A specialized metabolic pathway partitions citrate in hydroxyapatite to impact mineralization of bones and teeth.

Citrate is a critical metabolic substrate and key regulator of energy metabolism in mammalian cells. It has been known for decades that the skeleton contains most (>85%) of the body's citrate, but the question of why and how this metabolite should be partitioned in bone has received singularly little attention. Here, we show that osteoblasts use a specialized metabolic pathway to regulate uptake, endogenous production, and the deposition of citrate into bone. Osteoblasts express high levels of the membranous Na+-dependent citrate transporter solute carrier family 13 member 5 (Slc13a5) gene. Inhibition or genetic disruption of Slc13a5 reduced osteogenic citrate uptake and disrupted mineral nodule formation. Bones from mice lacking Slc13a5 globally, or selectively in osteoblasts, showed equivalent reductions in cortical thickness, with similarly compromised mechanical strength. Surprisingly, citrate content in mineral from Slc13a5-/- osteoblasts was increased fourfold relative to controls, suggesting the engagement of compensatory mechanisms to augment endogenous citrate production. Indeed, through the coordinated functioning of the apical membrane citrate transporter SLC13A5 and a mitochondrial zinc transporter protein (ZIP1; encoded by Slc39a1), a mediator of citrate efflux from the tricarboxylic acid cycle, SLC13A5 mediates citrate entry from blood and its activity exerts homeostatic control of cytoplasmic citrate. Intriguingly, Slc13a5-deficient mice also exhibited defective tooth enamel and dentin formation, a clinical feature, which we show is recapitulated in primary teeth from children with SLC13A5 mutations. Together, our results reveal the components of an osteoblast metabolic pathway, which affects bone strength by regulating citrate deposition into mineral hydroxyapatite.

Infigratinib, a selective FGFR1-3 tyrosine kinase inhibitor, alters dentoalveolar development at high doses.

BACKGROUND: Fibroblast growth factor receptor-3 (FGFR3) gain-of-function mutations are linked to achondroplasia. Infigratinib, a FGFR1-3 tyrosine kinase inhibitor, improves skeletal growth in an achondroplasia mouse model. FGFs and their receptors have critical roles in developing teeth, yet effects of infigratinib on tooth development have not been assessed. Dentoalveolar and craniofacial phenotype of Wistar rats dosed with low (0.1 mg/kg) and high (1.0 mg/kg) dose infigratinib were evaluated using micro-computed tomography, histology, and immunohistochemistry. RESULTS: Mandibular third molars were reduced in size and exhibited aberrant crown and root morphology in 100% of female rats and 80% of male rats at high doses. FGFR3 and FGF18 immunolocalization and extracellular matrix protein expression were unaffected, but cathepsin K (CTSK) was altered by infigratinib. Cranial vault bones exhibited alterations in dimension, volume, and density that were more pronounced in females. In both sexes, interfrontal sutures were significantly more patent with high dose vs vehicle. CONCLUSIONS: High dose infigratinib administered to rats during early stages affects dental and craniofacial development. Changes in CTSK from infigratinib in female rats suggest FGFR roles in bone homeostasis. While dental and craniofacial disruptions are not expected at therapeutic doses, our findings confirm the importance of dental monitoring in clinical studies.

Dental and craniofacial defects in the Crtap-/- mouse model of osteogenesis imperfecta type VII.

BACKGROUND: Inactivating mutations in the gene for cartilage-associated protein (CRTAP) cause osteogenesis imperfecta type VII in humans, with a phenotype that can include craniofacial defects. Dental and craniofacial manifestations have not been a focus of case reports to date. We analyzed the craniofacial and dental phenotype of Crtap-/- mice by skull measurements, micro-computed tomography (micro-CT), histology, and immunohistochemistry. RESULTS: Crtap-/- mice exhibited a brachycephalic skull shape with fusion of the nasofrontal suture and facial bones, resulting in mid-face retrusion and a class III dental malocclusion. Loss of CRTAP also resulted in decreased dentin volume and decreased cellular cementum volume, though acellular cementum thickness was increased. Periodontal dysfunction was revealed by decreased alveolar bone volume and mineral density, increased periodontal ligament (PDL) space, ectopic calcification within the PDL, bone-tooth ankylosis, altered immunostaining of extracellular matrix proteins in bone and PDL, increased pSMAD5, and more numerous osteoclasts on alveolar bone surfaces. CONCLUSIONS: Crtap-/- mice serve as a useful model of the dental and craniofacial abnormalities seen in individuals with osteogenesis imperfecta type VII.

