RECOMBINANT ADENOVIRUS-MEDIATED BONE MORPHOGENETIC PROTEIN 9 AND ERYTHROPOIETIN GENES CO-TRANSFECTION IN PROMOTING OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANGGuangde ¹ , SUChengshuai ² , JINXia ¹ , YANGShimao ³ ,  FANGDianji ⁴ , GUOYanwei ¹

1. Department of Oral and Maxillofacial Surgery, Jining Hospital of Stomatology, Jining Shandong, 232000, P.R.China;
2. Department of Oral Prosthodontics, Jining Hospital of Stomatology, Jining Shandong, 232000, P.R.China;
3. Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University;
4. Department of Oral and Maxillofacial Surgery, Dental Dispensary of Shanghai Minhang;

Corresponding?author：

FANGDianji, Email: dianjifang@aliyun.com

Keywords：

Adipose-derived stem cells; Recombinant adenovirus; Bone morphogenetic protein 9; Erythropoietin; Osteogenic differentiation; Rabbit

DOI：

10.7507/1002-1892.20160055

Video：

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Objective To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9 (BMP-9) and erythropoietin (EPO) genes co-transfection on osteogenic differentiation of adipose-derived stem cells (ADSCs) in vitro. Methods The inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups: normal cells (group A), empty plasmid control group (group B), BMP-9 or EPO recombinant adenovirus transfected cells (groups C and D), BMP-9 and EPO recombinant adenovirus co-transfected cells (group E). The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase (ALP) activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin (OPN) and osteocalcin (OCN) at 3 weeks. Results At 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot (P < 0.05). The ALP activity significantly increased in group E when compared with that in the other groups at 3, 7, and 14 days after osteogenic induction (P < 0.05); the number of calcium nodules in group E was significantly more than that in the other groups (P < 0.05). Real-time fluorescence quantitative PCR showed that OPN and OCN genes expressions were significantly higher in group E than other groups (P < 0.05), and in groups C and D than groups A and B (P < 0.05). Conclusion Recombinant adenovirus-mediated BMP-9 and EPO genes can transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the expressions of osteoblast-related genes and protein than non-transfected and single gene transfection.

Citation： ZHANGGuangde, SUChengshuai, JINXia, YANGShimao, FANGDianji, GUOYanwei. RECOMBINANT ADENOVIRUS-MEDIATED BONE MORPHOGENETIC PROTEIN 9 AND ERYTHROPOIETIN GENES CO-TRANSFECTION IN PROMOTING OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(3): 272-278. doi: 10.7507/1002-1892.20160055 Copy

