SIRT1/FOXO3a axis plays an important role in the prevention of mandibular bone loss induced by 1,25(OH)2D deficiency.

It has been reported that 1,25 dihydroxyvitamin D [1,25(OH)2D] deficiency leads to the loss of mandibular bone, however the mechanism is unclear. We investigated whether the Sirt1/FOXO3a signaling pathway is involved in this process. Using a 1,25(OH)2D deficiency model induced by genetic deletion in mice of 25-hydroxyvitamin D-1α hydroxylase [1α(OH)ase-/- mice]. We first documented a sharp reduction of expression levels of Sirt1 in the 1α(OH)ase-/- mice in vivo. Next, we demonstrated dose-dependent upregulation of Sirt1 by treatment with exogenous 1,25(OH)2D3in vitro. We then identified a functional VDR binding site in the Sirt1 promoter. By crossing Prx1-Sirt1 transgenic mice with 1α(OH)ase-/- mice we demonstrated that the overexpression of Sirt1 in mesenchymal stem cells (MSCs) greatly improved the 1α(OH)ase-/- mandibular bone loss phenotype by increasing osteoblastic bone formation and reducing osteoclastic bone resorption. In mechanistic studies, we showed, in 1α(OH)ase-/- mice, decreases of Sirt1 and FoxO3a, an increase in oxidative stress as reflected by a reduction of the antioxidant enzymes peroxiredoxin1 (Prdx1), SOD1 and SOD2 expression, and an increase of markers for osteocyte senescence and senescence associated secretory phenotypes (SASP), including ß-galactosidase (ß-gal), p16, p53 and p21. The targeted overexpression of Sirt1 in the 1α(OH)ase-/- mice restored the expression levels of these molecules. Finally, we demonstrated that a Sirt1 agonist can upregulate FOXO3a activity by increasing deacetylation and nuclear translocation. Overall, results from this study support the concept that targeted increases in Sirt1/FOXO3a signaling levels can greatly improve the bone loss caused by 1,25(OH)2D deficiency.

High-resolution isotopic evidence of specialised cattle herding in the European Neolithic.

Reconstructing stock herding strategies and land use is key to comprehending past human social organization and economy. We present laser-ablation strontium and carbon isotope data from 25 cattle (Bos taurus) to reconstruct mobility and infer herding management at the Swiss lakeside settlement of Arbon Bleiche 3, occupied for only 15 years (3384-3370 BC). Our results reveal three distinct isotopic patterns that likely reflect different herding strategies: 1) localized cattle herding, 2) seasonal movement, and 3) herding away from the site year-round. Different strategies of herding are not uniformly represented in various areas of the settlement, which indicates specialist modes of cattle management. The pressure on local fodder capacities and the need for alternative herding regimes must have involved diverse access to grazing resources. Consequently, the increasing importance of cattle in the local landscape was likely to have contributed to the progress of socio-economic differentiation in early agricultural societies in Europe.

Bone Degeneration and Its Recovery in SMP30/GNL-Knockout Mice.

Senescence marker protein-30 (SMP30) decreases androgen-independently with aging and is a lactone-hydrolyzing enzyme gluconolactonase (GNL) that is involved in vitamin C biosynthesis. In the present study, bone properties of SMP30/GNL knockout (KO) mice with deficiency in vitamin C synthesis were investigated to reveal the effects of SMP30/GNL and exogenous vitamin C supplementation on bone formation. Mineral content (BMC) and mineral density (BMD) of the mandible and femur of SMP30/GNL KO and wild-type mice at 2 and 3 months of age with or without vitamin C supplementation were measured by dual-energy X-ray absorptiometry. Body and bone weight of both age groups decreased and became significantly lower than those of wild-type mice. The bones of SMP30/GNL KO mice were rough and porous, with BMC and BMD significantly below wild-type. Oral supplementation with vitamin C eliminated differences in body weight, bone weight, BMC, and BMD between SMP30/GNL KO and wild-type mice at each age. These results indicate that bone degeneration in SMP30/GNL KO mice was caused by lack of vitamin C, and that this mouse strain is an appropriate model for bone metabolism in humans, which have no ability to synthesize vitamin C.

