EXPERIMENTAL STUDY ON OSTEOGENIC ACTIVITY OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY KLD-12 POLYPEPTIDE/RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 GEL_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WUBin , CHENLei , LIXiangchen , YUHongtao , YANGHai , LIANGXiangchen ,  SUNJianhua ,  DONGJinbo

Department of Orthopedics Center, the First Affiliated Hospital, College of Medicine, Shihezi University, Shihezi Xinjiang, 832008, P. R. China;

Corresponding author：

SUNJianhua, Email: 758719985@qq.com; DONGJinbo, Email: 56489005@qq.com

Keywords：

KLD-12; Bone morphogenetic protein 2; Bone marrow mesenchymal stem cells; Osteogenic activity; Rabbit

DOI：

10.7507/1002-1892.20160314

Video：

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Objective To investigate the effect of KLD-12 polypeptide complexed with recombinant human bone morphogenetic protein 2 (rhBMP-2) on osteogenic activity of rabbit bone marrow mesechymal stem cells (BMSCs). Methods Bone marrow was harvested from 3-month-old New Zealand white rabbit, and density gradient method was used to isolate and culture BMSCs. The third generation BMSCs were used for three-dimensional culture of KLD-12 polypetide/rhBMP-2 in vitro (experimental group) and KLD-12 polypeptide (control group). The morphology of the cells in the gel was observed by inverted phase contrast microscope at 7 days; alkaline phosphatase (ALP) and osteocalcin protein content were dectected at 3, 7, 10, 14, and 21 days; collagen type I immunofluorescence staining was done and real-time fluorescent quantitative PCR was performed to detect the relative expression of collagen type I and osteocalcin gene at 14 days. Results Under the inverted phase contrast microscope, the BMSCs in the gel of the experimental group and the control group showed circular growth, and the distribution was uniform at 7 days. There was no significant difference in the expressions of ALP and osteocalcin protein content between 2 groups at 3 and 7 days (P > 0.05); the above indexes in experimental group were significantly higher than those in the control group at 10-21 days (P < 0.05). Laser scanning confocal microscope observation showed that immunofluorescence staining for collagen type I was positive in the experimental group, and the expression was higher than that in the control group at 14 days. Real-time fluorescence quantitative PCR detection showed that the collagen type I and osteocalcin gene expressions were significantly higher than those in the control group (t=15.902, P=0.000; t=12.998, P=0.000). Conclusion BMSCs can normally grow and proliferate in the KLD-12 polypeptide, and KLD-12 polypeptide/rhBMP-2 has good biological activity to induce BMSCs differentiation into osteoblasts.

Citation： WUBin, CHENLei, LIXiangchen, YUHongtao, YANGHai, LIANGXiangchen, SUNJianhua, DONGJinbo. EXPERIMENTAL STUDY ON OSTEOGENIC ACTIVITY OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY KLD-12 POLYPEPTIDE/RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 GEL. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(12): 1518-1523. doi: 10.7507/1002-1892.20160314 Copy

