EFFECTS OF CO-TRANSFECTION BY BONE MORPHOGENETIC PROTEIN 14 AND Noggin SHORT HAIRPIN RNA LENTIVIRAL VECTORS ON OSTEOGENIC DIFFERENTIATION OF ADIPOSE DERIVED STEM CELLS IN RATS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIUHao ¹ , RUIYongjun ¹ , SUNZhenzhong ¹ , TANGFenglin ² ,  DINGTao ²

1. Department of Orthopedics, Wuxi 9th People's Hospital, Wuxi Jiangsu, 214000, P. R. China;
2. Department of Orthopedics, Wuxi People's Hospital;

Corresponding author：

DINGTao, Email: drdingtao@163.com

Keywords：

Bone morphogenetic protein 14; Noggin; Lentivirus; Co-transfection; Adipose derived stem cells; Rat

DOI：

10.7507/1002-1892.20160259

Video：

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Objective To explore the effects on osteogenic differentiation of adipose derived stem cells (ADSCs) by simultaneously down-regulating Noggin combined with up-regulating bone morphogenetic protein 14 (BMP-14) in vitro. Methods Primary ADSCs were isolated and expanded in vitro from 5 Sprague Dawley rats (weighing, 250-300 g). ADSCs were transfected with lentiviral (Lv)-enhanced green fluorescent protein in group A (control group), with Lv-BMP-14 in group B, and with Lv-BMP-14 and Lv-Noggin shRNA in group C. BMP-14 and osteogenesis-related genes[collagen type I, alkaline phosphatase (ALP), and osteocalcin (OCN)] mRNA expression levels were detected by real time fluorescence quantitative PCR at 3, 7, and 14 days after transfection. Alizarin red staining for calcium nodules was also employed to assess the osteogenic ability of co-transfected ADSCs. Results At 3 days after transfection, no significant difference was found in BMP-14 mRNA expression among groups P>0.05). At 7 and 14 days after transfection, BMP-14 mRNA expression was significantly higher in group C than groups A and B, and in group B than group A (P<0.05). At 3 days after transfection, collagen type I, ALP, and OCN mRNA expressions of group C were significantly higher than those of groups A and B (P<0.05), but no significant difference was shown between groups A and B P>0.05). At 7 and 14 days, collagen type I, ALP, and OCN mRNA expressions were higher in group C than groups A and B, and in group B than group A, showing significant difference (P<0.05) except collagen type I mRNA expression at 7 days between groups A and B P>0.05). The results of alizarin red staining showed that the amount of calcium nodules presented an increased tendency in the order of group A, group B, and group C. Conclusion BMP-14 is capable of enhancing osteogenic differentiation of ADSCs. A combination of inhibiting Noggin gene expression and enhancing BMP-14 gene expression in ADSCs can significantly strengthen osteogenic differentiation capability, showing significant synergistic effect.

Citation： LIU Hao, RUI Yongjun, SUN Zhenzhong, TANG Fenglin, DING Tao. EFFECTS OF CO-TRANSFECTION BY BONE MORPHOGENETIC PROTEIN 14 AND Noggin SHORT HAIRPIN RNA LENTIVIRAL VECTORS ON OSTEOGENIC DIFFERENTIATION OF ADIPOSE DERIVED STEM CELLS IN RATS. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(10): 1270-1275. doi: 10.7507/1002-1892.20160259 Copy

