Study on the correlation between the content of bone morphogenetic protein 2 in demineralized bone matrix and its osteogenic activity <i>in vitro</i> and <i>in vivo</i>_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LI Miao ¹ ,  BAI Yulong ¹ , PAN Xiaoliang ² , WANG Jingjing ³ , CHEN Weiming ³ , LUO Jingwan ³ , HU Kai ³ , CHEN Jinfa ³

1. Changzhou Gencong Biomechanics Research Center, Changzhou Jiangsu, 213164, P.R.China;
2. Ningxia Tissue and Organ Bank, Yinchuan Ningxia, 750004, P.R.China;
3. Shanghai Yapeng Biotechnology Co., Ltd, Shanghai, 201201, P.R.China;

Corresponding?author：

BAI Yulong, Email: long_yubai@sina.com

Keywords：

Demineralized bone matrix; bone morphogenetic protein 2; osteogenic activity; guanidine hydrochloride/ethylene diamine tetraacetic acid

DOI：

10.7507/1002-1892.202012006

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Objective To investigate the correlation between the content of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) and its osteogenic activity in vitro and in vivo, in order to choose a simple and convenient method to evaluate the osteogenic activity of DBM.Methods The left mid-femoral tissues of 9 donors were taken, and DBMs (S1-S9) were prepared by dynamic decalcification process, and inactivated DBM (control group) was prepared at the same time. Protease inhibitor method, collagenase method, guanidine hydrochloride/ethylene diamine tetraacetic acid (EDTA) method, and RIPA lysate method were used to extract BMP-2 in S1-S9 and inactivated DBMs. The BMP-2 content was measured and the differences between DBMs were compared. Then the S1-S9 and inactivated DBMs were co-cultured with mouse embryonic osteoblasts MC3T3-E1, respectively. The cell proliferation was detected by MTT method and fluorescence staining, and alkaline phosphatase (ALP) activity was detected at the same time. Thirty BALB/c male nude mice were divided into 10 groups, namely S1-S9 DBM groups (S1-S9 groups) and inactivated DBM group (control group), with 3 mice in each group. Muscle pockets of the middle thighs were prepared on both hindlimbs of mice in each group, and implanted corresponding DBM materials. At 4 weeks after operation, the samples were taken for HE staining observation and semi-quantitative evaluation, and the new bone formation score was calculated.Results The BMP-2 content of DBM derived from different donor bones was distinct. The BMP-2 content obtained by different extraction methods for DBM prepared from the same donor bone was also different, and the extraction efficiency of the guanidine hydrochloride/EDTA method was the highest. In vitro cell experiments, MTT test displayed that cell proliferations and ALP activity were significantly higher in S4 and S6 groups than in other groups at each time point after co-cultivation (P<0.05). Moreover, the cell proliferation of S4 group was the most significant at 7 days (P<0.05); fluorescence staining demonstrated that the osteoblasts of each group was in good condition, but the osteoblasts of S1, S2, S3, S4, and S6 groups were significantly more than other groups. In vivo ectopic osteogenesis experiments, the cartilage and new bone formation could be seen in the bone graft area of S1-S6 groups at 4 weeks after operation, and with the increase of BMP-2 content, the more new bone formation induced by the material, the higher the score of new bone formation of the material (P<0.05). Among them, S4 and S6 groups contained a large number of chondrocytes and osteoblasts in the osteogenesis area.Conclusion The osteogenic activity of DBM can be evaluated through BMP-2 quantitative detection combined with in vitro osteoblast proliferation and differentiation experiments.

