In vitro osteogenic induction of human adipose stem cells co-treated with betaine/osteogenesis differentiation medium

Document Type : Original article

Authors

Department of Cellular and Molecular Biology, School of Biology and Institute of Biological Sciences, Damghan University, Damghan, Iran

Abstract

Human adipose-derived stem cells (hADSCs) are widely used in regenerative medicine and affected by many biochemical and biophysical stimuli in vivo. Betaine has been reported to be a type of osteogenic stimulating biochemical factor. This study aimed to investigate the effects of betaine; on osteogenic differentiation of cultured hADSCs in osteogenesis differentiation medium. Mesenchymal stem cells were extracted from women undergoing liposuction after obtaining written consent and cultured in vitro. The cells at passage 4 were confirmed by flow cytometry and differentiated into osteocytes and adipocytes. Experimental groups were the cells cultured in osteogenesis differentiation medium (control), cultured in α-MEM and 10% serum-containing Betaine (BET) ,and cultured in osteogenesis differentiation medium containing 10 mM Betaine (OD+BET). After 14 and 21 days of treatment, osteogenic differentiation and the expression of RUNX2 and OCN genes were assessed by qualitative and quantitative Alizarin red staining and real-time PCR. There were significant increases in the calcium matrix deposits, alkaline phosphatase activity ,and expression of RUNX2 and OCN genes in the OD+BET group compared to the BET group. At the end of day 14, the calcium matrix formation was significantly decreased the in BET group compared to the control. Treatment of hADSCs with Betaine, and osteogenesis differentiation medium leads to increased alkaline phosphatase activity, matrix calcium deposits and expression of RUNX2 and OCN genes and finally stimulated osteogenesis. This kind of treatment could be used to support bone regeneration in the future of tissue engineering. 