High phosphate feeding promotes mineral and bone abnormalities in mice with chronic kidney disease.

BACKGROUND: Chronic kidney disease-mineral bone disorder (CKD-MBD) is a systemic syndrome characterized by imbalances in mineral homeostasis, renal osteodystrophy (ROD) and ectopic calcification. The mechanisms underlying this syndrome in individuals with chronic kidney disease (CKD) are not yet clear. METHODS: We examined the effect of normal phosphate (NP) or high phosphate (HP) feeding in the setting of CKD on bone pathology, serum biochemistry and vascular calcification in calcification-prone dilute brown non-agouti (DBA/2) mice. RESULTS: In both NP and HP-fed CKD mice, elevated serum parathyroid hormone and alkaline phosphatase (ALP) levels were observed, but serum phosphorus levels were equivalent compared with sham controls. CKD mice on NP diet showed trabecular alterations in the long bone consistent with high-turnover ROD, including increased trabecular number with abundant osteoblasts and osteoclasts. Despite trabecular bone and serum biochemical changes, CKD/NP mice did not develop vascular calcification. In contrast, CKD/HP mice developed arterial medial calcification (AMC), more severe trabecular bone alterations and cortical bone abnormalities that included decreased cortical thickness and density, and increased cortical porosity. Cortical bone porosity and trabecular number strongly correlated with the degree of aortic calcification. CONCLUSIONS: HP feeding was required to induce the full spectrum of CKD-MBD symptoms in CKD mice.

Catalysis-Independent ENPP1 Protein Signaling Regulates Mammalian Bone Mass.

Biallelic ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency induces vascular/soft tissue calcifications in generalized arterial calcification of infancy (GACI), and low bone mass with phosphate-wasting rickets in GACI survivors (autosomal hypophosphatemic rickets type-2). ENPP1 haploinsufficiency induces early-onset osteoporosis and mild phosphate wasting in adults. Both conditions demonstrate the unusual combination of reduced accrual of skeletal mineral, yet excess and progressive heterotopic mineralization. ENPP1 is the only enzyme that generates extracellular pyrophosphate (PPi), a potent inhibitor of both bone and heterotopic mineralization. Life-threatening vascular calcification in ENPP1 deficiency is due to decreased plasma PPi; however, the mechanism by which osteopenia results is not apparent from an understanding of the enzyme's catalytic activity. To probe for catalysis-independent ENPP1 pathways regulating bone, we developed a murine model uncoupling ENPP1 protein signaling from ENPP1 catalysis, Enpp1T238A mice. In contrast to Enpp1asj mice, which lack ENPP1, Enpp1T238A mice have normal trabecular bone microarchitecture and favorable biomechanical properties. However, both models demonstrate low plasma Pi and PPi, increased fibroblast growth factor 23 (FGF23), and by 23 weeks, osteomalacia demonstrating equivalent phosphate wasting in both models. Reflecting findings in whole bone, calvarial cell cultures from Enpp1asj mice demonstrated markedly decreased calcification, elevated transcription of Sfrp1, and decreased nuclear ß-catenin signaling compared to wild-type (WT) and Enpp1T238A cultures. Finally, the decreased calcification and nuclear ß-catenin signaling observed in Enpp1asj cultures was restored to WT levels by knockout of Sfrp1. Collectively, our findings demonstrate that catalysis-independent ENPP1 signaling pathways regulate bone mass via the expression of soluble Wnt inhibitors such as secreted frizzled-related protein 1 (SFRP1), whereas catalysis dependent pathways regulate phosphate homeostasis through the regulation of plasma FGF23. © 2022 American Society for Bone and Mineral Research (ASBMR).

Guidelines for Micro-Computed Tomography Analysis of Rodent Dentoalveolar Tissues.

