Recombinant human bone morphogenetic protein 2/porous calcium phosphate cement/autologous bone and combination of platelet-rich plasma in repairing of large bone defects in rabbits by Masquelet technique_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CONG Yuxuan , ZHANG Binfei , FU Yahui , WEI Xing , WANG Hu , DENG Hongli , ZHUANG Yan , ZHANG Kun ,  LEI Jinlai

Department of Orthopedic Trauma, Honghui Hospital, Xi’an Jiaotong University, Xi’an Shaanxi, 710054, P. R. China;

Corresponding author：

LEI Jinlai, Email: 13572407419@163.com

Keywords：

Bone defect; Masquelet technique; recombinant human bone morphogenetic protein 2; porous calcium phosphate cement; platelet-rich plasma; rabbit

DOI：

10.7507/1002-1892.202204112

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Objective To investigate the optimal mixing ratio of recombinant human bone morphogenetic protein 2 (rhBMP-2) with porous calcium phosphate cement (PCPC) and autologous bone as bone grafting material for the repair of large bone defects using Masquelet technique. The effect of platelet-rich plasma (PRP) on the healing of bone defects was evaluated under the optimal ratio of mixed bone. Methods Fifty-four New Zealand White rabbits were taken to establish a 2 cm long bone defect model of the ulna and treated using the Masquelet technique. Two parts of the experiment were performed in the second phase of the Masquelet technique. First, 36 modeled experimental animals were randomly divided into 4 groups (n=9) according to the mass ratio of autologous bone and rhBMP-2/PCPC. Group A: autologous bone (100%); group B: 25% autologous bone+75% rhBMP-2/PCPC; group C: 50% autologous bone+50% rhBMP-2/PCPC; group D: 75% autologous bone+25% rhBMP-2/PCPC. The animals were executed at 4, 8, and 12 weeks postoperatively for general observation, imaging observation, histological observation (HE staining), alkaline phosphatase (ALP) activity assay, and biomechanical assay (three-point bending test) were performed to assess the osteogenic ability and to determine the optimal mixing ratio. Then, 18 modeled experimental animals were randomly divided into 2 groups (n=9). The control group was implanted with the optimal mixture ratio of autologous bone+rhBMP-2/PCPC, and the experimental group was implanted with the optimal mixture ratio of autologous bone+rhBMP-2/PCPC+autologous PRP. The same method was used to observe the above indexes at 4, 8, and 12 weeks postoperatively. Results The bone healing process from callus formation to the cortical connection at the defected gap could be observed in each group after operation; new bone formation, bridging with the host bone, and bone remodeling to normal bone density were observed on imaging observation; new woven bone, new capillaries, bone marrow cavity, and other structures were observed on histological observation. The ALP activity of each group gradually increased with time (P<0.05); the ALP activity of group A was significantly higher than that of the other 3 groups at each time point after operation, and of groups C and D than group B (P<0.05); there was no significant difference between groups C and D (P>0.05). Biomechanical assay showed that the maximum load in three-point bending test of each group increased gradually with time (P<0.05), and the maximum loads of groups A and D were significantly higher than that of groups B and C at each time point after operation (P<0.05), but there was no significant difference between groups A and D (P>0.05). According to the above tests, the optimal mixing ratio was 75% autogenous bone+25% rhBMP-2/PCPC. The process of new bone formation in the experimental group and the control group was observed by gross observation, imaging examination, and histological observation, and the ability of bone formation in the experimental group was better than that in the control group. The ALP activity and maximum load increased gradually with time in both groups (P<0.05); the ALP activity and maximum load in the experimental group were significantly higher than those in the control group at each time point after operation (P<0.05), and the maximum load in the experimental group was also significantly higher than that in group A at 12 weeks after operation (P<0.05). Conclusion In the second phase of Masquelet technique, rhBMP-2/PCPC mixed with autologous bone to fill the bone defect can treat large bone defect of rabbit ulna, and it has the best osteogenic ability when the mixing ratio is 75% autologous bone+25% rhBMP-2/PCPC. The combination of PRP can improve the osteogenic ability of rhBMP-2/PCPC and autologous bone mixture.

