ABSTRACT:Introduction: Fibroblast growth factor (FGF)23 is produced primarily in bone and acts on kidney as a systemic phosphaturic factor; high levels result in rickets and osteomalacia. However, it remains unclear whether FGF23 acts locally and directly on bone formation. Materials and Methods: We overexpressed human FGF23 in a stage-specific manner during osteoblast development in fetal rat calvaria (RC) cell cultures by using the adenoviral overexpression system and analyzed its effects on osteoprogenitor proliferation, osteoid nodule formation, and mineralization. Bone formation was also measured by calcein labeling in parietal bone organ cultures. Finally, we addressed the role of tyrosine phosphorylation of FGF receptor (FGFR) in mineralized nodule formation. Results: Nodule formation and mineralization, but not osteoprogenitor proliferation, were independently suppressed by overexpression of FGF23 in RC cells. Increased FGF23 levels also suppressed bone formation in the parietal bone organ culture model. FGF23 overexpression enhanced phosphorylation of FGFR, whereas the impairment of mineralized nodule formation by FGF23 overexpression was abrogated by SU5402, an inhibitor of FGFR1 tyrosine kinase activity. Conclusions: These studies suggest that FGF23 overexpression suppresses not only osteoblast differentiation but also matrix mineralization independently of its systemic effects on Pi homeostasis.

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BackgroundEpidemiological studies have revealed a link between dental infection and preterm birth or low birth weight (PTB/LBW), however, the underlying mechanisms remain unclear. Progress in understanding the associated mechanisms has been limited in part by lack of an animal model for chronic infection-induced PTB/LBW, mimicking pregnancy under conditions of periodontitis. We aimed to establish a mouse model of chronic periodontitis in order to investigate the link between periodontitis and PTB/LBW.MethodsTo establish chronic inflammation beginning with dental infection, we surgically opened mouse (female, 8 weeks old) 1st molar pulp chambers and directly infected with w83 strain Porphyromonas gingivalis (P.g.), a keystone periodontal pathogen. Mating was initiated at 6 wks post-infection, by which time dental granuloma tissue had developed and live P.g. was cultured from extracted tooth root, which serves as a persistent source of P.g. The gestational day (gd) and birth weight were recorded during for P.g.-infected and control mice, and serum and placental tissues were collected at gd 15 to evaluate the systemic and local conditions during pregnancy.ResultsDental infection with P.g. significantly increased circulating TNF-α (2.5-fold), IL-17 (2-fold), IL-6 (2-fold) and IL-1β (2-fold). The P.g.-infected group delivered at gd 18.25 vs. gd 20.45 in the non-infected control (NC) group (p < 0.01), and pups exhibited LBW compared to controls (p < 0.01). P.g. was localized to placental tissues by immunohistochemistry and PCR, and defects in placental tissues of P.g. infected mice included premature rupture of membrane, placental detachment, degenerative changes in trophoblasts and endothelial cells, including necrotic areas. P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2. Further placental tissue damage was indicated in P.g. infected mice by decreased CD-31 in endothelial cells, increased expression of 8OHdG, an indicator of oxidative DNA damage, and cleaved caspase-3, a marker of apoptosis. In vitro, P.g. lipopolysaccharide significantly increased expression of COX-2, IL-8 and TNF-α, in HTR-8 trophoblasts in an NF-κB-dependent fashion.ConclusionsOur novel mouse model supports previous epidemiological studies signifying dental infection as predisposing factor for PTB/LBW. We demonstrate PTB and LBW in infected mice, translocation of P.g to placental tissues, increased circulating and local pro-inflammatory markers, and the capability of P.g. LPS to directly induce cytokine production in trophoblasts, in vitro. These findings further underscore the importance of local and systemic infections and inflammation during pregnancy and suggest that prevention and/or elimination of dental infections such as marginal or periapical periodontitis before pregnancy may have a beneficial effect on PTB/LBW.

