Sustained release of recombinant human bone morphogenetic protein-2 combined with stromal vascular fraction cells in promoting posterolateral spinal fusion in rat model_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YUAN Wei ¹ ,  ZHENG Jun ² , QIAN Jinyu ² , ZHOU Xiaoxiao ¹ , WANG Minghui ¹ ,  WANG Xiuhui ¹

1. Department of Orthopedic Surgery, Affiliated Zhoupu Hospital, Shanghai University of Medicine & Health Sciences, Shanghai, 201318, P.R.China;
2. Department of Orthopedics and Traumatology, Yueyang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 200437, P.R.China;

Corresponding author：

ZHENG Jun, Email: dudu8238@163.com; WANG Xiuhui, Email: zpyygkwxh@sina.cn

Keywords：

Stromal vascular fraction cells; recombinant human bone morphogenetic protein-2; bone formation; spinal fusion; rat

DOI：

10.7507/1002-1892.201703043

Video：

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ObjectiveTo observe the effect of stromal vascular fraction cells (SVFs) from rat fat tissue combined with sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2) in promoting the lumbar fusion in rat model.MethodsSVFs were harvested from subcutaneous fat of bilateral inguinal region of 4-month-old rat through the collagenase I digestion. The sustained release carrier was prepared via covalent bond of the rhBMP-2 and β-tricalcium phosphate (β-TCP) by the biominetic apatite coating process. The sustained release effect was measured by BCA method. Thirty-two rats were selected to establish the posterolateral lumbar fusion model and were divided into 4 groups, 8 rats each group. The decalcified bone matrix (DBX) scaffold+PBS, DBX scaffold+rhBMP-2/β-TCP sustained release carrier, DBX scaffold+SVFs, and DBX scaffold+rhBMP-2/β-TCP sustained release carrier+SVFs were implanted in groups A, B, C, and D respectively. X-ray films, manual spine palpation, and high-resolution micro-CT were used to evaluate spinal fusion at 8 weeks after operation; bone mineral density (BMD) and bone volume fraction were analyzed; the new bone formation was evaluated by HE staining and Masson’s trichrome staining, osteocalcin (OCN) was detected by immunohistochemical staining.ResultsThe cumulative release amount of rhBMP-2 was about 40% at 2 weeks, indicating sustained release effect of rhBMP-2; while the control group was almost released within 2 weeks. At 8 weeks, the combination of manual spine palpation, X-ray, and micro-CT evaluation showed that group D had the strongest bone formation (100%, 8/8), followed by group B (75%, 6/8), group C (37.5%, 3/8), and group A (12.5%, 1/8). Micro-CT analysis showed BMD and bone volume fraction were significantly higher in group D than groups A, B, and C (P<0.05), and in group B than groups A and C (P<0.05). HE staining, Masson’s trichrome staining, and immunohistochemistry staining for OCN staining exhibited a large number of cartilage cells with bone matrix deposition, and an active osteogenic process similar to the mineralization of long bones in group D. The bone formation of group B was weaker than that of group D, and there was no effective new bone formation in groups A and C.ConclusionThe combination of sustained release of rhBMP-2 and freshly SVFs can significantly promote spinal fusion in rat model, providing a theoretical basis for further clinical applications.

Citation： YUAN Wei, ZHENG Jun, QIAN Jinyu, ZHOU Xiaoxiao, WANG Minghui, WANG Xiuhui. Sustained release of recombinant human bone morphogenetic protein-2 combined with stromal vascular fraction cells in promoting posterolateral spinal fusion in rat model. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(7): 862-869. doi: 10.7507/1002-1892.201703043 Copy