1.	Saulnier N, Puglisi MA, Lattanzi W, et al. Gene profiling of bone marrow-and adipose tissue-derived stromal cells: a key role of Kruppel-like factor 4 in cell fate regulation. Cytotherapy, 2011, 13(3): 329-340.
2.	Cramer C, Freisinger E, Jones RK, et al. Persistent high glucose concentrations alter the regenerative potential of mesenchymal stem cells. Stem Cells Dev, 2010, 19(12): 1875-1884.
3.	Cecchinato F, Karlsson J, Ferroni L, et al. Osteogenic potential of human adipose-derived stromal cells on 3-dimensional mesoporous TiO2 coating with magnesium impregnation. Mater Sci Eng C Mater Biol Appl, 2015, 52: 225-234.
4.	張嘉熙, 史冊, 劉姍姍, 等.促紅細胞生成素促進骨髓基質細胞成骨分化的實驗研究.口腔頜面外科雜志, 2014, 24(1): 21-26.
5.	張燕, 文巍, 羅進勇.骨形態發生蛋白9定向誘導多潛能干細胞成骨分化.生物化學與生物物理進展, 2009, 36(10): 1291-1298.
6.	張嘉熙, 鄧紅燕, 紀茗權, 等.促紅細胞生成素及其受體對成骨細胞的作用機制.國際口腔醫學雜志, 2015, 42(1): 102-105.
7.	A?il Y, Ghoniem AA, Wiltfang J, et al. Optimizing the osteogenic differentiation of human mesenchymal stromal cells by the synergistic action of growth factors. J Craniomaxillofac Surg, 2014, 42(8): 2002-2009.
8.	Barretto LS, Lessio C, Sawaki e Nakamura AN, et al. Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells. In Vitro Cell Dev Biol Anim, 2014, 50(9): 831-839.
9.	Lee HR, Kim HJ, Ko JS, et al. Comparative characteristics of porous bioceramics for an osteogenic response in vitro and in vivo. PLoS One, 2013, 8(12): e84272.
10.	Tsigkou O, Labbaf S, Stevens MM, et al. Monodispersed bioactive glass submicron particles and their effect on bone marrow and adipose tissue-derived stem cells. Adv Healthc Mater, 2014, 3(1): 115-125.
11.	Kang H, Dang AB, Joshi SK, et al. Novel mouse model of spinal cord injury-induced heterotopic ossification. J Rehabil Res Dev, 2014, 51(7): 1109-1118.
12.	Zhu D, Mackenzie NC, Shanahan CM, et al. BMP-9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway. Cell Mol Med, 2015, 19(1): 165-174.
13.	Huang J, Yuan SX, Wang DX, et al. The role of COX-2 in mediating the effect of PTEN on BMP9 induced osteogenic differentiation in mouse embryonic fibroblasts. Biomaterials, 2014, 35(36): 9649-9659.
14.	Wei Z, Salmon RM, Upton PD, et al. Regulation of bone morphogenetic protein 9 (BMP9) by redox-dependent proteolysis. J Biol Chem, 2014, 289(45): 31150-31159.
15.	Li C, Shi C, Kim J, et al. Erythropoietin promotes bone formation through EphrinB2/EphB4 signaling. J Dent Res, 2015, 94(3): 455-463.
16.	Li C, Ding J, Jiang L, et al. Potential of mesenchymal stem cells by adenovirus-mediated erythropoietin gene therapy approaches for bone defect. Cell Biochem Biophys, 2014, 70(2): 1199-1204.
17.	R?lfing JH, Baatrup A, Stiehler M, et al. The osteogenic effect of erythropoietin on human mesenchymal stromal cells is dose-dependent and involves non-hematopoietic receptors and multiple intracellular signaling pathways. Stem Cell Rev, 2014, 10(1): 69-78.
18.	Aksoy C, Guliyev A, Kilic E, et al. Bone marrow mesenchymal stem cells in patients with beta thalassemia major: molecular analysis with attenuated total reflection-Fourier transform infrared spectroscopy study as a novel method. Stem Cells Dev, 2012, 21(11): 2000-2011.
19.	Kim J, Jung Y, Sun H, et al. Erythropoietin mediated bone formation is regulated by mTOR signaling. Cell Biochem, 2012, 113(1): 220-228.
20.	McGee SJ, Havens AM, Shiozawa Y, et al. Effects of erythropoietin on the bone microenvironment. Growth Factors, 2012, 30(1): 22-28.