Citation： LI Miao, BAI Yulong, PAN Xiaoliang, WANG Jingjing, CHEN Weiming, LUO Jingwan, HU Kai, CHEN Jinfa. Study on the correlation between the content of bone morphogenetic protein 2 in demineralized bone matrix and its osteogenic activity in vitro and in vivo. Chinese Journal of Reparative and Reconstructive Surgery, 2021, 35(5): 620-626. doi: 10.7507/1002-1892.202012006 Copy

1.	陳雄, 尹東. 自體骨聯合同種異體骨骨移植的研究進展. 中國臨床新醫學, 2018, 11(3): 307-310.
2.	Wenhao W, Yeung KWK. Bone grafts and biomaterials substitutes for bone defect repair: A review. Bioactive materials, 2017, 2(4): 224-247.
3.	Honsawek S, Powers RM, Wolfinbarger L. Extractable bone morphogenetic protein and correlation with induced new bone formation in an in vivo assay in the athymic mouse model. Cell And Tissue Banking, 2005, 6(1): 13-23.
4.	李忠海, 劉謨震, 趙彥濤, 等. 三種植骨材料在腰椎后路椎間融合中的應用比較. 中國骨與關節雜志, 2018, 7(3): 230-235. doi: 10.3969/j.issn.2095-252X.2018.03.015.
5.	Urist MR. Bone: formation by autoinduction. Science, 1965, 150(3698): 893.
6.	Urist MR, Strates BS. The classic: bone morphogenetic protein. Clin Orthop Relat Res, 2009, 467(12): 3051-3062.
7.	Sampath TK, Coughlin JE, Whetstone RM, et al. Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily. J Biol Chem, 1990, 265(22): 13198-13205.
8.	Geiger M, Li RH, Friess W. Collagen sponges for bone regeneration with rhBMP-2. Adv Drug Deliv Rev, 2003, 55(12): 1613-1629.
9.	Kim HY, Lee JH, Yun JW, et al. Development of porous beads to provide regulated BMP-2 stimulation for varying durations: In vitro and in vivo studies for bone regeneration. Biomacromolecules, 2016, 17(5): 1633-1642.
10.	趙彥濤, 韓麗偉, 胡先同, 等. DBM中BMP-2檢測初步判斷骨庫供骨的活性. 中國矯形外科雜志, 2019, 27(20): 1896-1899.
11.	Blum B, Moseley J, Miller L, et al. Measurement of bone morphogenetic proteins and other growth factors in demineralized bone matrix. Orthopedics, 2004, 27(1 Suppl): s161-s165.
12.	Laitinen M, Jortikka L, Halttunen T, et al. Measurement of total and local bone morphogenetic protein concentration in bone tumours. Int Orthop, 1997, 21(3): 188-193.
13.	Sampath TK, Reddi AH. Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc Natl Acad Sci U S A, 1981, 78(12): 7599-7603.
14.	Pepene CE, Seck T, Diel I, et al. Concentration of insulin-like growth factor (IGF)-Ⅰ in iliac crest bone matrix in premenopausal women with idiopathic osteoporosis. Exp Clin Endocrinol Diabetes, 2004, 112(1): 38-43.
15.	國家藥品監督管理局. 同種異體修復材料脫礦骨材料的體內成骨誘導性能評價. YY/T 1680-2020: 1-12.
16.	Cook SD. Preclinical and clinical evaluation of osteogenic protein-1 (BMP-7) in bony sites. Orthopedics, 1999, 22(7): 669-671.
17.	趙彥濤, 李忠海, 胡先同, 等. 不同濃度rhBMP-2對MC3T3-E1細胞增殖、ALP活性及成骨分化的影響. 中國骨與關節損傷雜志, 2016, 31(3): 281-284.
18.	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.
19.	Raina DB, Matuszewski LM, Vater C, et al. A facile one-stage treatment of critical bone defects using a calcium sulfate/hydroxyapatite biomaterial providing spatiotemporal delivery of bone morphogenic protein-2 and zoledronic acid. Sci Adv, 2020, 6(48): eabc1779. doi: 10.1126/sciadv.abc1779.
20.	Marie PJ, Debiais F, Ha? E. Regulation of human cranial osteoblast phenotype by FGF-2, FGFR-2 and BMP-2 signaling. Histol Histopathol, 2002, 17(3): 877-885.

