All-trans retinoic acid and vascular endothelial growth factor induced the directional osteogenic differentiation of mouse embryonic fibroblasts_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

FENG Wei ,  TU Xiaolin

Life Science Institute, Chongqing Medical University, Chongqing, 400016, P.R.China;

Corresponding author：

TU Xiaolin, Email: xiaolint@hotmail.com

Keywords：

All-trans retinoic acid; vascular endothelial growth factor; mouse embryonic fibroblasts; osteogenic differentiation; angiogenesis

DOI：

10.7507/1002-1892.201906099

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Objective To investigate the effect of all-trans retinoic acid (ATRA) and vascular endothelial growth factor (VEGF) on the osteogenic differentiation of mouse embryonic fibroblasts (MEFs).Methods The fetal mice in the uterus of NIH pregnant mice (pregnancy 12-15 days) were collected, and the heads and hearts etc. were removed. Then MEFs were separated from the rest tissues of the fetal mice and cultured by trypsin digestion and adherent culture. HEK-293 cells were used to obtain recombinant adenovirus-red fluorescent protein (Ad-RFP) and Ad-VEGF by repeatedly freezing and thawing. Alkaline phosphatase (ALP) staining and quantitative detection were used to detect the changes of ALP activity in MEFs applied with ATRA or VEGF alone or combined use of ATRA and VEGF on the 3rd and 5th days. The cultured 3rd to 4th generation MEFs were divided into groups A, B, C, and D, and were cultured with DMSO plus Ad-RFP, ATRA, Ad-VEGF, ATRA plus Ad-VEGF, respectively. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the mRNA expressions of osteogenic markers including ALP, collagen type Ⅰ, osteopontin (OPN), osteocalcin (OCN), and angiogenic markers including VEGF, angiopoietin 1 (ANGPT1), and endomucin (EMCN) on the 3rd and 7th days. Immunohistochemical staining was used to detect the protein expressions of OPN and VEGF on the 3rd, 5th, and 7th days in each group. Alizarin red staining was used to detect calcium salt deposition levels in each group at 14 and 21 days after osteogenic induction. Fifteen athymic female nude mice aged 4 to 6 weeks were randomly divided into 3 groups and 5 mice in each group. Then MEFs treated with ATRA, Ad-VEGF, and ATRA plus Ad-VEGF were injected subcutaneously into the dorsal and ventral sides, respectively. X-ray observation, gross observation, and histological staining (Masson, HE, and Safranin O-fast green stainings) were performed at 5 weeks after implantation to observe the ectopic bone formation in nude mice in each group.Results MEFs were successfully isolated and cultured. The acquired Ad-RFP and Ad-VEGF were successfully transfected into MEFs with approximately 50% and 20% transfection rates. ALP activity tests showed that ATRA or Ad-VEGF could enhance ALP activity in MEFs (P<0.05), and ATRA had a stronger effect than Ad-VEGF; and the combined use of ATRA and Ad-VEGF significantly enhanced the ALP activity in MEFs (P<0.05). qRT-PCR test showed that the combined use of ATRA and Ad-VEGF also increased the relative mRNA expressions of early-stage osteogenesis-related markers ALP, OPN, and collagen type I (P<0.05); the relative mRNA expressions of angiogenesis-related markers VEGF, EMCN, and ANGPT1 increased at 7 days (P<0.05). Immunohistochemical staining showed that ATRA combined with Ad-VEGF not only enhanced OPN protein expression, but also increased VEGF protein expression on 7th day. Alizarin red staining showed that the application of ATRA or Ad-VEGF induced weak calcium salt deposition, and the combined use of ATRA and Ad-VEGF significantly enhanced the effect of calcium salt deposition in MEFs. The results of implantation experiments in nude mice showed that X-ray films observation revealed obvious bone mass in the ATRA plus Ad-VEGF group, and the bone was larger than that in other groups. Histological staining showed a large amount of collagen and mature bone trabeculae, bone matrix formation, and gray-green collagen bone tissue, indicating that the combined use of ATRA and Ad-VEGF significantly enhanced the osteogenic effect of MEFs in vivo.Conclusion The combined use of ATRA and VEGF can induce the osteogenic differentiation of MEFs.