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2.	Ito Z, Imagama S, Kanemura T, et al. Bone union rate with autologous iliac bone versus local bone graft in posterior lumbar interbody fusion (PLIF): a multicenter study. Eur Spine J, 2013, 22(5): 1158-1163.
3.	Tatara AM, Mikos AG. Tissue Engineering in Orthopaedics. J Bone Joint Surg (Am), 2016, 98(13): 1132-1139.
4.	Chou YF, Dunn JC, Wu BM. In vitro response of MC3T3-E1 pre-osteoblasts within three-dimensional apatite-coated PLGA scaffolds. J Biomed Mater Res B Appl Biomater, 2005, 75(1): 81-90.
5.	Akpancar S, Tatar O, Turgut H, et al. The Current Perspectives of Stem Cell Therapy in Orthopedic Surgery. Arch Trauma Res, 2016, 5(4): e37976.
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8.	Han S, Sun HM, Hwang KC, et al. Adipose-Derived Stromal Vascular Fraction Cells: Update on Clinical Utility and Efficacy. Crit Rev Eukaryot Gene Expr, 2015, 25(2): 145-152.
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10.	Varma MJ, Breuls RG, Schouten TE, et al. Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells. Stem Cells Dev, 2007, 16(1): 91-104.
11.	Shields LB, Raque GH, Glassman SD, et al. Adverse effects associated with high-dose recombinant human bone morphogenetic protein-2 use in anterior cervical spine fusion. Spine (Phila Pa 1976), 2006, 31(5): 542-547.
12.	Carragee EJ, Hurwitz EL, Weiner BK. A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. Spine J, 2011, 11(6): 471-491.
13.	Kaito T. Biologic enhancement of spinal fusion with bone morphogenetic proteins: current position based on clinical evidence and future perspective. J Spine Surg, 2016, 2(4): 357-358.
14.	Wang JC, Kanim LE, Yoo S, et al. Effect of regional gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats. J Bone Joint Surg (Am), 2003, 85-A(5): 905-911.
15.	Hsu WK, Wang JC, Liu NQ, et al. Stem cells from human fat as cellular delivery vehicles in an athymic rat posterolateral spine fusion model. J Bone Joint Surg (Am), 2008, 90(5): 1043-1052.
16.	Wang JC. Gene therapy for spinal fusion. Spine J, 2011, 11(6): 557-559.
17.	Lee M, Li W, Siu RK, et al. Biomimetic apatite-coated alginate/chitosan microparticles as osteogenic protein carriers. Biomaterials, 2009, 30(30): 6094-6101.
18.	Tessmar JK, Göpferich AM. Matrices and scaffolds for protein delivery in tissue engineering. Adv Drug Deliv Rev, 2007, 59(4-5): 274-291.
19.	Sohier J, Daculsi G, Sourice S, et al. Porous beta tricalcium phosphate scaffolds used as a BMP-2 delivery system for bone tissue engineering. J Biomed Mater Res A, 2010, 92(3): 1105-1114.
20.	Berendsen AD, Olsen BR. Bone development. Bone, 2015, 80: 14-18.
21.	Boden SD, Schimandle JH, Hutton WC. An experimental lumbar intertransverse process spinal fusion model. Radiographic, histologic, and biomechanical healing characteristics. Spine (Phila Pa 1976), 1995, 20(4): 412-420.
22.	Lin L, Shen Q, Wei X, et al. Comparison of osteogenic potentials of BMP4 transduced stem cells from autologous bone marrow and fat tissue in a rabbit model of calvarial defects. Calcif Tissue Int, 2009, 85(1): 55-65.