Streptococcus mutans and Streptococcus sobrinus are known to be associated with the development of dental caries. In this study these bacteria were detected in pre-school children (each with primary dentition, age range 3-5 years, n ¼ 60) using a PCR method, and then their presence was compared with the incidence of dental caries over a 1-year period. Plaque samples were collected from all erupted tooth sites using a sterile toothbrush. Dental examinations at the beginning of the study (baseline) and after 1 year were also performed to determine decayed, missing, filled teeth (dmft) scores using WHO caries diagnostic criteria. The prevalences of S. mutans and S. sobrinus across all the subjects were 61 . 7 % and 56 . 6 %, respectively; 13 subjects (21 . 7 %) were positive for S. mutans alone, 10 (16 . 6 %) were positive for S. sobrinus alone and 24 (40 . 0 %) were positive for both S. mutans and S. sobrinus, whereas 13 (21 . 7 %) were negative for both S. mutans and S. sobrinus. dmft scores of subjects positive for both S. mutans and S. sobrinus at baseline and after 1 year were significantly higher than of those positive for S. mutans alone at the same stages (P , 0 . 01 and P , 0 . 001, respectively). The caries incremental increase was also significantly greater in those with both bacteria detected (P , 0 . 05). Our results indicate that pre-school children harbouring both S. mutans and S. sobrinus have a significantly higher incidence of dental caries than those with S. mutans alone. INTRODUCTIONMutans streptococci (Streptococcus mutans and Streptococcus sobrinus) are considered to be major dental caries aetiologic agents. They are the most common pathogens isolated from human dental plaque and their prevalence has been reported in epidemiological studies (Hamada & Slade, 1980;Whiley & Beighton, 1998). S. mutans has been shown to be more prevalent than S. sobrinus in dental plaque samples (Loesche, 1986; Carlsson et al., 1987), while several epidemiological studies have shown that the prevalence of S. sobrinus is more closely associated with high caries activity (Fujiwara et al., 1991;Hirose et al., 1993).In several epidemiological studies, identification of S. mutans and S. sobrinus on selective media such as mitis-salivarius (MS) or MS-bacitracin (MSB) agar has been performed using colonial morphology (Wade et al., 1986;Svanberg & Krasse, 1990). However, accurate differentiation between S. mutans and S. sobrinus is not easy, and is also timeconsuming and laborious (de Soet et al., 1990). Further, it has been reported that S. sobrinus from dental plaque samples is especially difficult to culture directly on MSB selective medium (Jordan 1986;de Soet et al., 1990). Thus, it is of great importance to distinguish the presence of these two species separately in children for accurate prediction and effective prevention of dental caries.Thus far, several methods used for detecting and identifying mutans streptococci have been reported, including direct microscopy, cultivation, enzyme tests, mAbs, ELISAs and specie...

Streptococcus mutans and S. sobrinus are associated with the development of dental caries. These bacteria were detected by PCR and then their presence was compared with the incidence of dental caries in 77 Japanese pre-school children. Plaque samples were collected from all erupted tooth sites in the subjects, aged 3-5 years old and each with primary dentition, with a sterile toothbrush. A dental examination was performed for dmft (decayed, missing, filled, total) with the WHO caries diagnostic criteria. In all subjects, the prevalence of S. mutans and S. sobrinus was 72.8% and 61.1%, respectively; 19 (24.7%) were positive for S. mutans alone, 10 (13.0%) were positive for S. sobrinus alone, 37 (48.1%) were positive for both S. mutans and S. sobrinus, and 11 (14.3%) were negative for both S. mutans and S. sobrinus. The dmft scores of children positive for both S. mutans and S. sobrinus were significantly higher than those positive for S. mutans alone. These results indicate that children harbouring both S. mutans and S. sobrinus have a significantly higher incidence of dental caries than those with S. mutans alone.

