Adipogenesis and osteogenesis, a reciprocal relationship in bone marrow, are complex processes including proliferation of precursor cells, commitment to the specific lineage, and terminal differentiation. Accumulating evidence from in vitro and in vivo studies suggests that melatonin affects terminal differentiation of osteoblasts and adipocytes, but little is known about the effect of melatonin on the process of adipogenesis and osteogenesis, especially adipogenesis. This study was performed to determine the effect of melatonin on adipogenesis and osteogenesis in human mesenchymal stem cells (hMSCs). Cell proliferation assays demonstrated that melatonin had no apparent effect on the proliferation of hMSCs. When melatonin was added to the adipogenic/osteogenic medium, it directly inhibited adipogenesis and simultaneously promoted osteogenesis of hMSCs in a dose-dependent manner. Furthermore, quantitative RT-PCR demonstrated that melatonin significantly suppressed peroxisome proliferator-activated receptor gamma (PPARγ) expression (day 3, 25% decrease; day 6, 47% decrease), but promoted Runx2 expression (day 3, 87% increase; day 6, 56% increase) in the early stages of adipogenesis and osteogenesis of hMSCs. Moreover, melatonin down-regulated several markers of terminal adipocyte differentiation, including leptin (30%), lipoprotein lipase (LPL, 41%), adiponectin (51%), and adipocyte protein 2 (αP2, 45%). Meanwhile, melatonin up-regulated several markers of osteoblast differentiation, including alkaline phosphatase (110%), osteopontin (218%), and osteocalcin (310%). These results suggest that melatonin directly inhibits hMSCs adipogenic differentiation and significantly enhances hMSCs osteogenic differentiation by suppressing PPARγ expression and enhancing Runx2 expression; this provides further evidence for melatonin as an anti-osteoporosis drug.

High-density cell culture is pivotal for the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). Two high-density cell culture systems, micromass and pellet culture, have been used to induce chondrogenic differentiation of hMSCs. In micromass culture, the induced-cartilage tissues were larger, more homogenous and enriched in cartilage-specific collagen II but the fibrocartilage-like feature, collagen I, and hypertrophic chondrocyte feature, collagen X, were markedly decreased compared to those in pellet culture. Furthermore, real time RT-PCR analysis demonstrated that collagen II and aggrecan mRNA were up-regulated while collagen X and collagen I mRNA were down-regulated in micromass culture. Thus, the micromass culture system is a promising tool for in vitro chondrogenic studies.

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Intramembranous ossification and endochondral ossification are two ways through which bone formation and fracture healing occur. Accumulating amounts of evidence suggests that melatonin affects osteoblast differentiation, but little is known about the effects of melatonin on the process of chondrogenic differentiation. In this study, the effects of melatonin on human mesenchymal stem cells (MSCs) undergoing chondrogenic differentiation were investigated. Cells were induced along chondrogenic differentiation via high-density micromass culture in chondrogenic medium containing vehicle or 50 nm melatonin. Histological study and quantitative analysis of glycosaminoglycan (GAG) showed induced cartilage tissues to be larger and richer in GAG, collagen type II and collagen type X in the melatonin group than in the untreated controls. Real-time RT-PCR analysis demonstrated that melatonin treatment significantly up-regulated the expression of the genes involved in chondrogenic differentiation, including aggrecan (ACAN), collagen type II (COL2A1), collagen type X (COL10A1), SRY (sex-determining region Y)-box 9 (SOX9), runt-related transcription factor 2 (RUNX2) and the potent inducer of chondrogenic differentiation, bone morphogenetic protein 2 (BMP2). And the expression of melatonin membrane receptors (MT) MT1 and MT2 were detected in the chondrogenic-induced-MSCs by immunofluorescence staining. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to increase the size and GAG synthesis of the induced cartilage tissues, as well as to completely reverse the effect of melatonin on the gene expression of ACAN, COL2A1, COL10A1, SOX9 and BMP2 after 7 days of differentiation. These findings demonstrate that melatonin enhances chondrogenic differentiation of human MSCs at least partially through melatonin receptors.