Micro-computed tomography (µCT) has become essential for analysis of mineralized as well as nonmineralized tissues and is therefore widely applicable in the life sciences. However, lack of standardized approaches and protocols for scanning, analyzing, and reporting data often makes it difficult to understand exactly how analyses were performed, how to interpret results, and if findings can be broadly compared with other models and studies. This problem is compounded in analysis of the dentoalveolar complex by the presence of four distinct mineralized tissues: enamel, dentin, cementum, and alveolar bone. Furthermore, these hard tissues interface with adjacent soft tissues, the dental pulp and periodontal ligament (PDL), making for a complex organ. Drawing on others' and our own experience analyzing rodent dentoalveolar tissues by µCT, we introduce techniques to successfully analyze dentoalveolar tissues with similar or disparate compositions, densities, and morphological characteristics. Our goal is to provide practical guidelines for µCT analysis of rodent dentoalveolar tissues, including approaches to optimize scan parameters (filters, voltage, voxel size, and integration time), reproducibly orient samples, define regions and volumes of interest, segment and subdivide tissues, interpret findings, and report methods and results. We include illustrative examples of analyses performed on genetically engineered mouse models with phenotypes in enamel, dentin, cementum, and alveolar bone. The recommendations are designed to increase transparency and reproducibility, promote best practices, and provide a basic framework to apply µCT analysis to the dentoalveolar complex that can also be extrapolated to a variety of other tissues of the body. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Multiple Idiopathic Cervical Root Resorption: A Challenge for a Transdisciplinary Medical-Dental Team.

The goal of this perspective article is to use multiple idiopathic cervical root resorption (MICRR) as a model to demonstrate the need for transdisciplinary collaborations, from basic science to treatment planning, to improve the quality of health care for all. This is not a review of the literature on the current state of MICRR. Tooth root resorption is a normal physiological process required for resorption and exfoliation of primary teeth; however, root resorption of adult teeth is largely pathological. MICRR is an aggressive form of external root resorption, which occurs near the cemento-enamel junction (CEJ). The cause of MICRR remains elusive, however, it is mediated primarily by osteoclasts/odontoclasts. Accumulating case studies and experiments in animal models have provided insights into defining the etiologies and pathophysiological mechanisms for MICRR, which include: systemic conditions and syndromes, inherited genetic variants affecting osteoclast/odontoclast activity, altered periodontal structures, drug-induced root resorption and rebound effects after cessation of anti-resorptive treatment, chemotherapy, exposure to pets or viral infections, and other factors such as inflammatory conditions or trauma. To determine the causative factors for MICRR, as well as other oral-dental conditions, at minimum, a comprehensive health history should be collected for all patients by dental care providers, discussed with other health care providers and appropriate collaborations established. The examples highlighted in this perspective emphasize the need for transdisciplinary research collaborations coupled with integrated management strategies between medicine and dentistry in order to identify cause(s) early and improve clinical outcomes.

A Cross-Sectional Cohort Study of the Effects of FGF23 Deficiency and Hyperphosphatemia on Dental Structures in Hyperphosphatemic Familial Tumoral Calcinosis.

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare autosomal recessive disorder caused by mutations in FGF23, GALNT3, KLOTHO, or FGF23 autoantibodies. Prominent features include high blood phosphate and calcific masses, usually adjacent to large joints. Dental defects have been reported, but not systematically described. Seventeen patients with HFTC followed at the National Institutes of Health underwent detailed clinical, biochemical, molecular, and dental analyses. Studies of teeth included intraoral photos and radiographs, high-resolution µCT, histology, and scanning electron microscopy (SEM). A scoring system was developed to assess the severity of tooth phenotype. Pulp calcification was found in 13 of 14 evaluable patients. Short roots and midroot bulges with apical thinning were present in 12 of 13 patients. Premolars were most severely affected. µCT analyses of five HFTC teeth revealed that pulp density increased sevenfold, whereas the pulp volume decreased sevenfold in permanent HFTC teeth compared with age- and tooth-matched control teeth. Histology revealed loss of the polarized odontoblast cell layer and an obliterated pulp cavity that was filled with calcified material. The SEM showed altered pulp and cementum structures, without differences in enamel or dentin structures, when compared with control teeth. This study defines the spectrum and confirms the high penetrance of dental features in HFTC. The phenotypes appear to be independent of genetic/molecular etiology, suggesting hyperphosphatemia or FGF23 deficiency may be the pathomechanistic driver, with prominent effects on root and pulp structures, consistent with a role of phosphate and/or FGF23 in tooth development. Given the early appearance and high penetrance, cognizance of HFTC-related features may allow for earlier diagnosis and treatment. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Monocyte Subsets With High Osteoclastogenic Potential and Their Epigenetic Regulation Orchestrated by IRF8.

