The study objective was to investigate the effects of fluoride on intact parathyroid hormone (iPTH) secretion. Thyro-parathyroid complexes (TPC) from C3H (n = 18) and B6 (n = 18) mice were cultured in Ca²⁺-optimized medium. TPC were treated with 0, 250, or 500 µM NaF for 24 h and secreted iPTH assayed by ELISA. C3H (n = 78) and B6 (n = 78) mice were gavaged once with distilled or fluoride (0.001 mg [F^-]/g of body weight) water. At serial time points (0.5-96 h) serum iPTH, fluoride, total calcium, phosphorus, and magnesium levels were determined. Expression of genes involved in mineral regulation via the bone-parathyroid-kidney (BPK) axis, such as parathyroid hormone (Pth), calcium-sensing receptor (Casr), vitamin D receptor (Vdr), parathyroid hormone-like hormone (Pthlh), fibroblast growth factor 23 (Fgf23), α-Klotho (αKlotho), fibroblast growth factor receptor 1c (Fgf1rc), tumor necrosis factor 11 (Tnfs11), parathyroid hormone receptor 1 (Pth1r), solute carrier family 34 member 1 (Slc34a1), solute carrier 9 member 3 regulator 1 (Slc9a3r1), chloride channel 5 (Clcn5), and PDZ domain-containing 1 (Pdzk1), was determined in TPC, humeri, and kidneys at 24 h. An in vitro decrease in iPTH was seen in C3H and B6 TPC at 500 µM (p < 0.001). In vivo levels of serum fluoride peaked at 0.5 h in both C3H (p = 0.002) and B6 (p = 0.01). In C3H, iPTH decreased at 24 h (p < 0.0001), returning to baseline at 48 h. In B6, iPTH increased at 12 h (p < 0.001), returning to baseline at 24 h. Serum total calcium, phosphorus, and magnesium levels did not change significantly. Pth, Casr,αKlotho,Fgf1rc,Vdr, and Pthlh were significantly upregulated in C3H TPC compared to B6. In conclusion, the effects of fluoride on TPC in vitro were equivalent between the 2 mouse strains. However, fluoride demonstrated an early strain-dependent effect on iPTH secretion in vivo. Both strains demonstrated differences in the expression of genes involved in the BPK axis, suggesting a possible role in the physiologic handling of fluoride.

RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment, and cell proliferation. During amelogenesis, ameloblasts (which produce the enamel proteins) undergo dramatic cytoskeletal changes and the RhoA protein level is up‐regulated. Transgenic mice were generated that express a dominant‐negative RhoA transgene in ameloblasts using amelogenin gene‐regulatory sequences. Transgenic and wild‐type (WT) molar tooth germs were incubated with sodium fluoride (NaF) or sodium chloride (NaCl) in organ culture. Filamentous actin (F‐actin) stained with phalloidin was elevated significantly in WT ameloblasts treated with NaF compared with WT ameloblasts treated with NaCl or with transgenic ameloblasts treated with NaF, thereby confirming a block in the RhoA/Rho‐associated protein kinase (ROCK) pathway in the transgenic mice. Little difference in quantitative fluorescence (an estimation of fluorosis) was observed between WT and transgenic incisors from mice provided with drinking water containing NaF. We subsequently found reduced transgene expression in incisors compared with molars. Transgenic molar teeth had reduced amelogenin, E‐cadherin, and Ki67 compared with WT molar teeth. Hypoplastic enamel in transgenic mice correlates with reduced expression of the enamel protein, amelogenin, and E‐cadherin and cell proliferation are regulated by RhoA in other tissues. Together these findings reveal deficits in molar ameloblast function when RhoA activity is inhibited.