1.	Greenough CG, Talyor LJ, Fraser RD. Anterior lumbar fusion:results, assessment techniques and prognostic factor. Eur Spine J, 1994, 3(4):225-230.
2.	Molinari RW, Sloboda JF, Arrington EC. Low-grade isthmic spondylolisthesis treated with instrumented posterior lumbar interbody fusion in U.S. servicemen. J Spinal Disord Tech, 2005, 18 Suppl:S 24-29.
3.	Fritzell P, Hägg O, Wessberg P, et al. Chronic low back pain and fusion:a comparison of three surgical techniques:a prospective multicenter randomized study from the Swedish lumbar spine study group. Spine, 2002, 27(11):1131-1141.
4.	Scheufler KM, Diesing D. Use of bone graft replacement in spinal fusions. Orthopade, 2015, 44(2):146-153.
5.	Banwart JC, Asher MA, Hassanein RS. Iliac crest bone graft harvest donor site morbidity. A statistical evaluation. Spine (Phila Pa 1976), 1995, 20(9):1055-1060.
6.	Kanemura T, Ishikawa Y, Matsumoto A, et al. The maturation of grafted bone after posterior lumbar interbody fusion with an interbody carbon cage:a prospective five-year study. J Bone Joint Surg (Br), 2011, 93(12):1638-1645.
7.	Heida K Jr, Ebraheim M, Siddiqui S, et al. Effects on clinical outcomes of grafts and spacers used in transforaminal lumbar interbody fusion:a critical review. Orthop Surg, 2013, 5(1):13-17.
8.	Mobbs RJ, Chung M, Rao PJ. Bone graft substitutes for anterior lumbar interbody fusion. Orthop Surg, 2013, 5(2):77-85.
9.	Lee P, Tran K, Chang W, et al. Influence of chondroitin sulfate and hyaluronic acid presence in nanofibers and its alignment on the bone marrow stromal cells:cartilage regeneration. J Biomed Nanotechnol, 2014, 10(8):1469-1479.
10.	You M, Jing J, Tian D, et al. Dioscin stimulates differentiation of mesenchymal stem cells towards hypertrophic chondrocytes in vitro and endochondral ossification in vivo. Am J Transl Res, 2016, 8(9):3930-3938.
11.	Zhang K, Ikeda Y, Kasugai S, et al. Extended Culture Conditions for Multipotent Bone Marrow-Derived Mesenchymal Stem Cells. Kokubyo Gakkai Zasshi, 2016, 83(1):13-24.
12.	Khanmohammadi M, Khanjani S, Bakhtyari MS, et al. Proliferation and chondrogenic differentiation potential of menstrual blood-and bone marrow-derived stem cells in two-dimensional culture. Int J Hematol, 2012, 95(5):484-493.
13.	周永春, 鲁超, 宋宗让, 等.兔骨髓间充质干细胞分离培养及体外诱导分化为成骨细胞.医学临床研究, 2015, 32(4):702-706.
14.	Urist MR. Bone:formation by auto induction. Science, 1965, 150(3698):893-899.
15.	Wozney JM, Rosen V, Celeste AJ, et al. Novel regulators of bone formation:molecular clones and activities. Science, 1988, 242(4885):1528-1534.
16.	Chatzinikolaidou M, Pontikoglou C, Terzaki K, et al. Recombinant human bone morphogenetic protein 2(rhBMP-2) immobilized on laser-fabricated 3D scaffolds enhance osteogenesis. Colloids Surf B Biointerfaces, 2016, 149:233-242.
17.	金捷, 周少怀, 卞峰, 等.基质细胞衍生因子-1/骨形成发生蛋白-2壳聚糖缓释微体促进兔骨髓间充质干细胞成骨分化.中华实验外科杂志, 2014, 31(7):1424-1427.
18.	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, 2016.[Epub ahead of print].
19.	Balseiro S, Nottmeier EW. Vertebral osteolysis originating from subchondral cyst end plate defects in transforaminal lumbar interbody fusion using rhBMP-2. Report of two cases. Spine J, 2010, 10(7):e6-e10.
20.	Rihn JA, Patel R, Makda J, et al. Complications associated with single-level transforaminal lumbar interbody fusion. Spine J, 2009, 9(8):623-629.
21.	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.
22.	Kisiday J, Jin M, Kurz B, et al. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division:implications for cartilage tissue repair. Proc Natl Acad Sci U S A, 2002, 99(15):9996-10001.
23.	Sun J, Zheng Q. Experimental study on self-assembly of KLD-12 peptide hydrogel and 3-D culture of MSC encapsulated within hydrogel in vitro. J Huazhong Univ Sci Technol Med Sci, 2009, 29(4):512-516.
24.	Sun J, Zheng Q, Wu Y, et al. Biocompatibility of KLD-12 peptide hydrogel as a scaffold in tissue engineering of intervertebral discs in rabbits. J Huazhong Univ Sci Technol Med Sci, 2010, 30(2):173-177.
25.	Bian Z, Sun J. Development of a KLD-12 polypeptide/TGF-β1-tissue scaffold promoting the differentiation of mesenchymal stem cell into nucleus pulposus-like cells for treatment of intervertebral disc degeneration. Int J Clin Exp Pathol, 2015, 8(2):1093-1103.
26.	Kanazawa I, Yamaguchi T, Yamamoto M, et al. Serum osteocalcin/bone-specific alkaline phosphatase ratio is a predictor for the presence of vertebral fractures in men with type 2 diabetes. Calcified Tissue Int, 2009, 85(3):228-234.
27.	Mizuno M, Kuboki Y. Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen. J Biochem, 2001, 129(1):133-138.

