Ectopic osteogenesis of bone marrow stromal stem cells under bone morphogenetic protein 2/vascular endothelial growth factor 165 co-transfections

doi:10.3969/j.issn.2095-4344.2017.09.005

Abstract

Abstract:

BACKGROUND: Double gene transfection using bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor 165 (VEGF165) for bone marrow stromal stem cells (BMSCs) to induce osteogenesis provides experimental basis for the study on tissue engineering bone.

OBJECTIVE: To investigate the ability of BMP2 and VEGF165 double gene modified rat BMSCs to induce osteogenesis.

METHODS: BMSCs were isolated from the femur and tibia of four 4-week-old Sprague-Dawley rats by whole bone marrow adherent culture. Passage 3 BMSCs were randomized into five groups: non-transfection group, empty plasmid group, BMP2 transfection group, VEGF165 transfection group, BMP2 and BMP2/VEGF165 transfection group (co-transfection group). Then, western blot assay was used to detect expression of BMP2 and VEGF165 at 48 hours after transfection, and the activity of alkaline phosphatase was detected in each group at 7 days after transfection.

RESULTS AND CONCLUSION: Highly expressed BMP2 in BMP2 and co-transfection groups and highly expressed VEGF165 in VEGF165 and co-transfection groups were found after transfection. The expression of BMP2 or VEGF165 in the co-transfection group was significantly higher than that in the BMP2 or VEGF165 transfection group after transfection, respectively (P < 0.05). Western blot results showed that the activity of alkaline phosphatase was ranked as: co-transfection group > BMP2 transfection group > VEGF165 transfection group > empty plasmid group and non-transfection group. There was a significant difference in the activity of alkaline phosphatase between co-transfection group and any of single gene transfection groups (P < 0.05). To conclude, BMP2/VEGF165 co-transfection promotes the ectopic osteogenesis of BMSCs.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Bone Marrow, Bone Morphogenetic Proteins, Vascular Endothelial Growth Factors, Alkaline Phosphatase, Tissue Engineering

CLC Number:

R394.2

Zhou Hang-yu, Xia De-lin, Gan Sheng-yuan, Shao Xue-lei. Ectopic osteogenesis of bone marrow stromal stem cells under bone morphogenetic protein 2/vascular endothelial growth factor 165 co-transfections[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1334-1339.

Figures/Tables 3

References

[1] Langer R, Vacanti J. Advances in tissue engineering. J Pediatr Surg. 2016;51(1):8-12.

[2] Kumar V, Khare T, Sharma M, et al. Engineering Crops for Future: A Phosphoproteomics Approach. Curr Protein Pept Sci. 2017 Feb 9. [Epub ahead of print]

[3] Ansari T, Lange P, Southgate A, et al. Stem Cell-Based Tissue-Engineered Laryngeal Replacement. Stem Cells Transl Med. 2017;6(2):677-687.

[4] Sánchez P, Pedraz JL, Orive G. Biologically active and biomimetic dual gelatin scaffolds for tissue engineering. Int J Biol Macromol. 2017;98:486-494.

[5] Paschos NK, Brown WE, Eswaramoorthy R, et al. Advances in tissue engineering through stem cell-based co-culture. J Tissue Eng Regen Med. 2015;9(5):488-503.

[6] Ehnert S, Aspera-Werz RH, Freude T, et al. Distinct Gene Expression Patterns Defining Human Osteoblasts' Response to BMP2 Treatment: Is the Therapeutic Success All a Matter of Timing. Eur Surg Res. 2016;57(3-4): 197-210.

[7] McBride-Gagyi SH, McKenzie JA, Buettmann EG, et al. Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice. Bone. 2015;81:533-543.

[8] Rogers MB, Shah TA, Shaikh NN. Turning Bone Morphogenetic Protein 2 (BMP2) on and off in Mesenchymal Cells. J Cell Biochem. 2015;116(10):2127-2138.

[9] Jang CH, Choi CH, Cho YB. Effect of BMP2-Platelet-rich Plasma-Biphasic Calcium Phosphate Scaffold on Accelerated Osteogenesis in Mastoid Obliteration. In Vivo. 2016;30(6): 835-839.

[10] Kawakami Y, Takayama K, Matsumoto T, et al. Anterior Cruciate Ligament-Derived Stem Cells Transduced With BMP2 Accelerate Graft-Bone Integration After ACL Reconstruction. Am J Sports Med. 2016 Nov 30. [Epub ahead of print]

[11] Liu K, Jing Y, Zhang W, et al. Silencing miR-106b accelerates osteogenesis of mesenchymal stem cells and rescues against glucocorticoid-induced osteoporosis by targeting BMP2. Bone. 2017;97:130-138.

