OSTEODIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS AFTER TRANSFECTED BY LENTIVIRAL VECTOR MEDIATED BONE MORPHOGENETIC PROTEIN 2_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XU Shenggui ¹ , LIN Jianhua ² , LIU Weinan ³ , WU Chaoyang ²

1Department of Orthopedics, Mindong Hospital Affiliated to Fujian Medical University, Ningde Fujian, 355000, P.R.China;;
2Department of Orthopedics, the First Affiliated Hospital of Fujian Medical University;;
3Department of Orthopedics, the Affiliated People’s Hospital of Fujian University of Traditional Chinese Medicine. Corresponding author: LIN Jianhua, E-mail: jianhua0918@126.com;

Corresponding author：

Keywords：

Osteochondral tissue engineering; Bone marrow mesenchymal stem cells; Bone morphogenetic protein 2 Lentiviral vector; Osteogenesis differentiation; Porcine

DOI：

10.7507/1002-1892.20130299

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Objective To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 (BMP-2) gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells (BMSCs) osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering. Methods BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines (weighing, 15 kg), and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection (MOI) of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group (BMP-2 vector group); BMSCs transfected by empty vector (empty vector group), and non-transfected BMSCs (non-transfection group) were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase (ALP) staining, ALP activities, and Alizarin red staining. Results The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentiviral vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed. Conclusion The porcine BMP- 2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.

Citation： XU Shenggui,LIN Jianhua,LIU Weinan,WU Chaoyang. OSTEODIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS AFTER TRANSFECTED BY LENTIVIRAL VECTOR MEDIATED BONE MORPHOGENETIC PROTEIN 2. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(11): 1380-1385. doi: 10.7507/1002-1892.20130299 Copy