1. Greenough CG, Talyor LJ, Fraser RD. Anterior lumbar fusion:results, assessment techniques and prognostic factor. Eur Spine J, 1994, 3(4):225-230.
2. Molinari RW, Sloboda JF, Arrington EC. Low-grade isthmic spondylolisthesis treated with instrumented posterior lumbar interbody fusion in U.S. servicemen. J Spinal Disord Tech, 2005, 18 Suppl:S 24-29.
3. Fritzell P, Hägg O, Wessberg P, et al. Chronic low back pain and fusion:a comparison of three surgical techniques:a prospective multicenter randomized study from the Swedish lumbar spine study group. Spine, 2002, 27(11):1131-1141.
4. Scheufler KM, Diesing D. Use of bone graft replacement in spinal fusions. Orthopade, 2015, 44(2):146-153.
5. Banwart JC, Asher MA, Hassanein RS. Iliac crest bone graft harvest donor site morbidity. A statistical evaluation. Spine (Phila Pa 1976), 1995, 20(9):1055-1060.
6. Kanemura T, Ishikawa Y, Matsumoto A, et al. The maturation of grafted bone after posterior lumbar interbody fusion with an interbody carbon cage:a prospective five-year study. J Bone Joint Surg (Br), 2011, 93(12):1638-1645.
7. Heida K Jr, Ebraheim M, Siddiqui S, et al. Effects on clinical outcomes of grafts and spacers used in transforaminal lumbar interbody fusion:a critical review. Orthop Surg, 2013, 5(1):13-17.
8. Mobbs RJ, Chung M, Rao PJ. Bone graft substitutes for anterior lumbar interbody fusion. Orthop Surg, 2013, 5(2):77-85.
9. Lee P, Tran K, Chang W, et al. Influence of chondroitin sulfate and hyaluronic acid presence in nanofibers and its alignment on the bone marrow stromal cells:cartilage regeneration. J Biomed Nanotechnol, 2014, 10(8):1469-1479.
10. You M, Jing J, Tian D, et al. Dioscin stimulates differentiation of mesenchymal stem cells towards hypertrophic chondrocytes in vitro and endochondral ossification in vivo. Am J Transl Res, 2016, 8(9):3930-3938.
11. Zhang K, Ikeda Y, Kasugai S, et al. Extended Culture Conditions for Multipotent Bone Marrow-Derived Mesenchymal Stem Cells. Kokubyo Gakkai Zasshi, 2016, 83(1):13-24.
12. Khanmohammadi M, Khanjani S, Bakhtyari MS, et al. Proliferation and chondrogenic differentiation potential of menstrual blood-and bone marrow-derived stem cells in two-dimensional culture. Int J Hematol, 2012, 95(5):484-493.
13. 周永春, 鲁超, 宋宗让, 等.兔骨髓间充质干细胞分离培养及体外诱导分化为成骨细胞.医学临床研究, 2015, 32(4):702-706.
14. Urist MR. Bone:formation by auto induction. Science, 1965, 150(3698):893-899.
15. Wozney JM, Rosen V, Celeste AJ, et al. Novel regulators of bone formation:molecular clones and activities. Science, 1988, 242(4885):1528-1534.
16. Chatzinikolaidou M, Pontikoglou C, Terzaki K, et al. Recombinant human bone morphogenetic protein 2(rhBMP-2) immobilized on laser-fabricated 3D scaffolds enhance osteogenesis. Colloids Surf B Biointerfaces, 2016, 149:233-242.
17. 金捷, 周少怀, 卞峰, 等.基质细胞衍生因子-1/骨形成发生蛋白-2壳聚糖缓释微体促进兔骨髓间充质干细胞成骨分化.中华实验外科杂志, 2014, 31(7):1424-1427.
18. 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, 2016.[Epub ahead of print].
19. Balseiro S, Nottmeier EW. Vertebral osteolysis originating from subchondral cyst end plate defects in transforaminal lumbar interbody fusion using rhBMP-2. Report of two cases. Spine J, 2010, 10(7):e6-e10.
20. Rihn JA, Patel R, Makda J, et al. Complications associated with single-level transforaminal lumbar interbody fusion. Spine J, 2009, 9(8):623-629.
21. 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.
22. Kisiday J, Jin M, Kurz B, et al. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division:implications for cartilage tissue repair. Proc Natl Acad Sci U S A, 2002, 99(15):9996-10001.
23. Sun J, Zheng Q. Experimental study on self-assembly of KLD-12 peptide hydrogel and 3-D culture of MSC encapsulated within hydrogel in vitro. J Huazhong Univ Sci Technol Med Sci, 2009, 29(4):512-516.
24. Sun J, Zheng Q, Wu Y, et al. Biocompatibility of KLD-12 peptide hydrogel as a scaffold in tissue engineering of intervertebral discs in rabbits. J Huazhong Univ Sci Technol Med Sci, 2010, 30(2):173-177.
25. Bian Z, Sun J. Development of a KLD-12 polypeptide/TGF-β1-tissue scaffold promoting the differentiation of mesenchymal stem cell into nucleus pulposus-like cells for treatment of intervertebral disc degeneration. Int J Clin Exp Pathol, 2015, 8(2):1093-1103.
26. Kanazawa I, Yamaguchi T, Yamamoto M, et al. Serum osteocalcin/bone-specific alkaline phosphatase ratio is a predictor for the presence of vertebral fractures in men with type 2 diabetes. Calcified Tissue Int, 2009, 85(3):228-234.
27. Mizuno M, Kuboki Y. Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen. J Biochem, 2001, 129(1):133-138.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON OSTEOGENIC ACTIVITY OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY KLD-12 POLYPEPTIDE/RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 GEL

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