EXPERIMENTAL STUDY ON TISSUE ENGINEERED BONES CONSTRUCTED BY HUMAN BONE MORPHOGENETIC PROTEIN 2 GENE-MODIFIED HUMAN BONE MARROW MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

 YULimin , MAJunxuan , YUBinsheng

Shenzhen Key Laboratory of Spine Surgery, Department of Spine Surgery, Peking University Shenzhen Hospital, Shenzhen Guangdong, 518036, P. R. China;

Corresponding author：

YULimin, Email: yuinwater@163.com

Keywords：

Tissue engineered bone; Bone marrow mesenchymal stem cells; Bone morphogenetic protein 2; Gene therapy; Transfection

DOI：

10.7507/1002-1892.20160313

Video：

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Objective To investigate the bone regeneration potential of cell-tissue engineered bone constructed by human bone marrow mesenchymal stem cells (hBMSCs) expressing the transduced human bone morphogenetic protein 2 (hBMP-2) gene stably. Methods The full-length hBMP-2 gene was cloned from human muscle tissues by RT-PCR and connected into a vector to consturct a eukaryotic expression system. And then the gene expression system was transduced to hBMSCs with lipidosome. hBMSCs were transfected by hBMP-2 gene (experimental group) and by empty plasmid (negative control group), untransfected hBMP-2 served as blank control group. RT-PCR, dot-ELISA, immunohistochemical analysis and ALP activity were performed to compare and evaluate the situation of hBMP-2 expression and secretion after transfection. hBMSCs transfected by hBMP-2 gene were seeded on hydroxyapatite (HA) and incubated for 4 days to construct the hBMP-2 gene modified tissue engineered bone, and then the tissue engineered bone was observed by the inverted phase contrast microscope and scanning electron microscope. Then the hBMP-2 gene modified tissue engineered bone (group A, n=3), empty plasmid transfected hBMSCs seeded on HA (group B, n=3), hBMSCs suspension transfected by hBMP-2 gene (group C, n=3), and hBMP-2 plasmids and lipidosome (group D, n=3) were implanted into bilateral back muscles of nude mice. The osteogenic activity was detected by HE staining and alcian blue staining after 4 weeks. Results At 48 hours and 3 weeks after transfection, RT-PCR and dot-ELISA results indicated that the transfected hBMSCs could express and secrete active and exogenous hBMP-2 stably. The immunohistochemical staining was positive, and the ALP activity in the transfected hBMSCs was significantly higher than that in two control groups (P < 0.05). The transfected hBMSCs had a good attaching and growing on the three-demension suface of HA under inverted phase contrast microscope and scanning electron microscope. In vivo study indicated that a lot of new bone formation was obviously found at 4 out of 6 sides of back muscles in group A. Some new bone formation at both sides of back muscles was observed in 1 of 3 mice in group B. No new bone formation was found in group C. A few new bone formation was observed at one side of back muscles in group D. Conclusion The tissue engineered bone constructed by hBMP-2 gene modified hBMSCs and HA is able to express and secrete active hBMP2 stably and can promote new bone formation effectively in muscles of nude mice.

Citation： YULimin, MAJunxuan, YUBinsheng. EXPERIMENTAL STUDY ON TISSUE ENGINEERED BONES CONSTRUCTED BY HUMAN BONE MORPHOGENETIC PROTEIN 2 GENE-MODIFIED HUMAN BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(12): 1512-1517. doi: 10.7507/1002-1892.20160313 Copy

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15.	Shi S, Kirk M, Kahn AJ. The role of type I collagen in the regulation of the osteoblast phenotype. J Bone Miner Res, 1996, 11(8):1139-1145.
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17.	郝增涛, 冯卫, 郝廷, 等. BMSCs来源成骨细胞和内皮细胞复合壳聚糖-羟基磷灰石多孔支架构建血管化组织工程骨研究.中国修复重建外科杂志, 2012, 26(4):489-494.
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21.	Kay MA. State-of-the-art gene-based therapies:the road ahead. Nat Rev Genet, 2011, 12(5):316-328.