1.	孔劲松, 阮建伟, 黄杨, 等. rhBMP-2、VEGF165双基因转染脂肪干细胞复合支架对体外成骨分化的影响. 浙江创伤外科, 2016, 21(4):608-610.
2.	Urist MR. Bone:formation by autoinduction. Science, 1965, 150 (3698):893-899.
3.	Chen G, Deng C, Li YP. TGF-beta and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci, 2012, 8(2):272-288.
4.	Jin L, Li X. Growth differentiation factor 5 regulation in bone regeneration. Curr Pharm Des, 2013, 19(19):3364-3373.
5.	Mulloy B, Rider CC. The bone morphogenetic proteins and their antagonists. Vitam Horm, 2015, 99:63-90.
6.	Fan J, Im CS, Guo M, et al. Enhanced osteogenesis of adipose-derived stem cells by regulating bone morphogenetic protein signaling antagonists and agonists. Stem Cells Transl Med, 2016, 5(4):539-551.
7.	雷蕙嘉, 孙建军, 彭本刚, 等. 大鼠脂肪干细胞的分离培养及鉴定的初步研究. 中国实验诊断学, 2011, 15(8):1245-1248.
8.	Venkatesan J, Bhatnagar I, Manivasagan P, et al. Alginate composites for bone tissue engineering:a review. Int J Biol Macromol, 2015, 72:269-281.
9.	Chen G, Dong C, Yang L, et al. 3D scaffolds with different stiffness but the same microstructure for bone tissue engineering. ACS Appl Mater Interfaces, 2015, 7(29):15790-15802.
10.	Hernigou P. Bone transplantation and tissue engineering. Part Ⅱ:bone graft and osteogenesis in the seventeenth, eighteenth and nineteenth centuries (Duhamel, Haller, Ollier and MacEwen). Int Orthop, 2015, 39(1):193-204.
11.	Lu CH, Chang YH, Lin SY, et al. Recent progresses in gene delivery-based bone tissue engineering. Biotechnol Adv, 2013, 31(8):1695-1706.
12.	Lei Q, Chen J, Huang W, et al. Proteomic analysis of the effect of extracellular calcium ions on human mesenchymal stem cells:Implications for bone tissue engineering. Chem Biol Interact, 2015, 233:139-146.
13.	Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies. Tissue Eng, 2001, 7(2):211-228.
14.	张淦, 程迅生. 骨组织工程的研究进展. 安徽医学, 2016, 8:1057-1061.
15.	Jin L, Li X. Growth differentiation factor 5 regulation in bone regeneration. Curr Pharm Des, 2013, 19(19):3364-3373.
16.	Gruber R, Mayer C, Schulz W, et al. Stimulatory effects of cartilage-derived morphogenetic proteins 1 and 2 on osteogenic differentiation of bone marrow stromal cells. Cytokine, 2000, 12(11):1630-1638.
17.	Zeng Q, Li X, Choi L, et al. Recombinant growth/differentiation factor-5 stimulates osteogenic differentiation of fat-derived stromal cells in vitro. Connect Tissue Res, 2006, 47(5):264-270.
18.	Murphy MK, Huey DJ, Hu JC, et al. TGF-beta1, GDF-5, and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells. Stem Cells, 2015, 33(3):762-773.
19.	Gruber R, Mayer C, Bobacz K, et al. Effects of cartilage-derived morphogenetic proteins and osteogenic protein-1 on osteochondrogenic differentiation of periosteum-derived cells. Endocrinology, 2001, 142(5):2087-2094.
20.	方成, 胡永成, 陈晓鹏. 基因调控技术在骨组织工程中应用的研究进展. 中国矫形外科杂志, 2014, 22(20):1861-1867.
21.	Mulligan RC. Development of gene transfer technology. Hum Gene Ther, 2014, 25(12):995-1002.
22.	肖振涛, 郭中凯, 郭立新. 基因强化骨组织工程中的病毒载体. 中国组织工程研究, 2015, 19(2):272-276.
23.	Sukul M, Nguyen TB, Min YK, et al. Effect of local sustainable release of BMP2-VEGF from nano-cellulose loaded in sponge biphasic calcium phosphate on bone regeneration. Tissue Eng Part A, 2015, 21(11-12):1822-1836.
24.	Zhang W, Zhang X, Ling J, et al. Osteo-/odontogenic differentiation of BMP2 and VEGF gene-co-transfected human stem cells from apical papilla. Mol Med Rep, 2016, 13(5):3747-3754.
25.	Ramasubramanian A, Shigi S, Lee GK, et al. Non-viral delivery of inductive and suppressive genes to adipose-derived stem cells for osteogenic differentiation. Pharm Res, 2011, 28(6):1328-1337.
26.	Barati D, Shariati SR, Moeinzadeh S, et al. Spatiotemporal release of BMP-2 and VEGF enhances osteogenic and vasculogenic differentiation of human mesenchymal stem cells and endothelial colony-forming cells co-encapsulated in a patterned hydrogel. J Control Release, 2016, 223:126-136.