Citation： FENG Wei, TU Xiaolin. All-trans retinoic acid and vascular endothelial growth factor induced the directional osteogenic differentiation of mouse embryonic fibroblasts. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(2): 246-255. doi: 10.7507/1002-1892.201906099 Copy

1.	才越, 王绪凯. 骨组织工程化构建中复合支架的研究进展. 口腔生物医学, 2016, 7(4): 200-203.
2.	王福科, 张红, 李彦林, 等. 联合培养血管内皮细胞与脂肪干细胞构建组织工程骨异位成骨. 中国修复重建外科杂志, 2019, 33(10): 1310-1319.
3.	Arthur A, Zannettino A, Gronthos S. The therapeutic applications of multipotential mesenchymal/stromal stem cells in skeletal tissue repair. J Cell Physiol, 2009, 218(2): 237-245.
4.	赵斌, 马剑雄, 马信龙. 组织工程周围神经血管化研究进展. 中国修复重建外科杂志, 2019, 33(8): 1029-1032.
5.	Weng Z, Wang C, Zhang C, et al. All-trans retinoic acid promotes osteogenic differentiation and bone consolidation in a rat distraction osteogenesis model. Calcif Tissue Int, 2019, 104(3): 320-330.
6.	Pourjafar M, Saidijam M, Mansouri K, et al. All-trans retinoic acid preconditioning enhances proliferation, angiogenesis and migration of mesenchymal stem cell in vitro and enhances wound repair in vivo. Cell Prolif, 2017, 50(1).doi: 10.1111/cpr.12315.
7.	刘子铭. HIF1α 上调 Runx2 促进 BMP9 诱导的间充质干细胞成骨分化与血管形成机制研究. 重庆: 重庆医科大学, 2019.
8.	Siddikuzzaman, Guruvayoorappan C, Berlin Grace VM. All trans retinoic acid and cancer. Immunopharmacol Immunotoxicol, 2011, 33(2): 241-249.
9.	Clagett-Dame M, McNeill EM, Muley PD. Role of all-trans retinoic acid in neurite outgrowth and axonal elongation. J Neurobiol, 2006, 66(7): 739-756.
10.	Vaughan MR, Pippin JW, Griffin SV, et al. ATRA induces podocyte differentiation and alters nephrin and podocin expression in vitro and in vivo. Kidney Int, 2005, 68(1): 133-144.
11.	Zhang W, Deng ZL, Chen L, et al. Retinoic acids potentiate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. PLoS One, 2010, 5(7): e11917.
12.	Yan Q, Li Y, Cheng N, et al. Effect of retinoic acid on the function of lipopolysaccharide-stimulated bone marrow stromal cells grown on titanium surfaces. Inflamma Res, 2015, 64(1): 63-70.
13.	Henning P, Conaway HH, Lerner UH. Retinoid receptors in bone and their role in bone remodeling. Front Endocrinol (Lausanne), 2015, 6: 31.
14.	Wan DC, Siedhoff MT, Kwan MD, et al. Refining retinoic acid stimulation for osteogenic differentiation of murine adipose-derived adult stromal cells. Tissue Eng, 2007, 13(7): 1623-1631.
15.	Song HM, Nacamuli RP, Xia W, et al. High-dose retinoic acid modulates rat calvarial osteoblast biology. J Cell Physiol, 2005, 202(1): 255-262.
16.	Feskanich D, Singh V, Willett WC, et al. Vitamin A intake and hip fractures among postmenopausal women. JAMA, 2002, 287(1): 47-54.
17.	Michaëlsson K, Lithell H, Vessby B, et al. Serum retinol levels and the risk of fracture. N Engl J Med, 2003, 348(4): 287-294.
18.	Iba K, Chiba H, Yamashita T, et al. Phase-independent inhibition by retinoic acid of mineralization correlated with loss of tetranectin expression in a human osteoblastic cell line. Cell Struct Funct, 2001, 26(4): 227-233.
19.	Wiper-Bergeron N, St-Louis C, Lee JM. CCAAT/Enhancer binding protein beta abrogates retinoic acid-induced osteoblast differentiation via repression of Runx2 transcription. Mol Endocrinol, 2007, 21(9): 2124-2135.
20.	Hisada K, Hata K, Ichida F, et al. Retinoic acid regulates commitment of undifferentiated mesenchymal stem cells into osteoblasts and adipocytes. J Bone Miner Metab, 2013, 31(1): 53-63.
21.	Wan DC, Shi YY, Nacamuli RP, et al. Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor typeⅠB signaling. Proc Natl Acad Sci U S A, 2006, 103(33): 12335-12340.
22.	Jabalee J, Franz-Odendaal TA. Vascular endothelial growth factor signaling affects both angiogenesis and osteogenesis during the development of scleral ossicles. Dev Biol, 2015, 406(1): 52-62.
23.	Street J, Bao M, deGuzman L, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci U S A, 2002, 99(15): 9656-9661.
24.	Liu Y, Berendsen AD, Jia S, et al. Intracellular VEGF regulates the balance between osteoblast and adipocyte differentiation. J Clin Invest, 2012, 122(9): 3101-3113.
25.	Chung R, Foster BK, Xian CJ. The potential role of VEGF-induced vascularisation in the bony repair of injured growth plate cartilage. J Endocrinol, 2014, 221(1): 63-75.