1. 陳雄, 尹東. 自體骨聯合同種異體骨骨移植的研究進展. 中國臨床新醫學, 2018, 11(3): 307-310.
2. Wenhao W, Yeung KWK. Bone grafts and biomaterials substitutes for bone defect repair: A review. Bioactive materials, 2017, 2(4): 224-247.
3. Honsawek S, Powers RM, Wolfinbarger L. Extractable bone morphogenetic protein and correlation with induced new bone formation in an in vivo assay in the athymic mouse model. Cell And Tissue Banking, 2005, 6(1): 13-23.
4. 李忠海, 劉謨震, 趙彥濤, 等. 三種植骨材料在腰椎后路椎間融合中的應用比較. 中國骨與關節雜志, 2018, 7(3): 230-235. doi: 10.3969/j.issn.2095-252X.2018.03.015.
5. Urist MR. Bone: formation by autoinduction. Science, 1965, 150(3698): 893.
6. Urist MR, Strates BS. The classic: bone morphogenetic protein. Clin Orthop Relat Res, 2009, 467(12): 3051-3062.
7. Sampath TK, Coughlin JE, Whetstone RM, et al. Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily. J Biol Chem, 1990, 265(22): 13198-13205.
8. Geiger M, Li RH, Friess W. Collagen sponges for bone regeneration with rhBMP-2. Adv Drug Deliv Rev, 2003, 55(12): 1613-1629.
9. Kim HY, Lee JH, Yun JW, et al. Development of porous beads to provide regulated BMP-2 stimulation for varying durations: In vitro and in vivo studies for bone regeneration. Biomacromolecules, 2016, 17(5): 1633-1642.
10. 趙彥濤, 韓麗偉, 胡先同, 等. DBM中BMP-2檢測初步判斷骨庫供骨的活性. 中國矯形外科雜志, 2019, 27(20): 1896-1899.
11. Blum B, Moseley J, Miller L, et al. Measurement of bone morphogenetic proteins and other growth factors in demineralized bone matrix. Orthopedics, 2004, 27(1 Suppl): s161-s165.
12. Laitinen M, Jortikka L, Halttunen T, et al. Measurement of total and local bone morphogenetic protein concentration in bone tumours. Int Orthop, 1997, 21(3): 188-193.
13. Sampath TK, Reddi AH. Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc Natl Acad Sci U S A, 1981, 78(12): 7599-7603.
14. Pepene CE, Seck T, Diel I, et al. Concentration of insulin-like growth factor (IGF)-Ⅰ in iliac crest bone matrix in premenopausal women with idiopathic osteoporosis. Exp Clin Endocrinol Diabetes, 2004, 112(1): 38-43.
15. 國家藥品監督管理局. 同種異體修復材料脫礦骨材料的體內成骨誘導性能評價. YY/T 1680-2020: 1-12.
16. Cook SD. Preclinical and clinical evaluation of osteogenic protein-1 (BMP-7) in bony sites. Orthopedics, 1999, 22(7): 669-671.
17. 趙彥濤, 李忠海, 胡先同, 等. 不同濃度rhBMP-2對MC3T3-E1細胞增殖、ALP活性及成骨分化的影響. 中國骨與關節損傷雜志, 2016, 31(3): 281-284.
18. 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.
19. Raina DB, Matuszewski LM, Vater C, et al. A facile one-stage treatment of critical bone defects using a calcium sulfate/hydroxyapatite biomaterial providing spatiotemporal delivery of bone morphogenic protein-2 and zoledronic acid. Sci Adv, 2020, 6(48): eabc1779. doi: 10.1126/sciadv.abc1779.
20. Marie PJ, Debiais F, Ha? E. Regulation of human cranial osteoblast phenotype by FGF-2, FGFR-2 and BMP-2 signaling. Histol Histopathol, 2002, 17(3): 877-885.

Chinese Journal of Reparative and Reconstructive Surgery

Study on the correlation between the content of bone morphogenetic protein 2 in demineralized bone matrix and its osteogenic activity in vitro and in vivo

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