1. 孔劲松, 阮建伟, 黄杨, 等. rhBMP-2、VEGF165双基因转染脂肪干细胞复合支架对体外成骨分化的影响. 浙江创伤外科, 2016, 21(4):608-610.
2. Urist MR. Bone:formation by autoinduction. Science, 1965, 150 (3698):893-899.
3. Chen G, Deng C, Li YP. TGF-beta and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci, 2012, 8(2):272-288.
4. Jin L, Li X. Growth differentiation factor 5 regulation in bone regeneration. Curr Pharm Des, 2013, 19(19):3364-3373.
5. Mulloy B, Rider CC. The bone morphogenetic proteins and their antagonists. Vitam Horm, 2015, 99:63-90.
6. Fan J, Im CS, Guo M, et al. Enhanced osteogenesis of adipose-derived stem cells by regulating bone morphogenetic protein signaling antagonists and agonists. Stem Cells Transl Med, 2016, 5(4):539-551.
7. 雷蕙嘉, 孙建军, 彭本刚, 等. 大鼠脂肪干细胞的分离培养及鉴定的初步研究. 中国实验诊断学, 2011, 15(8):1245-1248.
8. Venkatesan J, Bhatnagar I, Manivasagan P, et al. Alginate composites for bone tissue engineering:a review. Int J Biol Macromol, 2015, 72:269-281.
9. Chen G, Dong C, Yang L, et al. 3D scaffolds with different stiffness but the same microstructure for bone tissue engineering. ACS Appl Mater Interfaces, 2015, 7(29):15790-15802.
10. Hernigou P. Bone transplantation and tissue engineering. Part Ⅱ:bone graft and osteogenesis in the seventeenth, eighteenth and nineteenth centuries (Duhamel, Haller, Ollier and MacEwen). Int Orthop, 2015, 39(1):193-204.
11. Lu CH, Chang YH, Lin SY, et al. Recent progresses in gene delivery-based bone tissue engineering. Biotechnol Adv, 2013, 31(8):1695-1706.
12. Lei Q, Chen J, Huang W, et al. Proteomic analysis of the effect of extracellular calcium ions on human mesenchymal stem cells:Implications for bone tissue engineering. Chem Biol Interact, 2015, 233:139-146.
13. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies. Tissue Eng, 2001, 7(2):211-228.
14. 张淦, 程迅生. 骨组织工程的研究进展. 安徽医学, 2016, 8:1057-1061.
15. Jin L, Li X. Growth differentiation factor 5 regulation in bone regeneration. Curr Pharm Des, 2013, 19(19):3364-3373.
16. Gruber R, Mayer C, Schulz W, et al. Stimulatory effects of cartilage-derived morphogenetic proteins 1 and 2 on osteogenic differentiation of bone marrow stromal cells. Cytokine, 2000, 12(11):1630-1638.
17. Zeng Q, Li X, Choi L, et al. Recombinant growth/differentiation factor-5 stimulates osteogenic differentiation of fat-derived stromal cells in vitro. Connect Tissue Res, 2006, 47(5):264-270.
18. Murphy MK, Huey DJ, Hu JC, et al. TGF-beta1, GDF-5, and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells. Stem Cells, 2015, 33(3):762-773.
19. Gruber R, Mayer C, Bobacz K, et al. Effects of cartilage-derived morphogenetic proteins and osteogenic protein-1 on osteochondrogenic differentiation of periosteum-derived cells. Endocrinology, 2001, 142(5):2087-2094.
20. 方成, 胡永成, 陈晓鹏. 基因调控技术在骨组织工程中应用的研究进展. 中国矫形外科杂志, 2014, 22(20):1861-1867.
21. Mulligan RC. Development of gene transfer technology. Hum Gene Ther, 2014, 25(12):995-1002.
22. 肖振涛, 郭中凯, 郭立新. 基因强化骨组织工程中的病毒载体. 中国组织工程研究, 2015, 19(2):272-276.
23. Sukul M, Nguyen TB, Min YK, et al. Effect of local sustainable release of BMP2-VEGF from nano-cellulose loaded in sponge biphasic calcium phosphate on bone regeneration. Tissue Eng Part A, 2015, 21(11-12):1822-1836.
24. Zhang W, Zhang X, Ling J, et al. Osteo-/odontogenic differentiation of BMP2 and VEGF gene-co-transfected human stem cells from apical papilla. Mol Med Rep, 2016, 13(5):3747-3754.
25. Ramasubramanian A, Shigi S, Lee GK, et al. Non-viral delivery of inductive and suppressive genes to adipose-derived stem cells for osteogenic differentiation. Pharm Res, 2011, 28(6):1328-1337.
26. Barati D, Shariati SR, Moeinzadeh S, et al. Spatiotemporal release of BMP-2 and VEGF enhances osteogenic and vasculogenic differentiation of human mesenchymal stem cells and endothelial colony-forming cells co-encapsulated in a patterned hydrogel. J Control Release, 2016, 223:126-136.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECTS OF CO-TRANSFECTION BY BONE MORPHOGENETIC PROTEIN 14 AND Noggin SHORT HAIRPIN RNA LENTIVIRAL VECTORS ON OSTEOGENIC DIFFERENTIATION OF ADIPOSE DERIVED STEM CELLS IN RATS

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