Yang Z, Nimni M. Effects of moisture and temperature on the osteoinductivity of demineralized bone matrix. J Orthop Res, 2005, 23(4): 855-861.
16.	Kim S, Fan J, Lee CS, et al. Heparinized chitosan stabilizes the bioactivity of BMP-2 and potentiates the osteogenic efficacy of demineralized bone matrix. J Biol Eng, 2020, 14: 6.
17.	Kim SH, Choi HJ, Yoon DS, et al. Serial administration of rhBMP-2 and alendronate enhances the differentiation of osteoblasts. Int J Rheum Dis, 2021, 24(10): 1266-1272.
18.	Hughes-Fulford M, Li CF. The role of FGF-2 and BMP-2 in regulation of gene. induction, cell proliferation and mineralization. J Orthop Surg Res, 2011, 6: 8.
19.	Yin N, Zhu L, Ding L, et al. MiR-135-5p promotes osteoblast differentiation by targeting HIF1AN in MC3T3-E1 cells. Cell Mol Biol Lett, 2019, 24: 51.
20.	Thu HE, Mohamed IN, Hussain Z, et al. Eurycoma Longifolia as a potential adoptogen of male sexual health: a systematic review on clinical studies. Chin J Nat Med, 2017, 15(1): 71-80.
21.	Han B, Tang B, Nimni ME. Quantitative and sensitive in vitro assay for osteoinductive activity of demineralized bone matrix. J Orthop Res, 2003, 21(4): 648-654.
22.	Sampath TK, Maliakal JC, Hauschka PV, et al. Recombinant human osteogenic. protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J Biol Chem, 1992, 267(28): 20352-20362.
23.	Takuwa Y, Ohse C, Wang EA, et al. Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1. Biochem Biophys Res Commun, 1991, 174(1): 96-101.
24.	Cheng H, Jiang W, Phillips FM, et al. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg (Am), 2003, 85(8): 1544-1552.
25.	黄振明, 蔡卓, 钱静, 等. 微小RNA-335-5p调控BMP-2对人BMSCs成骨分化的影响. 中国修复重建外科杂志, 2020, 34(6): 781-786.

1. Nicholson JA, Makaram N, Simpson A, et al. Fracture nonunion in long bones: A literature review of risk factors and surgical management. Injury, 2021, 52(2): S3-S11.
2. Bhamb N, Kanim LEA, Drapeau S, et al. Comparative efficacy of commonly available human bone graft substitutes as tested for posterolateral fusion in an athymic rat model. Int J Spine Surg, 2019, 13(5): 437-458.
3. Urist MR. Bone: formation by autoinduction. Science, 1965, 150(3698): 893-899.
4. Oliveira Pinho F, Pinto Joazeiro P, Santos AR. Evaluation of the growth and differentiation of human fetal osteoblasts (hFOB) cells on demineralized bone matrix (DBM). Organogenesis, 2021, 17(3-4): 136-149.
5. Ku JK, Kim IH, Um IW, et al. Effect of gamma irradiation on the osteoinductivity of demineralized dentin matrix for allografts: A preliminary study. J Funct Biomater, 2022, 13(1): 14.
6. 李淼, 白玉龙, 潘小亮, 等. 脱钙骨基质中BMP-2含量与其体内/外成骨活性的相关性研究. 中国修复重建外科杂志, 2021, 35(5): 620-626.
7. 李矛, 白玉龙, 李淼, 等. 两种去抗原异种骨性能评价及修复大鼠颅骨缺损实验研究. 中国修复重建外科杂志, 2021, 35(10): 1303-1310.
8. Bae HW, Zhao L, Kanim LE, et al. Intervariability and intravariability of bone morphogenetic proteins in commercially available demineralized bone matrix products. Spine (Phila Pa 1976), 2006, 31(12): 1299-1306.
9. Bae H, Zhao L, Zhu D, et al. Variability across ten production lots of a single demineralized bone matrix product. J Bone Joint Surg (Am), 2010, 92(2): 427-435.
10. Zhang M, Powers RM, Wolfinbarger L. A quantitative assessment of osteoinductivity of human demineralized bone matrix. J Periodontol, 1997, 68(11): 1076-1084.
11. Murray SS, Brochmann EJ, Harker JO, et al. A statistical model to allow the phasing out of the animal testing of demineralised bone matrix products. Altern Lab Anim, 2007, 35(4): 405-409.
12. Hu ZM, Peel SA, Clokie CM. Osteoinductivity assay of the variability of repeated extractions of bone morphogenetic proteins from bovine bone at different times. Chin J Traumatol, 2004, 7(5): 301-307.
13. Ellis SL, Grassinger J, Jones A, et al. The relationship between bone, hemopoietic stem cells, and vasculature. Blood, 2011, 118(6): 1516-1524.
14. Siclari VA, Zhu J, Akiyama K, et al. Mesenchymal progenitors residing close to the bone surface are functionally distinct from those in the central bone marrow. Bone, 2013, 53(2): 575-586.
15. Han B, Yang Z, Nimni M. Effects of moisture and temperature on the osteoinductivity of demineralized bone matrix. J Orthop Res, 2005, 23(4): 855-861.
16. Kim S, Fan J, Lee CS, et al. Heparinized chitosan stabilizes the bioactivity of BMP-2 and potentiates the osteogenic efficacy of demineralized bone matrix. J Biol Eng, 2020, 14: 6.
17. Kim SH, Choi HJ, Yoon DS, et al. Serial administration of rhBMP-2 and alendronate enhances the differentiation of osteoblasts. Int J Rheum Dis, 2021, 24(10): 1266-1272.
18. Hughes-Fulford M, Li CF. The role of FGF-2 and BMP-2 in regulation of gene. induction, cell proliferation and mineralization. J Orthop Surg Res, 2011, 6: 8.
19. Yin N, Zhu L, Ding L, et al. MiR-135-5p promotes osteoblast differentiation by targeting HIF1AN in MC3T3-E1 cells. Cell Mol Biol Lett, 2019, 24: 51.
20. Thu HE, Mohamed IN, Hussain Z, et al. Eurycoma Longifolia as a potential adoptogen of male sexual health: a systematic review on clinical studies. Chin J Nat Med, 2017, 15(1): 71-80.
21. Han B, Tang B, Nimni ME. Quantitative and sensitive in vitro assay for osteoinductive activity of demineralized bone matrix. J Orthop Res, 2003, 21(4): 648-654.
22. Sampath TK, Maliakal JC, Hauschka PV, et al. Recombinant human osteogenic. protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J Biol Chem, 1992, 267(28): 20352-20362.
23. Takuwa Y, Ohse C, Wang EA, et al. Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1. Biochem Biophys Res Commun, 1991, 174(1): 96-101.
24. Cheng H, Jiang W, Phillips FM, et al. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg (Am), 2003, 85(8): 1544-1552.
25. 黄振明, 蔡卓, 钱静, 等. 微小RNA-335-5p调控BMP-2对人BMSCs成骨分化的影响. 中国修复重建外科杂志, 2020, 34(6): 781-786.

Chinese Journal of Reparative and Reconstructive Surgery

Content of bone morphogenetic protein 2 in demineralized bone matrix prepared from different long bones and study of the osteogenic properties in vitro

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