1. Kim DH, Rhim R, Li L, et al. Prospective study of iliac crest bone graft harvest site pain and morbidity. Spine J, 2009, 9(11):886-892.
2. Silber JS, Anderson DG, Daffner SD, et al. Donor site morbidity after anterior iliac crest bone harvest for single-level anterior cervical discectomy and fusion. Spine (Phila Pa 1976), 2003, 28(2):134-139.
3. Bueno EM, Glowacki J. Cell-free and cell-based approaches for bone regeneration. Nat Rev Rheumatol, 2009, 5(12):685-697.
4. Evans CH, Huard J. Gene therapy approaches to regenerating the musculoskeletal system. Nat Rev Rheumatol, 2015, 11(4):234-242.
5. Somia N, Verma IM. Gene therapy:trials and tribulations. Nat Rev Genet, 2000, 1(2):91-99.
6. 王庆德, 杨述华, 杨操, 等.成人骨髓间充质干细胞体外低氧培养及生物学特征.中国修复重建外科杂志, 2007, 21(10):1128-1132.
7. 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.
8. La WG, Kang SW, Yang HS, et al. The efficacy of bone morphogenetic protein-2 depends on its mode of delivery. Artif Organs, 2010, 34(12):1150-1153.
9. Mussano F, Ciccone G, Ceccarelli M, et al. Bone morphogenetic proteins and bone defects:a systematic review. Spine (Phila Pa 1976), 2007, 32(7):824-830.
10. Boden SD, Zdeblick TA, Sandhu HS, et al. The use of rhBMP-2 in interbody fusion cages. Definitive evidence of osteoinduction in humans:a preliminary report. Spine (Phila Pa 1976), 2000, 25(3):376-381.
11. Glover DJ, Lipps HJ, Jans DA. Towards safe, non-viral therapeutic gene expression in humans. Nat Rev Genet, 2005, 6(4):299-310.
12. Yin H, Kanasty RL, Eltoukhy AA, et al. Non-viral vectors for gene-based therapy. Nat Rev Genet, 2014, 15(8):541-555.
13. Anselme K. Osteoblast adhesion on biomaterials. Biomaterials, 2000, 21(7):667-681.
14. Lynch MP, Stein JL, Stein GS, et al. The influence of type I collagen on the development and maintenance of the osteoblast phenotype in primary and passaged rat calvarial osteoblasts:modification of expression of genes supporting cell growth, adhesion, and extracellular matrix mineralization. Exp Cell Res, 1995, 216(1):35-45.
15. Shi S, Kirk M, Kahn AJ. The role of type I collagen in the regulation of the osteoblast phenotype. J Bone Miner Res, 1996, 11(8):1139-1145.
16. Ohgushi H, Okumura M, Yoshikawa T, et al. Bone formation process in porous calcium carbonate and hydroxyapatite. J Biomed Mater Res, 1992, 26(7):885-895.
17. 郝增涛, 冯卫, 郝廷, 等. BMSCs来源成骨细胞和内皮细胞复合壳聚糖-羟基磷灰石多孔支架构建血管化组织工程骨研究.中国修复重建外科杂志, 2012, 26(4):489-494.
18. Matsuoka H, Akiyama H, Okada Y, et al. In vitro analysis of the stimulation of bone formation by highly bioactive apatite-and wollastonite-containing glass-ceramic:released calcium ions promote osteogenic differentiation in osteoblastic ROS17/2.8 cells. J Biomed Mater Res, 1999, 47(2):176-188.
19. Anitua E, Pinas L, Murias A, et al. Effects of calcium ions on titanium surfaces for bone regeneration. Colloids Surf B Biointerfaces, 2015, 130:173-181.
20. Cox DB, Platt RJ, Zhang F. Therapeutic genome editing:prospects and challenges. Nat Med, 2015, 21(2):121-131.
21. Kay MA. State-of-the-art gene-based therapies:the road ahead. Nat Rev Genet, 2011, 12(5):316-328.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON TISSUE ENGINEERED BONES CONSTRUCTED BY HUMAN BONE MORPHOGENETIC PROTEIN 2 GENE-MODIFIED HUMAN BONE MARROW MESENCHYMAL STEM CELLS

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