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2.	Bessa PC, Casal M, Reis RL. Bone morphogenetic proteins in tissue engineering: the road from the laboratory to the clinic, part I (basic concepts). J Tissue Eng Regn Med, 2008, 2(1): 1-13.
3.	Yoon ST, Boden SD. Osteoinductive molecules in orthopaedics: basic science and preclinical studies. Clin Orthop Relat Res, 2002, (395): 33-43.
4.	林建华, 赖建明, 林文平, 等. 胶原-壳聚糖/胶原-纳米羟基磷灰石仿生支架材料的制备及其生物相容性检测. 中国修复重建外科杂志, 2012, 26(8): 1001-1006.
5.	赖建明, 林建华, 林文平, 等. 猪TGF-β1基因重组慢病毒载体的构建及其在BMSCs中的表达. 中国修复重建外科杂志, 2012, 26(7): 849-854.
6.	林朝贵, 范林, 陈良龙. 利用In-Fusion技术构建存活素-增强型绿色荧光蛋白融合基因重组慢病毒表达载体. 心血管康复医学杂志, 2010, 19(1): 10-14.
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8.	Frosch KH, Drengk A, Krause P, et al. Stem cell-coated titanium implants for the partial joint resurfacing of the knee. Biomaterials, 2006, 27(12): 2542-2549.
9.	Mithoefer K, McAdams T, Williams RJ, et al. Clinical efficacy of the microfracture technique for articular cartilage repair in the knee: an evidence-based systematic analysis. Am J Sport Med, 2009, 37(10): 2053-2063.
10.	Asheesh B, Williams RJ III. Cartilage resurfacing using synthetic composite plugs. Techniques in Knee Surgery, 2009, 8(1): 29-36.
11.	刘明, 项舟, 裴福兴, 等. BMSCs 种植双相复合支架修复兔关节软骨及软骨下骨缺损. 中国修复重建外科杂志, 2010, 24(1): 87-93.
12.	Caplan AI. Mesenchymal stem cells: cell-based reconstructive therapy in orthopedics. Tissue Eng, 2005, 11(7-8): 1198-2211.
13.	Yamaguchi A, Ishizuya T, Kintou N, et al. Effects of BMP-2, BMP- 4, and BMP-6 on osteoblastic differentiation of bone marrowderived stromal cell lines, ST2 and MC3T3-G2/PA6. Biochem Biophys Res Commun, 1996, 220(2): 366-371.
14.	Xie G, Sun J, Zhong G, et al. Hydroxyapatite nanoparticles as a controlled-release carrier of BMP-2: absorption and release kinetics in vitro. J Mater Sci Mater Med, 2010, 21(6): 1875-1880.
15.	Grgurevic L, Macek B, Mercep M, et al. Bone morphogenetic protein (BMP)1-3 enhances bone repair. Biochem Biophys Res Commun, 2011, 408(1): 25-31.
16.	Yazici C, Takahata M, Reynolds DG, et al. Self-complementary AAV2.5-BMP-2 -coated femoral allografts mediated superior bone healing versus live autografts in mice with equivalent biomechanics to unfractured femur. Mol Ther, 2011, 19(8): 1416-1425.
17.	Guillot PV, De Bari C, Dell’Accio F, et al. Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources. Differentiation, 2008, 76(9): 946-957．.
18.	Handschel J, Berr K, Depprich RA, et al. Induction of osteogenic markers in differentially treated cultures of embryonic stem cells. Head Face Med, 2008, 4: 10．.
19.	Maegawa N, Kawamura K, Hirose M, et al. Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2). J Tissue Eng Regen Med, 2007, 1(4): 306-313.
20.	Verkey M, Kllcharski C, Haque T, et al. In vitro osteogenic response of rat bone marrow cells to bFGF and BMP-2 treatments. Clin Orthop Relat Res, 2006, (443): 113-123.
21.	Wyatt LE, Chung CY, Carlsen B, et al. Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) alter connexin 43 phosphorylation in MC3T3-E1 cells. BMC Cell Biol, 2001, 2: 14.
22.	Cockrell AS, Kafri T. Gene delivery by lentivirus vectors. Mol Biotechnol, 2007, 36(3): 184-204.
23.	Gould DJ, Favorov P. Vectors for the treatment of autoimmune diseases. Gene Therapy, 2003, 10(10): 912-927.
24.	D’Costa J, Mansfield SG, Humeau LM. Lentiviral vectors in clinical trials: Current status. Curr Opin Mol Ther, 2009, 11(5): 554-564.
25.	张慧, 王贵超, 韩兴龙, 等. 去分化间充质干细胞成骨再分化潜能的实验研究. 南京医科大学学报: 自然科学版, 2013, 33(8): 1044-1048.

