Melatonin is known to regulate a variety of physiological processes including control of circadian rhythms, regulation of seasonal reproductive function, regulation of body temperature, and so forth. Accumulating evidence from in vitro and in vivo experiments using rodent and chicken has also suggested that melatonin may have an influence on skeletal growth and bone formation. However, little is known about the effects of melatonin on human osteoblasts, which thus remains to be elucidated. This study was performed to determine whether melatonin could affect the proliferation and differentiation of human osteoblasts in vitro and to demonstrate the possibility that melatonin could be applied as a pharmaceutical agent to shorten the treatment period of bone fracture, various osteotomies, and bone distraction. Reverse transcription-polymerase chain reaction and Western blot analysis showed that human osteoblasts expressed melatonin 1a receptor and that its expression levels decreased gradually with the age of the hosts. Melatonin stimulated the proliferation and alkaline phosphatase activity of human osteoblasts in a dose-dependent manner at the pharmacological concentrations. Melatonin also promotes gene expression of type I collagen, osteopontin, bone sialoprotein, and osteocalcin in a dose-dependent manner, and stimulated the mineralized matrix formation in vitro. Moreover, intraperitoneal administration of melatonin to mice increased the volume of newly formed cortical bone of femora. These results demonstrated that melatonin directly accelerated the differentiation of osteoblasts of human as well as rodent and chicken and also suggested that melatonin could be applied as a pharmaceutical agent to promote bone regeneration.

Maspin, a serine protease inhibitor, is expressed by formative osteoblasts. The repression of maspin expression in osteoblastic cells decreased the level of latent TGF-␤ in the extracellular matrix, whereas the overexpression of maspin increased latent TGF-␤. These findings suggest that maspin plays an important role in bone matrix formation, particularly in the accumulation of latent TGF-␤.Introduction: Maspin is a serine protease inhibitor that exhibits tumor suppressive and anti-angiogenic activities. This study was performed to elucidate a possible role for maspin in bone formation. Materials and Methods:We performed immunohistochemical analysis of the expression of maspin during endochondral ossification. We evaluated the expression of maspin mRNA and protein in ROS 17/2.8 cells and primary rat osteoblastic cells by RT-PCR, immunocytochemistry, and Western blot analysis. We also examined the accumulation of TGF-␤ in the extracellular matrix of cultured ROS 17/2.8 cells after transfection with vectors expressing either maspin or maspin antisense. Results: We observed expression of maspin by active osteoblasts in vivo. Rat osteoblastic cells also expressed maspin mRNA and protein in vitro. Moreover, the accumulation of latent TGF-␤ in the extracellular matrix significantly decreased in cultures exposed to an anti-maspin antibody and when cells were transfected with a maspin antisense-expressing vector. In contrast, accumulation of latent TGF-␤ in the extracellular matrix increased after transfection of cells with a vector expressing maspin. Conclusions: These findings suggest that maspin expressed in active osteoblasts plays an important physiological role during maturation of the bone matrix, and in particular, during the process of accumulation of latent TGF-␤ in the extracellular matrix.