Osteoclasts (OCs) are bone-resorbing cells formed by the serial fusion of monocytes. In mice and humans, three distinct subsets of monocytes exist; however, it is unclear if all of them exhibit osteoclastogenic potential. Here we show that in wild-type (WT) mice, Ly6Chi and Ly6Cint monocytes are the primary source of OC formation when compared to Ly6C- monocytes. Their osteoclastogenic potential is dictated by increased expression of signaling receptors and activation of preestablished transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of interferon regulatory factor 8 (IRF8), a transcription factor important for myelopoiesis and osteoclastogenesis, all three monocyte subsets are programmed to display higher osteoclastogenic potential. Enhanced NFATc1 nuclear translocation and amplified transcriptomic and epigenetic changes initiated at early developmental stages direct the increased osteoclastogenesis in Irf8-deficient mice. Collectively, our study provides novel insights into the transcription factors and active cis-regulatory elements that regulate OC differentiation. © 2020 American Society for Bone and Mineral Research (ASBMR).

Mitigating osteonecrosis of the jaw (ONJ) through preventive dental care and understanding of risk factors.

It is well established that alterations in phosphate metabolism have a profound effect on hard and soft tissues of the oral cavity. The present-day clinical form of osteonecrosis of the jaw (ONJ) was preceded by phosphorus necrosis of the jaw, ca. 1860. The subsequent removal of yellow phosphorus from matches in the early 20th century saw a parallel decline in "phossy jaw" until the early 2000s, when similar reports of unusual jaw bone necrosis began to appear in the literature describing jaw necrosis in patients undergoing chemotherapy and concomitant steroid and bisphosphonate treatment. Today, the potential side effect of ONJ associated with medications that block osteoclast activity (antiresorptive) is well known, though the mechanism remains unclear and the management and outcomes are often unsatisfactory. Much of the existing literature has focused on the continuing concerns of appropriate use of bisphosphonates and other antiresorptive medications, the incomplete or underdeveloped research on ONJ, and the use of drugs with anabolic potential for treatment of osteoporosis. While recognizing that ONJ is a rare occurrence and ONJ-associated medications play an important role in fracture risk reduction in osteoporotic patients, evidence to date suggests that health care providers can lower the risk further by dental evaluations and care prior to initiating antiresorptive therapies and by monitoring dental health during and after treatment. This review describes the current clinical management guidelines for ONJ, the critical role of dental-medical management in mitigating risks, and the current understanding of the effects of predominantly osteoclast-modulating drugs on bone homeostasis.

Inactivating Mutation in IRF8 Promotes Osteoclast Transcriptional Programs and Increases Susceptibility to Tooth Root Resorption.

This is the first study to our knowledge to report a novel mutation in the interferon regulatory factor 8 gene (IRF8G388S ) associated with multiple idiopathic tooth root resorption, a form of periodontal disease. The IRF8G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. Functional assays demonstrated that the IRF8G388S mutant promoted osteoclastogenesis and failed to inhibit NFATc1-dependent transcriptional activation when compared with IRF8WT control. Further, similar to subjects with heterozygous IRF8G388S mutation, Irf8+/- mice exhibited increased osteoclast activity in the mandibular alveolar bone surrounding molar teeth. Immunohistochemistry illustrated increased NFATc1 expression in the dentoalveolar region of Irf8-/- and Irf8+/- mice when compared with Irf8+/+ controls. Genomewide analyses revealed that IRF8 constitutively bound to regulatory regions of several thousand genes in osteoclast precursors, and genetic aberration of IRF8 significantly enhanced many osteoclast-specific transcripts. Collectively, this study delineates the critical role of IRF8 in defining osteoclast lineage and osteoclast transcriptional program, which may help in better understanding of various osteoclast-mediated disorders, including periodontal disease. © 2019 American Society for Bone and Mineral Research.