Keywords

References

1.  Cheng KH, Kuo TL, Kuo KK, Hsiao GM. Human adipose-derived stem cells: Isolation, characterization and current application in regeneration medicine. Genomic  Medicine, Biomarker and Health Society 2011;3:53-62.
2.  Roobrouck VD, Ulloa-Montoya F, Verfaillie CMJEcr. Self-renewal and differentiation capacity of young and aged stem cells. Exp Cell Res 2008;314:1937-1944.
3.  Ciuffi S, Zonefrati R, Brandi ML. Adipose stem cells for bone tissue repair. Clin Cases Miner Bone Metab 2017;14:217-226.
4.  Locke M, Windsor J, Dunbar PR. Human adipose-derived stem cells: isolation, characterization and applications in surgery. ANZ J Surg 2009;79:235-244.
5.  Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13:4279-4295.
6.  Stamatopoulos A, Stamatopoulos T, Gamie Z, Kenanidis E, Ribeiro RDC, Rankin KS, Gerrand C, Dalgarno K, Tsiridis E. Mesenchymal stromal cells for bone sarcoma treatment: Roadmap to clinical practice. J Bone Oncol 2019:100231.
7.  Safwani WKZW, Makpol S, Sathapan S, Chua K. Impact of adipogenic differentiation on stemness and osteogenic gene expression in extensive culture of human adipose-derived stem cells. Arch Med Sci 2014;10:597-606.
8.  Chen HT, Lee MJ, Chen CH, Chuang SC, Chang LF, Ho ML, Hung SH, Fu YC, Wang YH, Wang HI, Wang GJ, Kang L, Cheng JK. Proliferation and differentiation potential of human adipose‐derived mesenchymal stem cells isolated from elderly patients with osteoporotic fractures. J Cell Mol Med 2012;16:582-593.
9.  Hattori H, Masuoka K, Sato M, Ishihara M, Asazuma T, Takase B, Kikuchi M, Nemoto K, Ishihara M. Bone formation using human adipose tissue-derived stromal cells and a biodegradable scaffold. J Biomed Mater Res B Appl Biomater 2006;76:230-239.
10. Marędziak M, Marycz K, Tomaszewski KA, Kornicka K, Henry BM. The influence of aging on the regenerative potential of human adipose derived mesenchymal stem cells. Stem Cells Int 2016;1-15.
11. Barba M, Cicione C, Bernardini C, Michetti F, Lattanzi W. Adipose-derived mesenchymal cells for bone regereneration: state of the art. Biomed Res Int  2013;2013.416391.
12. Li X, Yao J, Wu L, Jing W, Tang W, Lin Y, Tian W, Liu L. Osteogenic induction of adipose‐derived stromal cells: not a requirement for bone formation in vivo. Artif Organs 2010;34:46-54.
13. Lever M, Slow S. The clinical significance of betaine, an osmolyte with a key role in methyl group metabolism. Clin biochem 2010;43:732-744.
14. Craig SA. Betaine in human nutrition. Am J Clin Nutr 2004;80:539-549.
15. Parikh NR, Vaughn CL, Williams LL, Kempson SA. Acute activation of the renal betaine/GABA transporter in response to a decrease in extracellular calcium. ISRN Physiol  2012;2013.
16. Waditee R, Hibino T, Tanaka Y, Nakamura T, Incharoensakdi A, Hayakawa S, Suzuki S, Futsuhara Y, Kawamitsu Y, Takabe T, Takabe T. Functional characterization of betaine/proline transporters in betaine-accumulating mangrove. J Biol Chem 2002;277: 18373-18382.
17. Zhou Y, Holmseth S, Hua R, Lehre AC, Olofsson AM, Poblete-Naredo I, Kempson SA, Danbolt NC. The betaine-GABA transporter (BGT1, slc6a12) is predominantly expressed in the liver and at lower levels in the kidneys and at the brain surface. Am J Physiol Renal Physiol 2011;302:F316-F328.
18. Slow S, Lever M, Chambers ST, George PM. Plasma dependent and independent accumulation of betaine in male and female rat tissues. Physiol Res 2009;58:403-410.
19. Kharbanda KK, Rogers II DD, Mailliard ME, Siford GL, Barak AJ, Beckenhauer HC, Sorrell MF, Tuma DJ. Role of elevated S-adenosylhomocysteine in rat hepatocyte apoptosis: protection by betaine. Biochem Pharmacol 2005;70:1883-1890.
20. Villa I, Senesi P, Montesano A, Ferraretto A, Vacante F, Spinello A, Bottani M, Bolamperti S, Rubinacci A, Luzi L, Terruzzi I. Betaine promotes cell differentiation of human osteoblasts in primary culture. J Transl Med 2017;15:132.
21. Lee MS, Kim MS, Park SY, Kang CW. Effects of betaine on ethanol-stimulated secretion of IGF-I and IGFBP-1 in rat primary hepatocytes: involvement of p42/44 MAPK activation. World J Gastroenterol 2006;12:1718-1722.
22. Song Z, Deaciuc I, Zhou Z, Song M, Chen T, Hill D, McClain CJ. Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis. Am J Physiol Gastrointest Liver Physiol 2007;293:G894-G902.
23. Wang Z, Yao T, Pini M, Zhou Z, Fantuzzi G, Song Z. Betaine improved adipose tissue function in mice fed a high-fat diet: a mechanism for hepatoprotective effect of betaine in nonalcoholic fatty liver disease. Am J Physiol Gastrointest Liver Physiol 2010;298:G634-G642.
24. Lee I. Betaine is a positive regulator of mitochondrial respiration. Biochem Biophys Res Commun 2015;456:621-625.
25. Apicella JM, Lee EC, Bailey BL, Saenz C, Anderson JM, Craig SA, Kraemer WJ, Volek JS, Maresh CM. Betaine supplementation enhances anabolic endocrine and Akt signaling in response to acute bouts of exercise. Eur J Appl Physiol 2013;113:793-802.
26. Yang Q, Yin W, Chen Y, Zhu D, Yin J, Zhang C, Gao Y. Betaine alleviates alcohol-induced osteonecrosis of the femoral head via mTOR signaling pathway regulation. Biomed Pharmacother 2019;120:109486.
