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Abstract  Physiological root resorption is a phenomenon that normally takes place in deciduous teeth; root resorption of permanent teeth occurs only under pathological conditions. The molecular mechanisms underlying these processes are still unclear. Our previous study showed that osteoclasts cultured on deciduous dentine exhibited a higher degree of resorption and higher levels of cathepsin K and MMP-9 mRNA than osteoclasts cultured on permanent dentine. These results could be because of different susceptibilities to acid and the different organic matrices between deciduous and permanent dentine. Thus, the purpose of this study was to investigate the effect of dentine extracts from bovine deciduous and permanent dentine on osteoclast activity. Osteoclasts, obtained from mouse bone marrow cells co-cultured with an osteoblast-rich fraction in the presence of 1,25-(OH)2-vitamin D3 and PGE2, were incubated with or without 0.6 M HCl extracts from bovine deciduous or permanent dentine for 48 h. TRAP positive cell number, TRAP activity, the areas of resorption pits, and mRNA levels of TRAP, v-ATPase, calcitonin receptor, cathepsin K, and MMP-9 were examined. The results illustrated that TRAP activity, the resorbed area, and the mRNA levels of osteoclast marker genes seemed to be suppressed by both deciduous and permanent dentine extracts. These findings indicate that some factors that suppress osteoclast activity are contained in both deciduous and permanent dentine extracts. Although there was no significant difference in osteoclast activity between deciduous and permanent dentine extracts, osteoclasts incubated with permanent dentine extracts tend to exhibit less resorption activity than those incubated with deciduous dentine extracts. However, we could not clearly explain the causes of this. [ABSTRACT FROM AUTHOR]

Abstract  The classical NF-κB pathway plays an important role in osteoclast formation and differentiation; however, the role of NF-κB in osteoclast bone-resorbing activity is not well understood. To elucidate whether NF-κB is important for osteoclast bone-resorbing activity, we used a selective peptide inhibitor of the classical NF-κB pathway named the NBD peptide. Osteoclasts were generated using bone marrow macrophages in the presence of M-CSF and RANKL. The NBD peptide dose-dependently blocked the bone-resorbing activity of osteoclasts by reducing area, volume (p p  [ABSTRACT FROM AUTHOR]

Abstract  The release of tumor necrosis factor (TNF)-α from macrophages upon stimulation of lipopolysaccharide (LPS) is a major etiological factor of inflammatory bone disease and elicits the effects through TNF receptors type 1 and 2. Given the importance of TNF-α action on osteoclastic bone resorption, the role of TNF type 2 receptor (TNFR2) on bone resorption has not been elucidated well so far. The purpose of this study is to investigate the role of TNFR2 on LPS-induced inflammatory bone resorption in vivo. LPS at 10 mg/kg (Re 595) was injected subcutaneously on calvariae of wild-type (WT), TNF type 1 receptor (TNFR1)-deficient (KO), and TNFR2 KO mice, killed on day 5 after the LPS injection. The calvarial bone mineral density (BMD) was significantly decreased by LPS compared to the vehicle-injected control in WT mice, but not in TNFR1 KO mice. Interestingly, the decrease of calvarial BMD and the increase of the osteoclast number by LPS in TNFR2 KO mice seemed to be more than those in WT mice. Furthermore, the significant decrease by LPS on the BMD of tibiae, femurs, and lumber vertebrae were observed only in TNFR2 KO mice. Histomorphometric analysis of tibiae showed the significant increases of osteoclast number and surface in the LPS-injected TNFR2 KO mice, and the levels of urinary deoxypyridinoline reflected these increases of bone resorption parameters. The present data indicate that TNFR1 is critical for bone resorption at the site of LPS injection and that TNFR2 might have a protective role on the LPS-induced inflammatory bone resorption process. [ABSTRACT FROM AUTHOR]

Many clinical reports suggest that deciduous teeth exhibit a greater susceptibility to resorption than permanent ones. To examine the difference between deciduous and permanent dentine in their susceptibility to osteoclastic resorption, osteoclast-like cells (OCLs) were cultured on deciduous and permanent dentine slices. The number, area, depth, and volume of resorption pits were then measured, using image-analyzing systems. We measured the level of degraded collagen (cross-linked N-telopeptide of type I collagen; NTx) in culture medium using an enzyme-linked immunosorbent assay (ELISA). The levels of cathepsin K, matrix metallo proteinase (MMP)-9, and MMP-13 mRNAs in the cells attached to dentine were also analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). Deciduous dentine slices exhibited a significant (twofold) increase in resorbed area compared with the permanent slices. Three-dimensional analysis revealed that the volume of pits in deciduous dentine differed significantly (fourfold) compared to that in the permanent dentine. The depth of pits also followed the same trend. However, there was no significant difference in the number of pits or osteoclasts on the dentine slices. The NTx level in deciduous media was significantly more than that in permanent media. The mRNA levels also followed the same trend. These results suggest that deciduous dentine is more susceptible to resorption than permanent dentine and signals from the substrate play an important role in physiological resorption. [ABSTRACT FROM AUTHOR]