1. Saulnier N, Puglisi MA, Lattanzi W, et al. Gene profiling of bone marrow-and adipose tissue-derived stromal cells: a key role of Kruppel-like factor 4 in cell fate regulation. Cytotherapy, 2011, 13(3): 329-340.
2. Cramer C, Freisinger E, Jones RK, et al. Persistent high glucose concentrations alter the regenerative potential of mesenchymal stem cells. Stem Cells Dev, 2010, 19(12): 1875-1884.
3. Cecchinato F, Karlsson J, Ferroni L, et al. Osteogenic potential of human adipose-derived stromal cells on 3-dimensional mesoporous TiO2 coating with magnesium impregnation. Mater Sci Eng C Mater Biol Appl, 2015, 52: 225-234.
4. 張嘉熙, 史冊, 劉姍姍, 等.促紅細胞生成素促進骨髓基質細胞成骨分化的實驗研究.口腔頜面外科雜志, 2014, 24(1): 21-26.
5. 張燕, 文巍, 羅進勇.骨形態發生蛋白9定向誘導多潛能干細胞成骨分化.生物化學與生物物理進展, 2009, 36(10): 1291-1298.
6. 張嘉熙, 鄧紅燕, 紀茗權, 等.促紅細胞生成素及其受體對成骨細胞的作用機制.國際口腔醫學雜志, 2015, 42(1): 102-105.
7. A?il Y, Ghoniem AA, Wiltfang J, et al. Optimizing the osteogenic differentiation of human mesenchymal stromal cells by the synergistic action of growth factors. J Craniomaxillofac Surg, 2014, 42(8): 2002-2009.
8. Barretto LS, Lessio C, Sawaki e Nakamura AN, et al. Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells. In Vitro Cell Dev Biol Anim, 2014, 50(9): 831-839.
9. Lee HR, Kim HJ, Ko JS, et al. Comparative characteristics of porous bioceramics for an osteogenic response in vitro and in vivo. PLoS One, 2013, 8(12): e84272.
10. Tsigkou O, Labbaf S, Stevens MM, et al. Monodispersed bioactive glass submicron particles and their effect on bone marrow and adipose tissue-derived stem cells. Adv Healthc Mater, 2014, 3(1): 115-125.
11. Kang H, Dang AB, Joshi SK, et al. Novel mouse model of spinal cord injury-induced heterotopic ossification. J Rehabil Res Dev, 2014, 51(7): 1109-1118.
12. Zhu D, Mackenzie NC, Shanahan CM, et al. BMP-9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway. Cell Mol Med, 2015, 19(1): 165-174.
13. Huang J, Yuan SX, Wang DX, et al. The role of COX-2 in mediating the effect of PTEN on BMP9 induced osteogenic differentiation in mouse embryonic fibroblasts. Biomaterials, 2014, 35(36): 9649-9659.
14. Wei Z, Salmon RM, Upton PD, et al. Regulation of bone morphogenetic protein 9 (BMP9) by redox-dependent proteolysis. J Biol Chem, 2014, 289(45): 31150-31159.
15. Li C, Shi C, Kim J, et al. Erythropoietin promotes bone formation through EphrinB2/EphB4 signaling. J Dent Res, 2015, 94(3): 455-463.
16. Li C, Ding J, Jiang L, et al. Potential of mesenchymal stem cells by adenovirus-mediated erythropoietin gene therapy approaches for bone defect. Cell Biochem Biophys, 2014, 70(2): 1199-1204.
17. R?lfing JH, Baatrup A, Stiehler M, et al. The osteogenic effect of erythropoietin on human mesenchymal stromal cells is dose-dependent and involves non-hematopoietic receptors and multiple intracellular signaling pathways. Stem Cell Rev, 2014, 10(1): 69-78.
18. Aksoy C, Guliyev A, Kilic E, et al. Bone marrow mesenchymal stem cells in patients with beta thalassemia major: molecular analysis with attenuated total reflection-Fourier transform infrared spectroscopy study as a novel method. Stem Cells Dev, 2012, 21(11): 2000-2011.
19. Kim J, Jung Y, Sun H, et al. Erythropoietin mediated bone formation is regulated by mTOR signaling. Cell Biochem, 2012, 113(1): 220-228.
20. McGee SJ, Havens AM, Shiozawa Y, et al. Effects of erythropoietin on the bone microenvironment. Growth Factors, 2012, 30(1): 22-28.

Chinese Journal of Reparative and Reconstructive Surgery

RECOMBINANT ADENOVIRUS-MEDIATED BONE MORPHOGENETIC PROTEIN 9 AND ERYTHROPOIETIN GENES CO-TRANSFECTION IN PROMOTING OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN VITRO

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