Citation： CONG Yuxuan, ZHANG Binfei, FU Yahui, WEI Xing, WANG Hu, DENG Hongli, ZHUANG Yan, ZHANG Kun, LEI Jinlai. Recombinant human bone morphogenetic protein 2/porous calcium phosphate cement/autologous bone and combination of platelet-rich plasma in repairing of large bone defects in rabbits by Masquelet technique. Chinese Journal of Reparative and Reconstructive Surgery, 2022, 36(10): 1288-1295. doi: 10.7507/1002-1892.202204112 Copy

1.	赵轶男, 刘建, 刘昌盛, 等. 不同孔径多孔CPC材料修复兔大段骨缺损的实验研究. 中国矫形外科杂志, 2012, 20(10): 924-927, 963.
2.	李保亮, 刘雷, 赵文博, 等. 异种骨治疗骨缺损的瓶颈及发展趋势. 中国骨伤, 2015, 28(12): 1166-1170.
3.	Chadayammuri V, Hake M, Mauffrey C. Innovative strategies for the management of long bone infection: a review of the Masquelet technique. Patient Saf Surg, 2015, 9: 32. doi: 10.1186/s13037-015-0079-0.
4.	Lee GS, Park JH, Shin US, et al. Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone tissue engineering. Acta Biomater, 2011, 7(8): 3178-3186.
5.	Li RH, Bouxsein ML, Blake CA, et al. rhBMP-2 injected in a calcium phosphate paste (alpha-BSM) accelerates healing in the rabbit ulnar osteotomy model. J Orthop Res, 2003, 21(6): 997-1004.
6.	Li M, Liu X, Liu X, et al. Calcium phosphate cement with BMP-2-loaded gelatin microspheres enhances bone healing in osteoporosis: a pilot study. Clin Orthop Relat Res, 2010, 468(7): 1978-1985.
7.	Moore DC, Ehrlich MG, McAllister SC, et al. Recombinant human platelet-derived growth factor-BB augmentation of new-bone formation in a rat model of distraction osteogenesis. J Bone Joint Surg (Am), 2009, 91(8): 1973-1984.
8.	He F, Chen Y, Li J, et al. Improving bone repair of femoral and radial defects in rabbit by incorporating PRP into PLGA/CPC composite scaffold with unidirectional pore structure. J Biomed Mater Res A, 2015, 103(4): 1312-1324.
9.	Podaropoulos L, Veis AA, Papadimitriou S, et al. Bone regeneration using beta-tricalcium phosphate in a calcium sulfate matrix. J Oral Implantol, 2009, 35(1): 28-36.
10.	袁冰, 韦卓. 骨缺损修复的研究进展. 生物骨科材料与临床研究, 2014, 11(3): 38-41.
11.	肖琦科, 魏玉珊, 赵轶男, 等. 多孔磷酸钙骨水泥与转染骨形态发生蛋白2基因的骨髓间充质干细胞复合修复股骨髁骨缺损. 中国组织工程研究, 2016, 20(43): 6403-6408.
12.	Seeherman HJ, Bouxsein M, Kim H, et al. Recombinant human bone morphogenetic protein-2 delivered in an injectable calcium phosphate paste accelerates osteotomy-site healing in a nonhuman primate model. J Bone Joint Surg (Am), 2004, 86(9): 1961-1972.