Phosphate: known and potential roles during development and regeneration of teeth and supporting structures.

Inorganic phosphate (P(i)) is abundant in cells and tissues as an important component of nucleic acids and phospholipids, a source of high-energy bonds in nucleoside triphosphates, a substrate for kinases and phosphatases, and a regulator of intracellular signaling. The majority of the body's P(i) exists in the mineralized matrix of bones and teeth. Systemic P(i) metabolism is regulated by a cast of hormones, phosphatonins, and other factors via the bone-kidney-intestine axis. Mineralization in bones and teeth is in turn affected by homeostasis of P(i) and inorganic pyrophosphate (PPi), with further regulation of the P(i)/PP(i) ratio by cellular enzymes and transporters. Much has been learned by analyzing the molecular basis for changes in mineralized tissue development in mutant and knock-out mice with altered P(i) metabolism. This review focuses on factors regulating systemic and local P(i) homeostasis and their known and putative effects on the hard tissues of the oral cavity. By understanding the role of P(i) metabolism in the development and maintenance of the oral mineralized tissues, it will be possible to develop improved regenerative approaches.

Microporous nanofibrous fibrin-based scaffolds for bone tissue engineering.

The fibrotic response of the body to synthetic polymers limits their success in tissue engineering and other applications. Though porous polymers have demonstrated improved healing, difficulty in controlling their pore sizes and pore interconnections has clouded the understanding of this phenomenon. In this study, a novel method to fabricate natural polymer/calcium phosphate composite scaffolds with tightly controllable pore size, pore interconnection, and calcium phosphate deposition was developed. Microporous, nanofibrous fibrin scaffolds were fabricated using sphere-templating methods. Composite scaffolds were created by solution deposition of calcium phosphate on fibrin surfaces or by direct incorporation of nanocrystalline hydroxyapatite (nHA). The SEM results showed that fibrin scaffolds exhibited a highly porous and interconnected structure. Osteoblast-like cells, obtained from murine calvaria, attached, spread and showed a polygonal morphology on the surface of the biomaterial. Multiple cell layers and fibrillar matrix deposition were observed. Moreover, cells seeded on mineralized fibrin scaffolds exhibited significantly higher alkaline phosphatase activity as well as osteoblast marker gene expression compared to fibrin scaffolds and nHA incorporated fibrin scaffolds (0.25 and 0.5g). All types of scaffolds were degraded both in vitro and in vivo. Furthermore, these scaffolds promoted bone formation in a mouse calvarial defect model and the bone formation was enhanced by addition of rhBMP-2.

Transgenic overexpression of gremlin results in developmental defects in enamel and dentin in mice.

Bone morphogenetic proteins (BMPs) and BMP antagonists play a crucial role in the regulation of tooth development. One of the BMP extracellular antagonists, gremlin, is a highly conserved 20.7-kDa glycoprotein. Previously, researchers reported that transgenic mice overexpressing gremlin under the control of the osteocalcin promoter (gremlin OE) exhibit a skeletal phenotype and tooth fragility. To further define the tooth phenotype, teeth and surrounding supporting tissues, obtained from gremlin OE at ages of 4 weeks, 2 months, and 4 months, were examined. The histological results demonstrate that gremlin OE exhibit an enlarged pulp chamber with ectopic calcification and thinner dentin and enamel compared with wild-type control. In vitro studies using murine pulp cells revealed that gremlin inhibited BMP-4 mediated induction of Dspp. These data provide evidence that balanced interactions between BMP agonists/antagonists are required for proper development of teeth and surrounding tissues. It is clear that these interactions require further investigation to better define the mechanisms controlling tooth root formation (pulp, dentin, cementum, and surrounding tissue) to provide the information needed to successfully regenerate these tissues.

Enamel matrix derivative exhibits anti-inflammatory properties in monocytes.