The interfrontal bone (IF) is a minor skeletal trait residing between the frontal bones. IF is considered a quasi-continuous trait. Genetic and environmental factors appear to play roles in its development. The mechanism(s) underlying IF bone development are poorly understood. We sought to survey inbred strains of mice for the prevalence of IF and to perform QTL mapping studies. Archived mouse skulls from a mouse phenome project (MPP) were available for this study. 27 inbred strains were investigated with 6–20 mice examined for each strain. Skulls were viewed dorsally and the IF measured using a zoom stereomicroscope equipped with a calibrated reticle. A two generation cross between C3H/HeJ and C57BL/6J mice was performed to generate a panel of 468 F2 mice. F2 mice were phenotyped for presence or absence of IF bone and among mice with the IF bone maximum widths and lengths were measured. F2 mice were genotyped for 573 SNP markers informative between the two strains and subjected to linkage map construction and interval QTL mapping. Results: Strain dependent differences in the prevalence of IF bones were observed. Overall, 77.8% or 21/27, of the inbred strains examined had IF bones. Six strains (C3H/HeJ, MOLF/EiJ, NZW/LacJ, SPRET/EiJ, SWR/J, and WSB/EiJ) lack IF bones. Among the strains with IF bones, the prevalence ranged from 100% for C57BL/6J, C57/LJ, CBA/J, and NZB/B1NJ and down to 5% for strains such as CAST/Ei. QTL mapping for IF bone length and widths identifies for each trait one strong QTL detected on chromosome 14 along with several other significant QTLs on chromosomes 3, 4, 7, and 11. Strain dependent differences in IF will facilitate investigation of genetic factors contributing to IF development. IF bone formation may be a model to understand intrasutural bone formation.

Excessive fluoride ingestion during tooth development can cause dental fluorosis. Previously, we reported that fluoride activates histone acetyltransferase (HAT) to acetylate p53, promoting fluoride toxicity in mouse ameloblast-like LS8 cells. However, the roles of HAT and histone acetylation status in fluoride-mediated gene expression remain unidentified. Here, we demonstrate that fluoride-mediated histone modification causes gene expression alterations in LS8 cells. LS8 cells were treated with or without fluoride followed by ChIP-Seq analysis of H3K27ac. Genes were identified by differential H3K27ac peaks within ±1 kb from transcription start sites. The levels of mRNA of identified genes were assessed using rea-time PCR (qPCR). Fluoride increased H3K27ac peaks associated with Bax, p21, and Mdm2 genes and upregulated their mRNA levels. Fluoride decreased H3K27ac peaks and p53, Bad, and Bcl2 had suppressed transcription. HAT inhibitors (Anacardic acid or MG149) suppressed fluoride-induced mRNA of p21 and Mdm2, while fluoride and the histone deacetylase (HDAC) inhibitor sodium butyrate increased Bad and Bcl2 expression above that of fluoride treatment alone. To our knowledge, this is the first study that demonstrates epigenetic regulation via fluoride treatment via H3 acetylation. Further investigation is required to elucidate epigenetic mechanisms of fluoride toxicity in enamel development.

Aim: to evaluate the bone regeneration capacity of two alloplastic biomaterials in a critical-size rat calvarial defect model. Methods: A total of 80 rats were randomized into 8 groups of 10 animals each. An Ø8 mm, critical-size calvarial defect was created, and the following treatments were randomly allocated: sham surgery, deproteinized bovine bone mineral (DBBM) + collagen membrane (CM), poly-(lactic-co-glycolic-acid) (PLGA)-coated pure phase β-tricalcium phosphate (β-TCP), or PLGA-coated 60% hydroxyapatite (HA):40%β-TCP. Animals were allowed to heal for 2 and 6 weeks. Microcomputed tomography (μCT) was used to evaluate mineralized tissue and biomaterial displacement. Histological samples were used to evaluate new bone formation. Results: μCT analysis showed no significant differences among groups for total volume of mineralized tissue or residual biomaterials. DBBM + CM showed significantly increased horizontal biomaterial displacement at 2 weeks but not at 6 weeks. Histological analysis showed that sham surgery had a significantly higher percentage of bone area fraction than the DBBM + CM and PLGA + β-TCP at 2 weeks, but not at 6 weeks. Residual biomaterial area fraction showed no significant differences among experimental groups at any healing time. Conclusions: The alloplastic biomaterials showed suitable construct integrity and retention in the defect. All biomaterials were associated with limited new bone formation comparable to the sham surgery control.