Prenatal alcohol exposure reduces mandibular calcium and phosphorus concentrations in newborn rats.

Previous studies suggest that prenatal alcohol exposure affects fetal bone development, including bone quality. This study evaluated the chemical composition of mandibles from newborn rats after maternal 20% alcohol consumption before and throughout gestation. Nine rats were initially distributed into three groups: an Alcohol group, Pair-fed group, and Control group. The groups were fed prespecified diets for 8 weeks before and the 3 weeks during pregnancy. At age 5 days, eight newborns from each group were euthanized (total, n = 24). Using energy dispersive spectrometry, we evaluated samples of mandibles from newborns to identify changes in bone mineralization, specifically Ca and P concentrations. Ca and P concentrations were lower in the Alcohol group than in the Control and Pair-fed groups (P = 0.003 and P = 0.001, respectively). In summary, alcohol exposure before and throughout gestation reduces mandibular Ca and P concentrations in newborn rats. (J Oral Sci 58, 439-444, 2016).

Mandibular Jaw Bone Regeneration Using Human Dental Cell-Seeded Tyrosine-Derived Polycarbonate Scaffolds.

Here we present a new model for alveolar jaw bone regeneration, which uses human dental pulp cells (hDPCs) combined with tyrosine-derived polycarbonate polymer scaffolds [E1001(1k)] containing beta-tricalcium phosphate (ß-TCP) [E1001(1k)/ß-TCP]. E1001(1k)/ß-TCP scaffolds (5 mm diameter × 1 mm thickness) were fabricated to fit a 5 mm rat mandibular ramus critical bone defect. Five experimental groups were examined in this study: (1) E1001(1k)/ß-TCP scaffolds seeded with a high density of hDPCs, 5.0 × 10(5) hDPCs/scaffold (CH); (2) E1001(1k)/ß-TCP scaffolds seeded with a lower density of hDPCs, 2.5 × 10(5) hDPCs/scaffold (CL); (3) acellular E1001(1k)/ß-TCP scaffolds (SA); (4) acellular E1001(1k)/ß-TCP scaffolds supplemented with 4 µg recombinant human bone morphogenetic protein-2 (BMP); and (5) empty defects (EDs). Replicate hDPC-seeded and acellular E1001(1k)/ß-TCP scaffolds were cultured in vitro in osteogenic media for 1 week before implantation for 3 and 6 weeks. Live microcomputed tomography (µCT) imaging at 3 and 6 weeks postimplantation revealed robust bone regeneration in the BMP implant group. CH and CL groups exhibited similar uniformly distributed mineralized tissue coverage throughout the defects, but less than the BMP implants. In contrast, SA-treated defects exhibited sparse areas of mineralized tissue regeneration. The ED group exhibited slightly reduced defect size. Histological analyses revealed no indication of an immune response. In addition, robust expression of dentin and bone differentiation marker expression was observed in hDPC-seeded scaffolds, whereas, in contrast, BMP and SA implants exhibited only bone and not dentin differentiation marker expression. hDPCs were detected in 3-week but not in 6-week hDPC-seeded scaffold groups, indicating their survival for at least 3 weeks. Together, these results show that hDPC-seeded E1001(1k)/ß-TCP scaffolds support the rapid regeneration of osteo-dentin-like mineralized jaw tissue, suggesting a promising new therapy for alveolar jaw bone repair and regeneration.

Unique and reliable rat model for the assessment of cell therapy: bone union in the rat mandibular symphysis using bone marrow stromal cells.

Many kinds of bone graft materials have been developed and reported to repair various bone defects. The defects are usually created by surgical resection of pre-existing bone tissue. However, spontaneous healing of bone defects without implantation of materials could be seen, because bone tissue possesses inherent repairing property. The central portion of the lower jaw bone in many animals consists of fibrous tissue and is called the mandibular symphysis. It persists even in old animals and thus can be interpreted as a physiological bone gap or a non-healing bone defect. We implanted calcium phosphate porous ceramics alone or composites of the ceramics and bone marrow stromal cells (BMSCs) into the bone defect (mandibular symphysis) to examine whether it could be filled with new bone tissue, resulting in bone union. Eight weeks after implantation, micro-computed tomography (micro-CT) and histological and biomechanical analyses demonstrated that bone union of the mandibles occurred in all rats with composites but in none of those with ceramics alone. These results showed that the rat mandibular symphysis is a unique bone defect site for the evaluation of bone graft materials. These analyses demonstrated that ceramics alone could not contribute to bone healing in the defect; however, supplementation with BMSCs drastically changed the properties of the ceramics (turning them into osteogenic ceramics), which completely healed the defect. As BMSCs can be culture-expanded using small amounts of bone marrow, the use of the composites might have clinical significance for the reconstruction of various bone tissues, including facial bone.