BACKGROUND: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration with variable efficacy. EMD application results in significantly higher frequencies of sites without clinical signs of inflammation; additionally, patients receiving EMD therapy report significantly less post-treatment discomfort. However, there are few reports that focus on defining the biologic mechanisms for the observed anti-inflammatory effects of EMD. The aim of this study was to evaluate the influence of EMD on inflammatory-associated markers using an in vitro monocyte assay. METHODS: Rat monocytes were exposed to lipopolysaccharide (LPS; 100 ng/ml from Escherichia coli or Actinobacillus actinomycetemcomitans) along with EMD (0, 50, 100, or 200 microg/ml). Levels of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) (PGE(2)) in conditioned media were analyzed by enzyme-linked immunosorbent assay. In addition, the effects of exogenous PGE(2) on TNF-alpha production from LPS-stimulated monocytes were determined. RESULTS: LPS-stimulated monocytes exposed to EMD exhibited a decrease in TNF-alpha production (0.10- to 0.52-fold) and an increase in PGE(2) production (1.31- to 2.71-fold) compared to controls not treated with EMD. Exogenously applied PGE(2) decreased TNF-alpha production by LPS-stimulated monocytes in a dose-dependent manner, and EMD treatment enhanced this PGE(2)-mediated inhibition of TNF-alpha production. CONCLUSION: In addition to EMD's published role in inducing proliferation, migration, adhesion, mineralization, and differentiation of periodontal ligament cells, our results indicated that EMD modulates two inflammation-associated factors, TNF-alpha and PGE(2), in monocytes.

Prostaglandin E2 inhibits mineralization and enhances matrix metalloproteinase-13 in mature cementoblasts mainly via the EP4 pathway.

OBJECTIVES: Prostaglandin E(2) (PGE(2)) is an important factor in the pathogenesis of periodontal disease because of bone resorbing activity and association with attachment loss. PGE(2) and PGE receptor subtypes (EPs) play an important role in modulating bone metabolism via osteoblasts. However, little is known about the effects of PGE(2) on cementoblasts. The aims of this study were to determine the expression of EPs in mature cementoblasts, and to examine the effect of PGE(2) and EPs on their cellular function. DESIGN: Expression of EPs in immortalized mouse cementoblasts (OCCM-30 cells), which were characterized as mature cementoblasts, was determined using reverse transcriptase polymerase chain reaction (RT-PCR). The effects of PGE(2) and EP agonists on mineralization were examined by studying nodule formation with alizarin red S staining. Alkaline phosphatase (ALP) activity with PGE(2) and EP4 agonist was examined using the Bessey-Lowry enzymologic method. Effects of the PGE(2)-EP4 pathway on expression levels of osteocalcin (OCN) and matrix metalloproteinase-13 (MMP-13) mRNA were examined using real-time RT-PCR. RESULTS: OCCM-30 cells expressed EP1, EP2, EP3 and EP4 mRNA. PGE(2) and EP4 agonist led to downregulation of mineralized nodule formation and ALP activity in OCCM-30 cells. OCN mRNA expression was suppressed and MMP-13 mRNA expression was stimulated via the PGE(2)-EP4 pathway in OCCM-30 cells. CONCLUSIONS: Cementoblasts may downregulate their mineralization ability and upregulate MMP-13 production through the PGE(2)-EP4 pathway, and may contribute to destruction of connective tissue attachment under inflammatory conditions.

Autotherapies: Enhancing Endogenous Healing and Regeneration.

The promise of tissue engineering and regenerative medicine to reduce the burden of disease and improve quality of life are widely acknowledged. Traditional tissue engineering and regenerative medicine approaches rely on generation of tissue constructs in vitro for subsequent transplantation or injection of exogenously manipulated cells into a host. While promising, few such therapies have succeeded in clinical practice. Here, we propose that recent advances in stem cell and developmental biology, immunology, bioengineering, and material sciences, position us to develop a new generation of in vivo regenerative medicine therapies, which we term autotherapies. Autotherapies are strategies based on optimizing endogenous tissue responses and capitalizing on manipulation of stem cell niches and endogenous tissue microenvironments to enhance tissue healing and regeneration.