BACKGROUND:Studies have indicated that melatonin can promote the differentiation of adipose-derived stem cels into neurons, and the effect of melatonin on the osteoblasts form adipose-derived stem cels is rarely reported. OBJECTIVE:To observe the osteogenic differentiation of adipose-derived stem cels and the effect of melatonin on the bio-viability of differentiated cels. METHODS:(1) Adipose-derived stem cels were isolated and purified from the inguinal fat of Kunming mice by type I colagenase 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 cels. 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 cels 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 cels and detect alkaline phosphatase activity after 3 days and 6 days respectively. RESULTS AND CONCLUSION: (1) After seeding for 48 hours, most cels were adherent, and after 4 days, the cels displayed multiple shapes and colonies of different sizes formed. After subculture, cel morphology homogenized as spindle shape. Cels positive for CD44 were brownish yelow, and localized mainly on the cel membrane. (2) Differentiated cels were positive for von Kossa staining and black sediments scattered in the extracelular matrix. Alkaline phosphatase expressed positively, and brown-black particles, appeared within cels. (3) Melatonin supplement improved the viability of differentiated cels; and 1, 10 and 100 μmol/L was observed as the optimal concentrations both at 24 and 48 hours. (4) The intracelular alkaline phosphatatse activity was increased with time in al the groups (P < 0.05). Compared with the blank group, the intracelular alkaline phosphatase activity in Melatonin groups (1, 10 and 100 μmol/L) had nochanges 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 cels, and improve the activity of intracelular alkaline phosphatase.