Effects of Er-Zhi-Wan on microarchitecture and regulation of Wnt/ß-catenin signaling pathway in alveolar bone of ovariectomized rats.

Recent studies have shown that Er-Zhi-Wan (EZW), a traditional Chinese medicine consisting of Herba Ecliptae (HE) and Fructus Ligustri Lucidi (FLL), had a definite antiosteoporotic effect on osteoporotic femur, but its effect on osteoporosis of alveolar bone remains unknown. In the present study, we investigated the effects of Er-Zhi-Wan (EZW) on the microarchitecture and the regulation of Wnt/ß-catenin signaling pathway in the alveolar bone of ovariectomized rats. Thirty Sprague-Dawley rats were randomly divided into three groups: sham operation group (sham, n=10), ovariectomy (OVX) group (n=10), and OVX with EZW treatment group (EZW group, n=10). From one week after ovariectomy, EZW (100 mg/mL) or vehicle (distilled water) was fed (1 mL/100 g) once per day for 12 weeks until the sacrifice of the rats. The body weights were measured weekly. After sacrifice, the sera and mandible were collected and routinely prepared for the measurement of alveolar trabecular microarchitecture, serum levels of E2, bone-specific alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase 5b (TRAP5b), as well as mandibular mRNA expression of Wnt/ß-catenin signaling pathway molecules wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5), ß-catenin and dickkopf homolog 1 (DKK1). The results showed that EZW treatment significantly prevented the body weight gain, degradation of alveolar trabecular microarchitecture and alveolar bone loss in the OVX rats. Furthermore, we observed that EZW could increase the serum levels of E2 and BALP, and decrease levels of serum TRAP5b in EZW group compared with vehicle group. In addition, RT-PCR results revealed that EZW upregulated the expression levels of wnt3a, LRP5 and ß-catenin, and reduced the expression of DKK1 in OVX rats. Taken together, our results suggested that EZW may have potential anti-osteoporotic effects on osteoporotic alveolar bone by stimulating Wnt/LRP5/ß-catenin signaling pathway.

[Effect of acidophilic multiprobiotic symbiter on pathological changes in bone periodontal tissue under hypo-acidic condition].

The omeprazole-induced hypoacidity leads to metabolic disorders in the bone periodontal tissue: a disbalance of NO-ergic system, activation of collagen and noncollagen proteins catabolism. We have shown that such disorders are corrected by a multiprobiotic of new generation -- symbiter acidophilic.

Methyl farnesoate synthesis in the lobster mandibular organ: the roles of HMG-CoA reductase and farnesoic acid O-methyltransferase.

Eyestalk ablation (ESA) increases crustacean production of methyl farnesoate (MF), a juvenile hormone-like compound, but the biochemical steps involved are not completely understood. We measured the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and farnesoic acid O-methyl transferase (FAOMeT), an early step and the last step in MF synthesis. ESA elevated hemolymph levels of MF in male lobsters. Enzyme activity suggested that increased MF production on day one was due largely to elevated HMGR activity while changes in FAOMeT activity closely paralleled changes in MF levels on day 14. Transcript levels for HMGR and FAOMeT changed little on day one, but both increased substantially on day 14. We treated ESA males with a partially purified mandibular organ-inhibiting hormone (MOIH) and observed a significant decline in MF levels, FAOMeT activity, and FAOMeT-mRNA levels after 5h. However, no effect was observed on HMGR activity or its mRNA indicating that they must be regulated by a separate sinus gland peptide. We confirmed that lobster HMGR was not a phosphoprotein and was not regulated by reversible phosphorylation, an important mechanism for regulating other HMGRs. Nevertheless, molecular modeling indicated that the catalytic mechanisms of lobster and mammalian HMGR were similar.