27. Veskovic M, Mladenovic D, Milenkovic M, Tosic J, Borozan S, Gopcevic K, Labudovic-Borovic M, Dragutinovic V, Vucevic D, Jorgacevic B, Isakovic A, Trajkovic V, Radosavljevic T. Betaine modulates oxidative stress, inflammation, apoptosis, autophagy, and Akt/mTOR signaling in methionine-choline deficiency-induced fatty liver disease. Eur J Pharmacol. 2019;848:39-48.
28. Li C, Wang Y, Li L, Han Z, Mao S, Wang G. Betaine protects against heat exposure–induced oxidative stress and apoptosis in bovine mammary epithelial cells via regulation of ROS production. Cell Stress Chaperones 2019;24:453-460.
29. Hosseinpur Z, Hashemi SM, Salehi E, Ghazanfari T. Comparison of TGF-β1 and NO production by mesenchymal stem cells isolated from murine lung and adipose tissues. Immunopharmacol Immunotoxicol 2016;38:214-220.
30. Wen L, Wang Y, Wang H, Kong L, Zhang L, Chen X, Ding Y. L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. Biochem Biophys Res Commun 2012;424:439-445.
31. Senesi P, Luzi L, Montesano A, Mazzocchi N, Terruzzi I. Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation. J Transl Med 2013;1:174.
32. Palumbo P, Lombardi F, Siragusa G, Cifone MG, Cinque B, Giuliani M. Methods of isolation, characterization and expansion of human adipose-derived stem cells (ASCs): an overview. Int J Mol Sci 2018;19:1897.
33. Gregory CA, Gunn WG, Peister A, Prockop DJ. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 2004;329:77-84.
34. Seyedjafari E, Soleimani M, Ghaemi N, Shabani I. Nanohydroxyapatite-coated electrospun poly (l-lactide) nanofibers enhance osteogenic differentiation of stem cells and induce ectopic bone formation. Biomacromolecules 2010;11:3118-3125.
35. Arjmand M, Ardeshirylajimi A, Maghsoudi H, Azadian E. Osteogenic differentiation potential of mesenchymal stem cells cultured on nanofibrous scaffold improved in the presence of pulsed electromagnetic field. J Cell Physiol 2018;233:1061-1070.
36. Su CY, Fang T, Fang HW. Effects of Electrostatic Field on Osteoblast Cells for Bone Regeneration Applications. Biomed Res Int  2017;2017:7124817.
37. Tsai MT, Li WJ, Tuan RS, Chang WH. Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation. J Orthop Res 2009;27:1169-1174.
38. Martins JM, Neves JA, Freitas A, Tirapicos JL. Effect of long-term betaine supplementation on chemical and physical characteristics of three muscles from the Alentejano pig. J Sci Food Agric 2012;92:2122-2127.
39. Devol DL, Rotwein P, Sadow JL, Novakofski J, Bechtel PJ. Activation of insulin-like growth factor gene expression during work-induced skeletal muscle growth. Am J Physiol 1990;259:E89-E95.
40. Conejo R, Lorenzo M. Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts. J Cell Physiol 2001;187:96-108.
41. Burattini S, Ferri P, Battistelli M, Curci R, Luchetti F, Falcieri E. C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. Eur J Histochem  2004:223-234.
42. Zahanich I, Graf EM, Heubach JF, Hempel U, Boxberger S, Ravens U. Molecular and functional expression of voltage‐operated calcium channels during osteogenic differentiation of human mesenchymal stem cells. J Bone Miner Res 2005;20:1637-1646.
43. Yang S, Xu H, Yu S, Cao H, Fan J, Ge C, Fransceschi RT, Dong HH, Xiao G. Foxo1 mediates insulin-like growth factor 1 (IGF1)/insulin regulation of osteocalcin expression by antagonizing Runx2 in osteoblasts. J Biol Chem 2011;286:19149-19158.
44. Qiao M, Shapiro P, Kumar R, Passaniti A. Insulin-like growth factor-1 regulates endogenous RUNX2 activity in endothelial cells through a phosphatidylinositol 3-kinase/ERK-dependent and Akt-independent signaling pathway. J Biol Chem  2004;279: 42709-42718.
45. Xue P, Wu X, Zhou L, Ma H, Wang Y, Liu Y, Ma J, Li Y. IGF1 promotes osteogenic differentiation of mesenchymal stem cells derived from rat bone marrow by increasing TAZ expression. Biochem Biophys Res Commun 2013;433:226-231.
46. Chen J, Yuan K, Mao X, Miano JM, Wu H, Chen Y. Serum response factor regulates bone formation via IGF‐1 and Runx2 signals. J Bone Miner Res 2012;27:1659-1668.
47. Zhang M, Xuan S, Bouxsein ML, Von Stechow D, Akeno N, Faugere MC, Malluche H, Zhao G, Rosen CJ, Efstratiadis A, Clemens TL. Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J Biol Chem 2002;277:44005-44012.
48. Yarak S, Okamoto OK. Human adipose-derived stem cells: current challenges and clinical perspectives. An Bras Dermatol  2010;85:647-656.
49. Takemitsu H, Zhao D, Yamamoto I, Harada Y, Michishita M, Arai T. Comparison of bone marrow and adipose tissue-derived canine mesenchymal stem cells. BMC Vet Res 2012; 8:150.
50. Varma MJO, Breuls RG, Schouten TE, Jurgens WJ, Bontkes HJ, Schuurhuis GJ, Ham Van SM, Milligen FJ. Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells. Stem Cells Dev 2007;16:91-104.
51. Zhu Y, Liu T, Song K, Fan X, Ma X, Cui Z. Adipose-derived stem cell: a better stem cell than BMSC. Cell Biochem Funct 2008;26:664-675.
52. Tapp H, Hanley Jr EN, Patt JC, Gruber HE. medicine. Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair. Exp Biol Med 2009; 234:1-9.

Files

Share

How to cite

Statistics