Osteogenic differentiation of adipose-derived stem cells and the effect of melatonin on the bio-viability of differentiated cells

doi:10.3969/j.issn.2095-4344.2015.50.007

Abstract

Abstract:

BACKGROUND: Studies have indicated that melatonin can promote the differentiation of adipose-derived stem cells into neurons, and the effect of melatonin on the osteoblasts form adipose-derived stem cells is rarely reported.
OBJECTIVE: To observe the osteogenic differentiation of adipose-derived stem cells and the effect of melatonin on the bio-viability of differentiated cells.
METHODS: (1) Adipose-derived stem cells were isolated and purified from the inguinal fat of Kunming mice by type I collagenase digestion and differential adhesion method, respectively. Immunohistochemical staining of CD44 was used as a quality control. (2) Osteogenic induction medium was added to induce osteogenic
differentiation of passage 2 adipose-derived stem cells. Alkaline phosphatase staining and von Kossa method were combined to evaluate differentiation condition. (3) Melatonin at variable concentrations was added to treat mature osteocytes originated from adipose-derived stem cells and MTT was applied to determine their viability at 24 and 48 hours after culture respectively to find out optimal condition of melatonin treatment. (4) Melatonin at the optimal concentration was used to treat differentiated cells and detect alkaline phosphatase activity after 3 days and 6 days respectively.
RESULTS AND CONCLUSION: (1) After seeding for 48 hours, most cells were adherent, and after 4 days, the cells displayed multiple shapes and colonies of different sizes formed. After subculture, cell morphology homogenized as spindle shape. Cells positive for CD44 were brownish yellow, and localized mainly on the cell membrane. (2) Differentiated cells were positive for von Kossa staining and black sediments scattered in the extracellular matrix. Alkaline phosphatase expressed positively, and brown-black particles, appeared within cells. (3) Melatonin supplement improved the viability of differentiated cells; and 1, 10 and 100 μmol/L was observed as the optimal concentrations both at 24 and 48 hours. (4) The intracellular alkaline phosphatatse activity was increased with time in all the groups (P < 0.05). Compared with the blank group, the intracellular alkaline phosphatase activity in Melatonin groups (1, 10 and
100 μmol/L) had no changes at 3 days, but significantly increased at 6 days (P < 0.05). These findings indicate that melatonin can enhance the proliferation of osteocytes differentiated from adipose-derived stem cells, and improve the activity of intracellular alkaline phosphatase.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

CLC Number:

R394.2

Lu Tan, Wei Na, Zhang Chao, Dong Yu-zhen. Osteogenic differentiation of adipose-derived stem cells and the effect of melatonin on the bio-viability of differentiated cells[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(50): 8072-8076.

Figures/Tables 6

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