Expression of phosphoproteins and amelotin in teeth.

The organic material of our teeth consists of collagens and a number of calcium-binding phosphoproteins. Six of these phosphoproteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins), namely osteopontin, bone sialoprotein, dentin matrix protein (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE) and enamelin. We prepared a cDNA library from rat incisors in order to identify the genes involved in tooth formation. The library was screened by subtractive hybridization with two probes; one specific for teeth, the other for bone. We found that the vast majority of the clones from our library were expressed at similar levels in bone and teeth, demonstrating the close relationship of the two tissues. Only 7% of all the clones were expressed in a tooth-specific fashion. These included clones for the enamel proteins; amelotin, amelogenin, ameloblastin and enamelin; for the dentin proteins DSPP and DMP1; and for the intermediate filament protein cytokeratin 13. Several typical bone proteins, including collagen I, osteocalcin, alkaline phosphatase and FATSO, were also expressed at significantly higher levels in teeth than in bone, probably due to the extreme growth rate of rat incisors. The amino acid sequence of rat amelotin showed 62% identity with the sequence from humans. It was expressed considerably later than the other enamel proteins, suggesting that amelotin may serve a function different from those of amelogenin, ameloblastin and enamelin.

Purification and characterization of a mandibular organ protein from the American lobster, Homarus americanus: a putative farnesoic acid O-methyltransferase.

Methyl farnesoate (MF) appears to have important roles in the development, morphogenesis, and reproduction of crustaceans. To better understand the regulation of MF synthesis, we studied farnesoic acid O-methyltransferase (FAOMeT, the final enzyme in the MF biosynthetic pathway) in the American lobster (Homarus americanus). FAOMeT purified from mandibular organ (MO) homogenates had a MW of approximately 38,000. The sequences of trypsin fragments of purified FAOMeT were used to design PCR primers to amplify a cDNA fragment, which was used to isolate a full-length cDNA containing a single open reading frame (ORF) of 828 bp encoding a protein of 276 amino acids. The deduced amino acid sequence of this putative FAOMeT protein contained two copies of a conserved approximately 135 amino acid domain we term the CF (CPAMD8/FAOMeT) domain; single copies of this domain also occur in the human CPAMD8 protein (a member of the alpha-2 macroglobulin family) and an uncharacterized Drosophila protein. The recombinant protein had no FAOMeT activity. However, its addition to MO homogenates from eyestalk ablated (ESA) lobsters increased enzyme activity by up to 75%, suggesting that FAOMeT may require an additional factor or modification (e.g., phosphorylation) for its activation. The mRNA for the putative FAOMeT was primarily found in the proximal region of the MO, the predominant site of MF synthesis. FAOMeT transcripts were found in muscle tissue from ESA animals, but not in green gland, hepatopancreas, or in muscle tissue from intact animals. FAOMeT mRNA was also detected in embryos and larval stages. This is the first comprehensive report of this protein in the lobster, and is an important step in elucidating the functions of MF in these animals.

Influence of tamoxifen on the mandible bone and hard teeth tissues of animals.

Tamoxifen, protactedly used, can cause disadvantageous changes in the bones and in the hard teeth tissues. The aim of this paper was to define to what extent tamoxifen given to the animals influences the structure of the mandible bone and the hard teeth tissues.

Putative membrane receptor for 1,25(OH)2 vitamin D3 in human mineralized tissues during prenatal development.

The calciotropic hormone, 1,25(OH)2vitamin D3[1,25(OH)2D3], controls the formation of dental and bone mineralized tissues. The role of nuclear 1,25(OH)2D3 receptor has been extensively studied in the diverse secretory cells, i.e., osteoblasts, chondrocytes, ameloblasts, and odontoblasts. A nongenomic pathway also has been characterized and related to the interactions of 1,25(OH)2D3 ligand with a putative cell membrane receptor. This recognition moiety called 1,25(OH)2vitamin D3 membrane-associated, rapid-response steroid-binding [1,25D3-MARRS] protein is investigated here in the craniofacial skeleton of human embryos and fetuses. Immunolocalization using a specific Ab099 against chick intestinal basolateral 1,25D3-MARRS protein was performed. The data show a complementary expression pattern of the membrane receptor when compared with published data on the nuclear receptor, notably during amelogenesis. In mandible, membrane receptors for 1,25(OH)2D3 were identified in the heterogenous bone cell population, including osteoblasts and osteoclasts. Differential 1,25D3-MARRS protein levels were observed in distinct developmental stages and anatomical sites of tooth and bone, suggesting the existence of cross-talk between local factors and 1,25D3-MARRS protein expression.

