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.

S U M M A R Y Leptin, a 16-kD circulating hormone secreted mainly by white adipose tissue, is a product of the obese (ob) gene. Leptin acts on human marrow stromal cells to enhance differentiation into osteoblasts and inhibit differentiation into adipocytes. Leptin also inhibits bone formation through a hypothalamic relay. To obtain a better understanding of the potential role of leptin in bone formation, the localization of leptin in endochondral ossification was examined immunohistochemically. High expression of leptin was identified in hypertrophic chondrocytes in the vicinity of capillary blood vessels invading hypertrophic cartilage and in a number of osteoblasts of the primary spongiosa beneath the growth plate. The hypertrophic chondrocytes far from the blood vessels were negative for leptin. Moreover, we detected the production and secretion of leptin by a mouse osteoblast cell line (MC3T3-E1) and a mouse chondrocyte cell line (MCC-5) by RT-PCR, immunocytochemistry, and Western blotting. Leptin enhanced the proliferation, migration, tube formation, and matrix metalloproteinase-2 (MMP-2) activity of human endothelial cells (HUVECs) in vitro. These findings suggest the possibility that leptin exerts its influence on endochondral ossification by regulating angiogenesis.

To apply osteoblasts to bone reconstruction, we proved that transplanted osteoblasts possessed the differentiated osteoblastic function and formed bonelike tissue in vivo after transplantation. First, we confirmed that dexamethasone (Dex) promoted the expression of osteoblastic phenotype in human osteoblast culture using reverse-transcription-polymerase chain reaction (RT-PCR). These osteoblasts were cultured for 10 days within collagen sponge, which consists of denatured type I collagen, in the presence or absence of 10 ؊7 M Dex. The osteoblasts along with collagen sponge were transplanted into the trapezius muscles of 8-week-old severe combined immunodeficiency (SCID) mice, and the transplants were harvested at 2, 4, 6, and 8 weeks.

Complete understanding of the physiology and pathology of the cartilage is essential to establish treatments for a variety of cartilage disorders and defects such as rheumatoid arthritis, congenital malformations, and tumors of cartilage. Although synthetic materials have been used in many cases, they possess inherent problems including wear of the materials and low mechanical strength. Autograft has been considered very effective to overcome these problems. However, the limitation of the transplant volume is a major problem in autograft to be overcome. The costal cartilage is the most serious candidate for donor site transplantation, since it is the largest permanent hyaline cartilage in the body. To investigate the possibility using the costal cartilage as a transplant source, we have established and characterized three mouse chondrocyte cell lines (MCC-2, MCC-5, and MCC-35) derived from the costal cartilage of 8-week-old male SV40 large T-antigen transgenic mice. At confluence, all the cell lines formed nodules that could be positively stained with alcian blue (pH 2.5). The size of nodules gradually increased during culturing time. After 2 and 6 weeks of culture, RT-PCR analysis demonstrated that all three cell lines expressed mRNA from the cartilage-specific genes for type II collagen, type XI collagen, aggrecan, and link protein. Furthermore, type X collagen expression was detected in MCC-5 and MCC-35 but not in MCC-2. Any phenotypic changes were not observed over 31 cell divisions. Immunocytochemistry showed further that MCC-2, MCC-5, and MCC-35 produced cartilage-specific proteins type II collagen and type XI collagen, while in addition MCC-5 and MCC-35 produced type X collagen. Treatment with 1alpha, 25-dihydroxyvitamin D(3) inhibited cell proliferation and differentiation of the three cell lines in a dose-dependent manner. These phenotypic characteristics have been found consistent with chondrocyte cell lines established from cartilage tissues other than costal cartilage. In conclusion, costal cartilage shows phenotypic similarities to other cartilages, i.e., articular cartilage and embryonic limbs, suggesting that costal cartilage may be very useful as the donor transplantation site for the treatment of cartilage disorders. Furthermore, the cell lines established in this study are also beneficial in basic research of cartilage physiology and pathology.

We describe a case of first and second branchial arch syndrome in a patient whose profile and occlusion improved in response to orthognatic surgery. A 2-year-9-month-old boy was referred to our clinic for the treatment of open bite. Because symptoms