1. 才越, 王绪凯. 骨组织工程化构建中复合支架的研究进展. 口腔生物医学, 2016, 7(4): 200-203.
2. 王福科, 张红, 李彦林, 等. 联合培养血管内皮细胞与脂肪干细胞构建组织工程骨异位成骨. 中国修复重建外科杂志, 2019, 33(10): 1310-1319.
3. Arthur A, Zannettino A, Gronthos S. The therapeutic applications of multipotential mesenchymal/stromal stem cells in skeletal tissue repair. J Cell Physiol, 2009, 218(2): 237-245.
4. 赵斌, 马剑雄, 马信龙. 组织工程周围神经血管化研究进展. 中国修复重建外科杂志, 2019, 33(8): 1029-1032.
5. Weng Z, Wang C, Zhang C, et al. All-trans retinoic acid promotes osteogenic differentiation and bone consolidation in a rat distraction osteogenesis model. Calcif Tissue Int, 2019, 104(3): 320-330.
6. Pourjafar M, Saidijam M, Mansouri K, et al. All-trans retinoic acid preconditioning enhances proliferation, angiogenesis and migration of mesenchymal stem cell in vitro and enhances wound repair in vivo. Cell Prolif, 2017, 50(1).doi: 10.1111/cpr.12315.
7. 刘子铭. HIF1α 上调 Runx2 促进 BMP9 诱导的间充质干细胞成骨分化与血管形成机制研究. 重庆: 重庆医科大学, 2019.
8. Siddikuzzaman, Guruvayoorappan C, Berlin Grace VM. All trans retinoic acid and cancer. Immunopharmacol Immunotoxicol, 2011, 33(2): 241-249.
9. Clagett-Dame M, McNeill EM, Muley PD. Role of all-trans retinoic acid in neurite outgrowth and axonal elongation. J Neurobiol, 2006, 66(7): 739-756.
10. Vaughan MR, Pippin JW, Griffin SV, et al. ATRA induces podocyte differentiation and alters nephrin and podocin expression in vitro and in vivo. Kidney Int, 2005, 68(1): 133-144.
11. Zhang W, Deng ZL, Chen L, et al. Retinoic acids potentiate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. PLoS One, 2010, 5(7): e11917.
12. Yan Q, Li Y, Cheng N, et al. Effect of retinoic acid on the function of lipopolysaccharide-stimulated bone marrow stromal cells grown on titanium surfaces. Inflamma Res, 2015, 64(1): 63-70.
13. Henning P, Conaway HH, Lerner UH. Retinoid receptors in bone and their role in bone remodeling. Front Endocrinol (Lausanne), 2015, 6: 31.
14. Wan DC, Siedhoff MT, Kwan MD, et al. Refining retinoic acid stimulation for osteogenic differentiation of murine adipose-derived adult stromal cells. Tissue Eng, 2007, 13(7): 1623-1631.
15. Song HM, Nacamuli RP, Xia W, et al. High-dose retinoic acid modulates rat calvarial osteoblast biology. J Cell Physiol, 2005, 202(1): 255-262.
16. Feskanich D, Singh V, Willett WC, et al. Vitamin A intake and hip fractures among postmenopausal women. JAMA, 2002, 287(1): 47-54.
17. Michaëlsson K, Lithell H, Vessby B, et al. Serum retinol levels and the risk of fracture. N Engl J Med, 2003, 348(4): 287-294.
18. Iba K, Chiba H, Yamashita T, et al. Phase-independent inhibition by retinoic acid of mineralization correlated with loss of tetranectin expression in a human osteoblastic cell line. Cell Struct Funct, 2001, 26(4): 227-233.
19. Wiper-Bergeron N, St-Louis C, Lee JM. CCAAT/Enhancer binding protein beta abrogates retinoic acid-induced osteoblast differentiation via repression of Runx2 transcription. Mol Endocrinol, 2007, 21(9): 2124-2135.
20. Hisada K, Hata K, Ichida F, et al. Retinoic acid regulates commitment of undifferentiated mesenchymal stem cells into osteoblasts and adipocytes. J Bone Miner Metab, 2013, 31(1): 53-63.
21. Wan DC, Shi YY, Nacamuli RP, et al. Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor typeⅠB signaling. Proc Natl Acad Sci U S A, 2006, 103(33): 12335-12340.
22. Jabalee J, Franz-Odendaal TA. Vascular endothelial growth factor signaling affects both angiogenesis and osteogenesis during the development of scleral ossicles. Dev Biol, 2015, 406(1): 52-62.
23. Street J, Bao M, deGuzman L, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci U S A, 2002, 99(15): 9656-9661.
24. Liu Y, Berendsen AD, Jia S, et al. Intracellular VEGF regulates the balance between osteoblast and adipocyte differentiation. J Clin Invest, 2012, 122(9): 3101-3113.
25. Chung R, Foster BK, Xian CJ. The potential role of VEGF-induced vascularisation in the bony repair of injured growth plate cartilage. J Endocrinol, 2014, 221(1): 63-75.

Chinese Journal of Reparative and Reconstructive Surgery

All-trans retinoic acid and vascular endothelial growth factor induced the directional osteogenic differentiation of mouse embryonic fibroblasts

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