YY1 activates Msx2 gene independent of bone morphogenetic protein signaling.

Msx2 is a homeobox gene expressed in multiple embryonic tissues which functions as a key mediator of numerous developmental processes. YY1 is a bi-functional zinc finger protein that serves as a repressor or activator to a variety of promoters. The role of YY1 during embryogenesis remains unknown. In this study, we report that Msx2 is regulated by YY1 through protein-DNA interactions. During embryogenesis, the expression pattern of YY1 was observed to overlap in part with that of Msx2. Most notably, during first branchial arch and limb development, both YY1 and Msx2 were highly expressed, and their patterns were complementary. To test the hypothesis that YY1 regulates Msx2 gene expression, P19 embryonal cells were used in a number of expression and binding assays. We discovered that, in these cells, YY1 activated endogenous Msx2 gene expression as well as Msx2 promoter-luciferase fusion gene activity. These biological activities were dependent on both the DNA binding and activation domains of YY1. In addition, YY1 bound specifically to three YY1 binding sites on the proximal promoter of Msx2 that accounted for this transactivation. Mutations introduced to these sites reduced the level of YY1 transactivation. As bone morphogenetic protein type 4 (BMP4) regulates Msx2 expression in embryonic tissues and in P19 cells, we further tested whether YY1 is the mediator of this BMP4 activity. BMP4 did not induce the expression of YY1 in early mouse mandibular explants, nor in P19 cells, suggesting that YY1 is not a required mediator of the BMP4 pathway in these tissues at this developmental stage. Taken together, these findings suggest that YY1 functions as an activator for the Msx2 gene, and that this regulation, which is independent of the BMP4 pathway, may be required during early mouse craniofacial and limb morphogenesis.

Bone mineral content and bone metabolism in young adults with severe periodontitis.

OBJECTIVES: To clarify in young adults with severe periodontitis (1) whether the bone mineral content (BMC) or density (BMD) in the mandible/other skeletal sites and the systemic bone metabolism differed from normal and (2) whether mandibular/forearm BMC did change during the 5 to 10-year follow-up. MATERIAL AND METHODS: 24 young otherwise normal patients with verified severe periodontitis were included, of which 20 attended the follow-up visit. Mandibular/forearm BMC was measured at both visits by dual-photon absorptiometry, supplemented with femoral neck/lumbar spine BMD measurements at follow-up visit by dual-energy X-ray absorptiometry. Serum alkaline phosphatase/ionized calcium, urinary excretion of pyridinoline/deoxy-pyridinoline were analysed at the follow-up visit. A conventional periodontal examination was performed at both visits. RESULTS: Mandibular BMC was significantly below normal mean BMC at both visits. The mandibular Z-scores were < or = -2.00 in 33.3% (8/24). BMC/BMD in the remaining sites and the values for bone markers did not differ from normal. Mandibular/forearm BMC was stable while a significant aggravation of alveolar bone loss occurred during the trial without change of probing depth. CONCLUSIONS: Severe periodontitis in young adults seems to be a local disorder associated with relatively low BMC in the jaws without systemic alterations of BMC/BMD and bone metabolism.

Influence of diet composition and malocclusion on masticatory organs in rats.