Many clinical reports suggest that deciduous teeth exhibit a greater susceptibility to resorption than permanent ones. To examine the difference between deciduous and permanent dentine in their susceptibility to osteoclastic resorption, osteoclast-like cells (OCLs) were cultured on deciduous and permanent dentine slices. The number, area, depth, and volume of resorption pits were then measured, using image-analyzing systems. We measured the level of degraded collagen (cross-linked N-telopeptide of type I collagen; NTx) in culture medium using an enzyme-linked immunosorbent assay (ELISA). The levels of cathepsin K, matrix metallo proteinase (MMP)-9, and MMP-13 mRNAs in the cells attached to dentine were also analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). Deciduous dentine slices exhibited a significant (twofold) increase in resorbed area compared with the permanent slices. Three-dimensional analysis revealed that the volume of pits in deciduous dentine differed significantly (fourfold) compared to that in the permanent dentine. The depth of pits also followed the same trend. However, there was no significant difference in the number of pits or osteoclasts on the dentine slices. The NTx level in deciduous media was significantly more than that in permanent media. The mRNA levels also followed the same trend. These results suggest that deciduous dentine is more susceptible to resorption than permanent dentine and signals from the substrate play an important role in physiological resorption. [ABSTRACT FROM AUTHOR]

Abstract Differentiation of mesenchymal stromal cells into osteoblasts is regulated by many factors including growth factors, cytokines, and hormones. Mechanical stress has been considered to be an important factor in bone modeling and remodeling. However, biological responses of stromal cells to mechanical stimuli are still unknown. To show the correlation between magnitude of mechanical strain and differentiation of stromal cells into osteoblasts, we investigated the proliferation and the expression of osteoblast-related genes in stromal cell line ST2 that is in the process of osteoblastic differentiation by treatment with ascorbic acid and ß-glycerophosphate, under 0.8%–15% elongation using the Flexercell Strain system. The expression of osteoblast-related genes was analyzed by real-time quantitative polymerase chain reaction (PCR). Cell proliferation significantly increased at 5%, 10%, and 15% elongation compared to that of unloaded controls. Alkaline phosphatase (ALPase) activity significantly increased at 0.8% and 5% elongation but decreased at 10% and 15% elongation. At 1?h and 6?h, mRNA level of Cbfa1/Runx2 increased at lower magnitudes of strain (0.8% and 5% elongation) but decreased at higher magnitude of strain (15% elongation). At 24 and 48?h, Cbfa1/Runx2 and osteocalcin mRNAs decreased at 5%, 10%, and 15% elongation, whereas cell proliferation and expression of type I collagen mRNA increased at the same elongation. These results indicate that mechanical strain stimulates osteoblastic differentiation of stromal cells at low magnitudes of strain. [ABSTRACT FROM AUTHOR]

Abstract. The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, unlike the limb bud cartilage. To elucidate the mechanism of chondrogenesis at the condylar cartilage, we analyzed the effects of basic fibroblast growth factor (bFGF) on the growth and development of mouse mandibular condyle using serum-free organ culture and on the expression of genes related to the chondrogenesis. Further, we investigated the localization of bFGF in cultured condyle by immunohistochemistry. The present immunohistochemical observations showed that bFGF is localized in the extracellular matrix of the mesenchymal condylar anlage, the perichondrium and the proliferative cell zone, and that immunostaining was diminished in the metachromatically stained area. In the condyle culture with added recombinant human bFGF (rhbFGF) for 5 days, the area occupied by hypertrophic chondrocytes in the mandibular condylar cartilage was reduced. A reverse transcription-polymerase chain reaction (RT-PCR) assay also showed that the mRNA levels of aggrecan and type X collagen were reduced compared with nontreated tissues. Treatment with rhbFGF for 2 days decreased cell proliferation in the perichondrium, and bFGF downregulated the Indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), bone morphogenetic protein 4 (BMP4), and core-binding factor α1 (Cbfa1) expression in the RT-PCR assay. These findings suggest that bFGF has the ability for inhibitory regulation of condylar growth, via the inhibition of proliferation and differentiation of chondrocytes, and that this inhibitory regulation is related to the downregulation of growth factors and transcription factors. [ABSTRACT FROM AUTHOR]