[12] Schliephake H, Rublack J, Förster A, et al. Functionalization of titanium implants using a modular system for binding and release of VEGF enhances bone-implant contact in a rodent model. J Clin Periodontol. 2015;42(3):302-310.

[13] Liu HJ, Hu RY, Dai WJ, et al. Culture and Identification of Bone Marrow Mesenchymal Stem Cells from Enhanced Green Fluorescent Protein-transgenic Rats. Zhongguo Yi Xue Ke Xue Yuan Xue Bao.2016;38(1):9-15.

[14] Nagasawa M, Cooper LF, Ogino Y, et al. Topography Influences Adherent Cell Regulation of Osteoclastogenesis. J Dent Res. 2016;95(3):319-326.

[15] Liu HJ, Hu RY, Dai WJ, et al. Culture and Identification of Bone Marrow Mesenchymal Stem Cells from Enhanced Green Fluorescent Protein-transgenic Rats. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2016;38(1):9-15.

[16] Xiang J, Zheng X, Zhu X, et al. Optimization of the protocols for in vitro culture and induction of hepatic differentiation of rat mesenchymal stem cells. Nan Fang Yi Ke Da Xue Xue Bao. 2015;35(8):1090-1096.

[17] Zhang W, Zhang F, Shi H, et al. Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro. PLoS One. 2014;9(2):e88794.

[18] 杨俊丽,韩霞,孙明启,等.兔骨髓间充质干细胞的生物学特征及原代培养[J].中国组织工程研究,2015,19(50):8043-8047.

[19] 刘伟,陈剑,宋佳,等.兔骨髓间充质干细胞的分离、培养及鉴定[J].中国实验诊断学,2013,17(8):1366-1369.

[20] Hopkins CR. Inhibitors of the bone morphogenetic protein (BMP) signaling pathway: a patent review (2008-2015). Expert Opin Ther Pat. 2016 Aug 4. [Epub ahead of print]

[21] Abdallah BM. Marrow adipocytes inhibit the differentiation of mesenchymal stem cells into osteoblasts via suppressing BMP-signaling. J Biomed Sci. 2017;24(1):11.

[22] Chen Z, Ni S, Han S, et al. Nanoporous microstructures mediate osteogenesis by modulating the osteo-immune response of macrophages. Nanoscale. 2017;9(2):706-718.

[23] Salazar VS, Gamer LW, Rosen V. BMP signalling in skeletal development, disease and repair. Nat Rev Endocrinol. 2016; 12(4):203-221.

[24] Itoi T, Harada Y, Irie H, et al. Escherichia coli-derived recombinant human bone morphogenetic protein-2 combined with bone marrow-derived mesenchymal stromal cells improves bone regeneration in canine segmental ulnar defects. BMC Vet Res. 2016;12:201.

[25] Sharma S, Sapkota D, Xue Y, et al. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation. PLoS One. 2016;11(1):e0147507.

[26] Hu T, Abbah SA, Toh SY, et al. Bone marrow-derived mesenchymal stem cells assembled with low-dose BMP-2 in a three-dimensional hybrid construct enhances posterolateral spinal fusion in syngeneic rats. Spine J. 2015;15(12):2552- 2563.

[27] Jin IG, Kim JH, Wu HG, et al. Effect of bone marrow-derived stem cells and bone morphogenetic protein-2 on treatment of osteoradionecrosis in a rat model. J Craniomaxillofac Surg. 2015;43(8):1478-1486.

[28] Yin C, Chen J, Chen Z, et al. hBMP-2 and hTGF-β1 expressed in implanted BMSCs synergistically promote the repairing of segmental bone defects. J Orthop Sci. 2015; 20(4):717-727.

[29] Wang X, Li Y, Han R, et al. Demineralized bone matrix combined bone marrow mesenchymal stem cells, bone morphogenetic protein-2 and transforming growth factor-β3 gene promoted pig cartilage defect repair. PLoS One. 2014; 9(12):e116061.

[30] Wang T, Liao TA, Zhong SB. Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome. Asian Pac J Trop Med. 2013;6(9):739-742.

[31] Liang W, Cai Q, Liu Y. Transfection of human vascular endothelial growth factor 165 gene into rat bone marrow mesenchymal stem cells in vitro. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2011;25(11):1383-1388.

[32] Hang D, Wang Q, Guo C, et al. Treatment of osteonecrosis of the femoral head with VEGF165 transgenic bone marrow mesenchymal stem cells in mongrel dogs. Cells Tissues Organs. 2012;195(6):495-506.