The present study was undertaken to determine the effects of different dietary consistencies and malocclusion induced by extraction of molar teeth on the masticatory organs of weaning and adult rats, by determining the biochemical properties of masseter muscle, and also Ca and P levels in mandibular bone. Male SD rats, 3 and 20 weeks old, were divided into 3 groups. Group one (G-1) was maintained on a solid diet, and Groups two (G-2) and three (G-3) on a semi-solid diet. Furthermore, the mandibular molar teeth of G-3 rats were extracted. The experimental period was 120 days. The masseter muscle and mandibular bone weights of G-1 in weaning rats were increased significantly in comparison with G-3, but not in adult rats. The CPK activities in weaning and adult rats of G-1 were higher than those in the other two groups. The order of LDH activity in weaning and adult rats was G-3 > G-2 > G-1. G-2 and particularly G-3 showed significantly lower glycogen contents than G-1. The Ca and P contents of the mandibular bone in G-2 and G-3 were lower than those in G-1. These results suggest that a different dietary consistency and malocclusion induced by extraction of mandibular molar teeth have a considerable influence on the development of masticatory organs, mandibular bone and masseter muscle.

Alterations in the expression of osteonectin, osteopontin and osteocalcin mRNAs during the development of skeletal tissues in vivo.

Heterogeneity in the expression of three members of non-collagenous matrix proteins in osteogenic and chondrogenic development in vivo were investigated by in situ hybridization. Sections of several skeletal tissues from mice at various stages of development were hybridized with digoxigenin-labeled complementary RNA probes encoding osteonectin (Osn), osteopontin (Osp) and osteocalcin (Osc). In calvariae and mandibulae, Osn messenger RNA (mRNA) was detected in cells in pre-osseous and osseous tissues before mineralization. Osp mRNA was found in cells attached to the mineralized bone matrix together with Osn mRNA followed by the expression Osc mRNA. In long bones, mRNAs for Osn, Osp and Osc were sequentially expressed with bone development from primary spongiosa to diaphyseal bone. In growth cartilage, Osn mRNA was observed in chondrocytes in non-mineralized cartilage, whereas Osp mRNA was detected in hypertrophic chondrocytes in mineralized cartilage matrix with a characteristic switch in expression. Osc mRNA was not detected in any chondrocytes. These results indicate that osteogenic differentiation in bone development in vivo is characterized by the sequential expression of these three genes, and suggest that these genes are expressed differentially and specifically, in association with extra-cellular matrix mineralization.

[The effect of mexidol on the rat periodontium during hypokinesia]. / Vliianie meksidola na parodont krys pri gipokinezii.

During 30-days-long hypokinesia changes in calcium-phosphoric metabolism, glycoprotein state, acid and alkaline phosphatase activity in bone tissue of the low jaw of rats proceed in some stages, that determines the nature of mexidol (3-oxipyridine derivative) effects. The remedy demonstrated the most expressed protective effect on parodontal tissues on the 15th day of animals' mobility limitation. The data obtained show that mexidol effecting hypokinesia develop in some stages and depend on the initial state of the organism. They also reflect indirectly the significance of the antioxidant status of the organism in mineralization of the parodontal bone tissue.

[The protein content and cathepsin D activity of the mandibular bone in rats during fracture healing]. / Soderzhanie belka i aktivnost' katepsina D v kosti nizhnei cheliusti krys pri zazhivlenii pereloma.

Soluble and total protein content and cathepsin D activity were measured in rat mandibular bone tissue during healing of a fracture treated with proteolytic enzymes (andecalin) or protease inhibitors (contrykal) electrophoresis. Contrykal essentially elevated collagen content in mandibular bone tissue and reduced lysosomal enzymes activity. Andecalin had no noticeable effect on the levels of total protein and collagen, but it significantly increased the activity of cathepsin D. The findings evidence that local contrykal electrophoresis in the focus of injury creates an effective concentration of the drug that enhances the fracture healing and prevents the development of pyoinflammatory complications.

Distribution of fluoride across cementum, dentine and alveolar bone in rats.

This study was undertaken to determine the fluoride distribution in cementum and neighboring hard tissues of the rat after different levels of fluoride administration via the drinking water. Specimens of cementum with underlying dentine and adjacent bone were removed from the distal roots of the first lower molars. The fluoride distribution in each specimen was determined in samples removed sequentially using an abrasive microsampling technique. Fluoride concentrations were highest at or near the surface and decreased towards the interior of cementum, dentine and alveolar bone in both control and experimental groups. With increasing fluoride intake, concentrations increased throughout the tissue. The distribution patterns of fluoride in cementum of contralateral teeth from the same animal were similar. Fluoride concentrations in cementum were higher than those of dentine and alveolar bone.