1. Ito Z, Matsuyama Y, Sakai Y, et al. Bone union rate with autologous iliac bone versus local bone graft in posterior lumbar interbody fusion. Spine (Phila Pa 1976), 2010, 35(21): E1101-1105.
2. Ito Z, Imagama S, Kanemura T, et al. Bone union rate with autologous iliac bone versus local bone graft in posterior lumbar interbody fusion (PLIF): a multicenter study. Eur Spine J, 2013, 22(5): 1158-1163.
3. Tatara AM, Mikos AG. Tissue Engineering in Orthopaedics. J Bone Joint Surg (Am), 2016, 98(13): 1132-1139.
4. Chou YF, Dunn JC, Wu BM. In vitro response of MC3T3-E1 pre-osteoblasts within three-dimensional apatite-coated PLGA scaffolds. J Biomed Mater Res B Appl Biomater, 2005, 75(1): 81-90.
5. Akpancar S, Tatar O, Turgut H, et al. The Current Perspectives of Stem Cell Therapy in Orthopedic Surgery. Arch Trauma Res, 2016, 5(4): e37976.
6. Witkowska-Zimny M, Walenko K. Stem cells from adipose tissue. Cell Mol Biol Lett, 2011, 16(2): 236-257.
7. Baer PC, Geiger H. Adipose-derived mesenchymal stromal/stem cells: tissue localization, characterization, and heterogeneity. Stem Cells Int, 2012, 2012: 812693.
8. Han S, Sun HM, Hwang KC, et al. Adipose-Derived Stromal Vascular Fraction Cells: Update on Clinical Utility and Efficacy. Crit Rev Eukaryot Gene Expr, 2015, 25(2): 145-152.
9. Saxer F, Scherberich A, Todorov A, et al. Implantation of Stromal Vascular Fraction Progenitors at Bone Fracture Sites: From a Rat Model to a First-in-Man Study. Stem Cells, 2016, 34(12): 2956-2966.
10. Varma MJ, Breuls RG, Schouten TE, et al. Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells. Stem Cells Dev, 2007, 16(1): 91-104.
11. Shields LB, Raque GH, Glassman SD, et al. Adverse effects associated with high-dose recombinant human bone morphogenetic protein-2 use in anterior cervical spine fusion. Spine (Phila Pa 1976), 2006, 31(5): 542-547.
12. Carragee EJ, Hurwitz EL, Weiner BK. A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. Spine J, 2011, 11(6): 471-491.
13. Kaito T. Biologic enhancement of spinal fusion with bone morphogenetic proteins: current position based on clinical evidence and future perspective. J Spine Surg, 2016, 2(4): 357-358.
14. Wang JC, Kanim LE, Yoo S, et al. Effect of regional gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats. J Bone Joint Surg (Am), 2003, 85-A(5): 905-911.
15. Hsu WK, Wang JC, Liu NQ, et al. Stem cells from human fat as cellular delivery vehicles in an athymic rat posterolateral spine fusion model. J Bone Joint Surg (Am), 2008, 90(5): 1043-1052.
16. Wang JC. Gene therapy for spinal fusion. Spine J, 2011, 11(6): 557-559.
17. Lee M, Li W, Siu RK, et al. Biomimetic apatite-coated alginate/chitosan microparticles as osteogenic protein carriers. Biomaterials, 2009, 30(30): 6094-6101.
18. Tessmar JK, Göpferich AM. Matrices and scaffolds for protein delivery in tissue engineering. Adv Drug Deliv Rev, 2007, 59(4-5): 274-291.
19. Sohier J, Daculsi G, Sourice S, et al. Porous beta tricalcium phosphate scaffolds used as a BMP-2 delivery system for bone tissue engineering. J Biomed Mater Res A, 2010, 92(3): 1105-1114.
20. Berendsen AD, Olsen BR. Bone development. Bone, 2015, 80: 14-18.
21. Boden SD, Schimandle JH, Hutton WC. An experimental lumbar intertransverse process spinal fusion model. Radiographic, histologic, and biomechanical healing characteristics. Spine (Phila Pa 1976), 1995, 20(4): 412-420.
22. Lin L, Shen Q, Wei X, et al. Comparison of osteogenic potentials of BMP4 transduced stem cells from autologous bone marrow and fat tissue in a rabbit model of calvarial defects. Calcif Tissue Int, 2009, 85(1): 55-65.

Chinese Journal of Reparative and Reconstructive Surgery

Sustained release of recombinant human bone morphogenetic protein-2 combined with stromal vascular fraction cells in promoting posterolateral spinal fusion in rat model

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