The purpose of this study was to investigate the intrafamilial distribution of mutans streptococci in Japanese families using chromosomal DNA fingerprinting with three endonucleases; EcoRI, HindIII and HaeIII. The analysis of 1,908 isolates cultured from the dental plaque of 76 subjects from 20 families (20 married couples and 36 of their children) resulted in the identification of 144 genotypes containing 114 strains of Streptococcus mutans (serotype c, 66.7%; e, 12.5%) and 30 strains of S. sobrinus (d, 13.2%; g, 7.6%). A mean of 1.89 genotypes (from one to four) was harbored in individual subjects, and a mean of 4.10 genotypes from two to seven was harbored in individual families. Among the 70 genotypes found in the children, 36 (51.4%) were in agreement with their mothers and 22 (31.4%) were in agreement with their fathers. The other genotypes (18.6%) did not correspond with the parents. Homologous strains between parents were found in only two couples. This result showed that fathers or others as well as mothers can be sources of transmission. Further, the serotype d, e and g strains showed significantly higher probabilities of transmission than serotype c.

Of the four prostaglandin (PG) E receptor subtypes (EP1-EP4), EP2 and EP4 have been proposed to mediate the anabolic action of PGE(2) on bone formation but comparative evaluation studies of EPs on bone formation do not necessarily share a common mechanism, implying that their additional features including downstream MAPK pathways may be beneficial to resolve this issue. We systematically assessed the roles of EPs in the rat calvaria (RC) cell culture model by using four selective EP agonists (EPAs). Consistent with relative expression levels of the respective receptors, multiple phenotypic traits of bone formation in vitro, including proliferation of nodule-associated cells, osteoblast marker expression and mineralized nodule formation were upregulated not only by PGE(2) but equally by EP2A and EP4A, but not by EP1A and EP3A. EP2A and EP4A were effective when cells were treated chronically or pulse-treated during nascent nodule formation. EP2A and EP4A equally stimulated the endogenous PGE(2) production, while EP2A caused a greater increase in cAMP production and c-Fos gene expression compared to EP4A. EP2A and EP4A activated predominantly p38 MAPK and ERK respectively, while c-Jun N-terminal kinase (JNK) was equally activated by both agonists. SB203580 (p38 MAPK inhibitor) blocked the PGE(2) effect on mineralized nodule formation, while U0126 (ERK inhibitor) and dicumarol (JNK inhibitor) were less effective. PGE(2)-dependent phosphorylation of the MAPKs was affected not only by protein kinase (PK)A and PKC inhibitors but also by adenylate cyclase and PKC activators. Co-treatment of RC cells with EP2A or EP4A and bone morphogenetic protein (BMP)2, whose effects on bone nodule formation is known to be, in part, mediated through the PKA and p38 MAPK pathways, resulted in an additive effect on mineralized nodule formation. Further, PGE(2), EP2A and EP4A did not increase BMP2/4 mRNA levels in RC cells, and EP2-induced phosphorylation of p38 MAPK was not eliminated by Noggin. These results suggest that, in the RC cell model, the anabolic actions of PGE(2) on mineralized nodule formation are mediated at least in part by activation of the EP2 and EP4 receptor subtype-specific MAPK pathways, independently of BMP signaling, in cells associated with nascent bone nodules.

Isolates of mutans streptococci were obtained from the dental plaque of ten subjects before and after the subjects had been free of detectable mutans streptococci for a mean period of 14.6 weeks (range, from two to 36 weeks). The mutans streptococci had been rendered undetectable by chlorhexidine varnish treatment. Examination of the restriction endonuclease analysis (REA) patterns of the isolates revealed that all ten subjects had one strain (REA type) after re-appearance of the mutans streptococci that was identical to one that had been present before the varnish treatment. In six of the ten subjects, only one strain was detected both before and after treatment. Each of the other four subjects appeared to gain a new strain after treatment; one of the four appeared to lose one strain, and another, four strains. The ability of strains to persist after the period of undetectability seemed unrelated to their resistance to chlorhexidine or to their ability to exhibit insoluble glucan-mediated adhesion. In the subjects harboring multiple REA types, one-seventh of the tooth surfaces sampled harbored two strains simultaneously, suggesting an inability of either strain to exclude the other aggressively. Overall, the study indicated that every subject receiving chlorhexidine varnish therapy had a primary strain of mutans streptococcus that re-emerged after treatment. In contrast, secondary strains were highly susceptible to being lost or gained.