[33] Zhang C, Ma Q, Qiang H, et al. Study on effect of recombinant adeno-associated virus vector co-expressing human vascular endothelial growth factor 165 and human bone morphogenetic protein 7 genes on bone regeneration and angiopoiesis in vivo. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 ;24(12):1449-1454.

[34] Zhang M, Wang D, Yin R. Histocompatibility of nano-hydroxyapatite/poly-co-glycolic acid tissue engineering bone modified by mesenchymal stem cells with vascular endothelial frowth factor. Zhonghua Yi Xue Za Zhi. 2015; 95(37):3061-3065.

[35] Liu S, Jin D, Wu JQ, et al. Neuropeptide Y stimulates osteoblastic differentiation and VEGF expression of bone marrow mesenchymal stem cells related to canonical Wnt signaling activating in vitro. Neuropeptides. 2016;56:105-113.

[36] Yadin D, Knaus P, Mueller TD. Structural insights into BMP receptors: Specificity, activation and inhibition. Cytokine Growth Factor Rev. 2016;27:13-34.

[37] Yadlapati M, Biguetti C, Cavalla F, et al. Characterization of a Vascular Endothelial Growth Factor-loaded Bioresorbable Delivery System for Pulp Regeneration. J Endod. 2017;43(1): 77-83.

[38] Payne SL, Peacock HM, Vickaryous MK. Blood vessel formation during tail regeneration in the leopard gecko (Eublepharis macularius): The blastema is not avascular. J Morphol. 2017;278(3):380-389.

[39] Liu Y, Olsen BR. Distinct VEGF functions during bone development and homeostasis. Arch Immunol Ther Exp (Warsz). 2014;62(5):363-368.

[40] Whitehouse MR, Howells NR, Parry MC, et al. Repair of Torn Avascular Meniscal Cartilage Using Undifferentiated Autologous Mesenchymal Stem Cells: From In Vitro Optimization to a First-in-Human Study. Stem Cells Transl Med. 2016 Dec 15. [Epub ahead of print].

[41] Chen K, Zhang C, Wang L, et al. Progress on strategies to promote vascularization in bone tissue engineering. Zhongguo Gu Shang. 2015;28(4):383-388.

[42] Li JJ, Zhao Q, Wang H, et al. Reconstruction of segmental bone defect by gene modified tissue engineering bone combined with vascularized periosteum. Zhonghua Zheng Xing Wai Ke Za Zhi. 2007;23(6):502-506.

[43] Han TY, Liu XW, Liang N, et al. In vitro effects of recombinant adenovirus-mediated bone morphogenetic protein 2/vascular endothelial growth factor 165 on osteogenic differentiation of bone marrow mesenchymal stem cells. Artif Cells Nanomed Biotechnol. 2017;45(1):108-114.

[44] Zhang C, Liu HM, Li QW, et al. Construction of recombinant adenovirus vector containing hBMP2 and hVEGF165 genes and its expression in rabbit Bone marrow mesenchymal stem cells. Tissue Cell. 2014;46(5):311-317.

[45] Lin Z, Wang JS, Lin L, et al. Effects of BMP2 and VEGF165 on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. Exp Ther Med. 2014;7(3):625-629.

[46] 管大凡,陈月芹,刘洪美,等. hBMP2/hVEGF165双基因腺病毒修饰BMSCs复合n-HA/PA66修复骨缺损影像学观察[J].中国矫形外科杂志,2015,23(20):1881-1887.

[47] 张聪,刘洪美,李庆伟,等.腺病毒载体介导人BMP-2与VEGF-165对骨髓间充质干细胞形态特征及增殖能力影响的实验研究[J].中国实验诊断学,2015,19(11):1813-1817.

[48] Silva E, Vasconcellos LM, Rodrigues BV, et al. PDLLA honeycomb-like scaffolds with a high loading of superhydrophilic graphene/multi-walled carbon nanotubes promote osteoblast in vitro functions and guided in vivo bone regeneration.Mater Sci Eng C Mater Biol Appl. 2017;73: 31-39.

[49] Liu DD, Zhang JC, Zhang Q, et al. TGF-β/BMP signaling pathway is involved in cerium-promoted osteogenic differentiation of mesenchymal stem cells. J Cell Biochem. 2013;114(5):1105-1114.

[50] 曹建华.年龄和性别与血清肝功能相关酶的关系探讨[J].检验医学与临床,2010,7(4):325-327.

[51] Hou L, Liu Y, Gao Q, et al. Spiroplasma eriocheiris Adhesin-Like Protein (ALP) Interacts with Epidermal Growth Factor (EGF) Domain Proteins to Facilitate Infection. Front Cell Infect Microbiol. 2017;7:13.

[52] 罗小娟.健康未成年人群干化学法血清酶水平分析[J].循证医学, 2015,15(4):232-236.