13.	Masquelet AC, Begue T. The concept of induced membrane for reconstruction of long bone defects. Orthop Clin North Am, 2010, 41(1): 27-37.
14.	张一, 田晓滨, 佘荣峰, 等. 膜诱导技术结合抗生素硫酸钙颗粒治疗下肢感染性骨缺损. 中华骨科杂志, 2017, 37(9): 513-519.
15.	Andrzejowski P, Giannoudis PV. The ‘diamond concept’ for long bone non-union management. J Orthop Traumatol, 2019, 20(1): 21. doi: 10.1186/s10195-019-0528-0.
16.	Haubruck P, Tanner MC, Vlachopoulos W, et al. Comparison of the clinical effectiveness of bone morphogenic protein (BMP)-2 and -7 in the adjunct treatment of lower limb nonunions. Orthop Traumatol Surg Res, 2018, 104(8): 1241-1248.
17.	Ahmed M, Reffat SA, Hassan A, et al. Platelet-rich plasma for the treatment of clean diabetic foot ulcers. Ann Vasc Surg, 2017, 38: 206-211.
18.	Kanthan SR, Kavitha G, Addi S, et al. Platelet-rich plasma (PRP) enhances bone healing in non-united critical-sized defects: a preliminary study involving rabbit models. Injury, 2011, 42(8): 782-789.
19.	Dong Z, Li B, Liu B, et al. Platelet-rich plasma promotes angiogenesis of prefabricated vascularized bone graft. J Oral Maxillofac Surg, 2012, 70(9): 2191-2197.
20.	崔旭, 张伯勋, 赵德伟. 三种不同方法制备的硫酸钙片植入修复桡骨节段性骨缺损的实验研究. 中华医学杂志, 2009, 89(11): 777-781.
21.	Dhillon RS, Schwarz EM, Maloney MD. Platelet-rich plasma therapy-future or trend? Arthritis Res Ther, 2012, 14(4): 219. doi:10.1186/ar3914.
22.	Moshiri A, Oryan A. Structural and functional modulation of early healing of full-thickness superficial digital flexor tendon rupture in rabbits by repeated subcutaneous administration of exogenous human recombinant basic fibroblast growth factor. J Foot Ankle Surg, 2011, 50(6): 654-662.
23.	Oryan A, Moshiri A. Recombinant fibroblast growth protein enhances healing ability of experimentally induced tendon injury in vivo. J Tissue Eng Regen Med, 2014, 8(6): 421-431.
24.	Kalén A, Wahlström O, Linder CH, et al. The content of bone morphogenetic proteins in platelets varies greatly between different platelet donors. Biochem Biophys Res Commun, 2008, 375(2): 261-264.
25.	Wahlström O, Linder C, Kalén A, et al. Acidic preparations of platelet concentrates release bone morphogenetic protein-2. Acta Orthop, 2008, 79(3): 433-437.