1. Khan Y, Yaszemski MJ, Mikos AG, et al. Tissue engineering of bone: material and matrix considerations. J Bone Joint Surg (Am), 2008, 90 Suppl 1: 36-42.
2. Bessa PC, Casal M, Reis RL. Bone morphogenetic proteins in tissue engineering: the road from the laboratory to the clinic, part I (basic concepts). J Tissue Eng Regn Med, 2008, 2(1): 1-13.
3. Yoon ST, Boden SD. Osteoinductive molecules in orthopaedics: basic science and preclinical studies. Clin Orthop Relat Res, 2002, (395): 33-43.
4. 林建华, 赖建明, 林文平, 等. 胶原-壳聚糖/胶原-纳米羟基磷灰石仿生支架材料的制备及其生物相容性检测. 中国修复重建外科杂志, 2012, 26(8): 1001-1006.
5. 赖建明, 林建华, 林文平, 等. 猪TGF-β1基因重组慢病毒载体的构建及其在BMSCs中的表达. 中国修复重建外科杂志, 2012, 26(7): 849-854.
6. 林朝贵, 范林, 陈良龙. 利用In-Fusion技术构建存活素-增强型绿色荧光蛋白融合基因重组慢病毒表达载体. 心血管康复医学杂志, 2010, 19(1): 10-14.
7. Rudert M. Histological evaluation of osteochondral defects: consideration of animal models with emphasis on the rabbit, experimental setup, follow-up and applied methods. Cells Tissues Organs, 2002, 171(4): 229-240.
8. Frosch KH, Drengk A, Krause P, et al. Stem cell-coated titanium implants for the partial joint resurfacing of the knee. Biomaterials, 2006, 27(12): 2542-2549.
9. Mithoefer K, McAdams T, Williams RJ, et al. Clinical efficacy of the microfracture technique for articular cartilage repair in the knee: an evidence-based systematic analysis. Am J Sport Med, 2009, 37(10): 2053-2063.
10. Asheesh B, Williams RJ III. Cartilage resurfacing using synthetic composite plugs. Techniques in Knee Surgery, 2009, 8(1): 29-36.
11. 刘明, 项舟, 裴福兴, 等. BMSCs 种植双相复合支架修复兔关节软骨及软骨下骨缺损. 中国修复重建外科杂志, 2010, 24(1): 87-93.
12. Caplan AI. Mesenchymal stem cells: cell-based reconstructive therapy in orthopedics. Tissue Eng, 2005, 11(7-8): 1198-2211.
13. Yamaguchi A, Ishizuya T, Kintou N, et al. Effects of BMP-2, BMP- 4, and BMP-6 on osteoblastic differentiation of bone marrowderived stromal cell lines, ST2 and MC3T3-G2/PA6. Biochem Biophys Res Commun, 1996, 220(2): 366-371.
14. Xie G, Sun J, Zhong G, et al. Hydroxyapatite nanoparticles as a controlled-release carrier of BMP-2: absorption and release kinetics in vitro. J Mater Sci Mater Med, 2010, 21(6): 1875-1880.
15. Grgurevic L, Macek B, Mercep M, et al. Bone morphogenetic protein (BMP)1-3 enhances bone repair. Biochem Biophys Res Commun, 2011, 408(1): 25-31.
16. Yazici C, Takahata M, Reynolds DG, et al. Self-complementary AAV2.5-BMP-2 -coated femoral allografts mediated superior bone healing versus live autografts in mice with equivalent biomechanics to unfractured femur. Mol Ther, 2011, 19(8): 1416-1425.
17. Guillot PV, De Bari C, Dell’Accio F, et al. Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources. Differentiation, 2008, 76(9): 946-957．.
18. Handschel J, Berr K, Depprich RA, et al. Induction of osteogenic markers in differentially treated cultures of embryonic stem cells. Head Face Med, 2008, 4: 10．.
19. Maegawa N, Kawamura K, Hirose M, et al. Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2). J Tissue Eng Regen Med, 2007, 1(4): 306-313.
20. Verkey M, Kllcharski C, Haque T, et al. In vitro osteogenic response of rat bone marrow cells to bFGF and BMP-2 treatments. Clin Orthop Relat Res, 2006, (443): 113-123.
21. Wyatt LE, Chung CY, Carlsen B, et al. Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) alter connexin 43 phosphorylation in MC3T3-E1 cells. BMC Cell Biol, 2001, 2: 14.
22. Cockrell AS, Kafri T. Gene delivery by lentivirus vectors. Mol Biotechnol, 2007, 36(3): 184-204.
23. Gould DJ, Favorov P. Vectors for the treatment of autoimmune diseases. Gene Therapy, 2003, 10(10): 912-927.
24. D’Costa J, Mansfield SG, Humeau LM. Lentiviral vectors in clinical trials: Current status. Curr Opin Mol Ther, 2009, 11(5): 554-564.
25. 张慧, 王贵超, 韩兴龙, 等. 去分化间充质干细胞成骨再分化潜能的实验研究. 南京医科大学学报: 自然科学版, 2013, 33(8): 1044-1048.

Chinese Journal of Reparative and Reconstructive Surgery

OSTEODIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS AFTER TRANSFECTED BY LENTIVIRAL VECTOR MEDIATED BONE MORPHOGENETIC PROTEIN 2

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