1. 赵轶男, 刘建, 刘昌盛, 等. 不同孔径多孔CPC材料修复兔大段骨缺损的实验研究. 中国矫形外科杂志, 2012, 20(10): 924-927, 963.
2. 李保亮, 刘雷, 赵文博, 等. 异种骨治疗骨缺损的瓶颈及发展趋势. 中国骨伤, 2015, 28(12): 1166-1170.
3. Chadayammuri V, Hake M, Mauffrey C. Innovative strategies for the management of long bone infection: a review of the Masquelet technique. Patient Saf Surg, 2015, 9: 32. doi: 10.1186/s13037-015-0079-0.
4. Lee GS, Park JH, Shin US, et al. Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone tissue engineering. Acta Biomater, 2011, 7(8): 3178-3186.
5. Li RH, Bouxsein ML, Blake CA, et al. rhBMP-2 injected in a calcium phosphate paste (alpha-BSM) accelerates healing in the rabbit ulnar osteotomy model. J Orthop Res, 2003, 21(6): 997-1004.
6. Li M, Liu X, Liu X, et al. Calcium phosphate cement with BMP-2-loaded gelatin microspheres enhances bone healing in osteoporosis: a pilot study. Clin Orthop Relat Res, 2010, 468(7): 1978-1985.
7. Moore DC, Ehrlich MG, McAllister SC, et al. Recombinant human platelet-derived growth factor-BB augmentation of new-bone formation in a rat model of distraction osteogenesis. J Bone Joint Surg (Am), 2009, 91(8): 1973-1984.
8. He F, Chen Y, Li J, et al. Improving bone repair of femoral and radial defects in rabbit by incorporating PRP into PLGA/CPC composite scaffold with unidirectional pore structure. J Biomed Mater Res A, 2015, 103(4): 1312-1324.
9. Podaropoulos L, Veis AA, Papadimitriou S, et al. Bone regeneration using beta-tricalcium phosphate in a calcium sulfate matrix. J Oral Implantol, 2009, 35(1): 28-36.
10. 袁冰, 韦卓. 骨缺损修复的研究进展. 生物骨科材料与临床研究, 2014, 11(3): 38-41.
11. 肖琦科, 魏玉珊, 赵轶男, 等. 多孔磷酸钙骨水泥与转染骨形态发生蛋白2基因的骨髓间充质干细胞复合修复股骨髁骨缺损. 中国组织工程研究, 2016, 20(43): 6403-6408.
12. Seeherman HJ, Bouxsein M, Kim H, et al. Recombinant human bone morphogenetic protein-2 delivered in an injectable calcium phosphate paste accelerates osteotomy-site healing in a nonhuman primate model. J Bone Joint Surg (Am), 2004, 86(9): 1961-1972.
13. Masquelet AC, Begue T. The concept of induced membrane for reconstruction of long bone defects. Orthop Clin North Am, 2010, 41(1): 27-37.
14. 张一, 田晓滨, 佘荣峰, 等. 膜诱导技术结合抗生素硫酸钙颗粒治疗下肢感染性骨缺损. 中华骨科杂志, 2017, 37(9): 513-519.
15. Andrzejowski P, Giannoudis PV. The ‘diamond concept’ for long bone non-union management. J Orthop Traumatol, 2019, 20(1): 21. doi: 10.1186/s10195-019-0528-0.
16. Haubruck P, Tanner MC, Vlachopoulos W, et al. Comparison of the clinical effectiveness of bone morphogenic protein (BMP)-2 and -7 in the adjunct treatment of lower limb nonunions. Orthop Traumatol Surg Res, 2018, 104(8): 1241-1248.
17. Ahmed M, Reffat SA, Hassan A, et al. Platelet-rich plasma for the treatment of clean diabetic foot ulcers. Ann Vasc Surg, 2017, 38: 206-211.
18. Kanthan SR, Kavitha G, Addi S, et al. Platelet-rich plasma (PRP) enhances bone healing in non-united critical-sized defects: a preliminary study involving rabbit models. Injury, 2011, 42(8): 782-789.
19. Dong Z, Li B, Liu B, et al. Platelet-rich plasma promotes angiogenesis of prefabricated vascularized bone graft. J Oral Maxillofac Surg, 2012, 70(9): 2191-2197.
20. 崔旭, 张伯勋, 赵德伟. 三种不同方法制备的硫酸钙片植入修复桡骨节段性骨缺损的实验研究. 中华医学杂志, 2009, 89(11): 777-781.
21. Dhillon RS, Schwarz EM, Maloney MD. Platelet-rich plasma therapy-future or trend? Arthritis Res Ther, 2012, 14(4): 219. doi:10.1186/ar3914.
22. Moshiri A, Oryan A. Structural and functional modulation of early healing of full-thickness superficial digital flexor tendon rupture in rabbits by repeated subcutaneous administration of exogenous human recombinant basic fibroblast growth factor. J Foot Ankle Surg, 2011, 50(6): 654-662.
23. Oryan A, Moshiri A. Recombinant fibroblast growth protein enhances healing ability of experimentally induced tendon injury in vivo. J Tissue Eng Regen Med, 2014, 8(6): 421-431.
24. Kalén A, Wahlström O, Linder CH, et al. The content of bone morphogenetic proteins in platelets varies greatly between different platelet donors. Biochem Biophys Res Commun, 2008, 375(2): 261-264.
25. Wahlström O, Linder C, Kalén A, et al. Acidic preparations of platelet concentrates release bone morphogenetic protein-2. Acta Orthop, 2008, 79(3): 433-437.

Chinese Journal of Reparative and Reconstructive Surgery

Recombinant human bone morphogenetic protein 2/porous calcium phosphate cement/autologous bone and combination of platelet-rich plasma in repairing of large bone defects in rabbits by Masquelet technique

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