Salivary proteomics in bisphosphonate-related osteonecrosis of the jaw.

OBJECTIVE: The objective of this study was to identify differentially expressed salivary proteins in bisphosphonate-related osteonecrosis of the jaw (BRONJ) patients that could serve as biomarkers for BRONJ diagnosis. SUBJECTS AND METHODS: Whole saliva obtained from 20 BRONJ patients and 20 controls were pooled within groups. The samples were analyzed using iTRAQ-labeled two-dimensional liquid chromatography-tandem mass spectrometry. RESULTS: Overall, 1340 proteins were identified. Of these, biomarker candidates were selected based on P-value (<0.001), changes in protein expression (≥1.5-fold increase or decrease), and unique peptides identified (≥2). Three comparisons made between BRONJ and control patients identified 200 proteins to be differentially expressed in BRONJ patients. A majority of these proteins were predicted to have a role in drug metabolism and immunological and dermatological diseases. Of all the differentially expressed proteins, we selected metalloproteinase-9 and desmoplakin for further validation. Immunoassays confirmed increased expression of metalloproteinase-9 in individual saliva (P = 0.048) and serum samples (P = 0.05) of BRONJ patients. Desmoplakin was undetectable in saliva. However, desmoplakin levels tended to be lower in BRONJ serum than controls (P = 0.157). CONCLUSIONS: Multiple pathological reactions are involved in BRONJ development. One or more proteins identified by this study may prove to be useful biomarkers for BRONJ diagnosis. The role of metalloproteinase-9 and desmoplakin in BRONJ requires further investigation.

Murine IRF8 Mutation Offers New Insight into Osteoclast and Root Resorption.

Interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, functions as a negative regulator of osteoclasts and helps maintain dental and skeletal homeostasis. Previously, we reported that a novel mutation in the IRF8 gene increases susceptibility to multiple idiopathic cervical root resorption (MICRR), a form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function in osteoclastogenesis, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 technique modeling the human IRF8G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature-inserted Het and Homo mice displayed significantly increased root resorption and osteoclast-mediated alveolar bone loss compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of IRF8G388S mutation to inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts, as well as its impact on autophagy-related pathways of osteoclast differentiation. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. IRF8G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. These insights extend beyond oral health and significantly advance our understanding of skeletal disorders mediated by increased osteoclast activity and IRF8's role in osteoclastogenesis.

Effect of Periodontitis and Periodontal Therapy on Oral and Gut Microbiota.

Mounting evidence indicates that periodontitis-related oral bacteria may contribute to gut microbial dysbiosis. This clinical study aimed to explore the oral-gut microbial signatures associated with periodontitis and to longitudinally evaluate the effect of periodontal treatment on the oral and gut microbial composition. Stool and saliva samples from generalized stage III/IV periodontitis patients (n = 47) were collected and analyzed by 16S ribosomal RNA gene amplicon sequencing, before and 3 mo after steps I to II of periodontal therapy. Periodontally healthy matched subjects (n = 47) were used as controls. Principal component analysis was carried out to identify oral-gut microbial profiles between periodontitis patients at baseline and healthy subjects; periodontitis samples were longitudinally compared before and after treatment. ß-Diversity of gut microbial profiles of periodontitis patients before treatment significantly differed from healthy controls (P < 0.001). Periodontal therapy was associated with a significant change in gut microbiota (P < 0.001), with post-treatment microbial profiles similar to healthy volunteers. A higher abundance of Bacteroides, Faecalibacterium, Fusobacterium, and Lachnospiraceae was noted in fecal samples of periodontitis patients at baseline compared to healthy controls. In contrast, Lactobacillus was the only genus more abundant in the latter. Additionally, periodontal therapy led to a parallel reduction in the salivary carriage of periodontal pathobionts, as well as gut Bacteroides, Lachnoclostridium, Lachnospiraceae, Oscillospiraceae, and Ruminococcaceae, to levels similar to healthy controls. Collectively, discriminating oral-gut microbial signatures of periodontitis were found. Periodontal treatment both mitigated oral dysbiosis and altered gut microbial composition, signifying potential broader implications for gastrointestinal health and disease.

Nociceptor Neurons Magnify Host Responses to Aggravate Periodontitis.

Periodontitis is a highly prevalent chronic inflammatory disease that progressively destroys the structures supporting teeth, leading to tooth loss. Periodontal tissue is innervated by abundant pain-sensing primary afferents expressing neuropeptides and transient receptor potential vanilloid 1 (TRPV1). However, the roles of nociceptive nerves in periodontitis and bone destruction are controversial. The placement of ligature around the maxillary second molar or the oral inoculation of pathogenic bacteria induced alveolar bone destruction in mice. Chemical ablation of nociceptive neurons in the trigeminal ganglia achieved by intraganglionic injection of resiniferatoxin decreased bone loss in mouse models of experimental periodontitis. Consistently, ablation of nociceptive neurons decreased the number of osteoclasts in alveolar bone under periodontitis. The roles of nociceptors were also determined by the functional inhibition of TRPV1-expressing trigeminal afferents using an inhibitory designer receptor exclusively activated by designer drugs (DREADD) receptor. Noninvasive chemogenetic functional silencing of TRPV1-expressing trigeminal afferents not only decreased induction but also reduced the progression of bone loss in periodontitis. The infiltration of leukocytes and neutrophils to the periodontium increased at the site of ligature, which was accompanied by increased amount of proinflammatory cytokines, such as receptor activator of nuclear factor κΒ ligand, tumor necrosis factor, and interleukin 1ß. The extents of increase in immune cell infiltration and cytokines were significantly lower in mice with nociceptor ablation. In contrast, the ablation of nociceptors did not alter the periodontal microbiome under the conditions of control and periodontitis. Altogether, these results indicate that TRPV1-expressing afferents increase bone destruction in periodontitis by promoting hyperactive host responses in the periodontium. We suggest that specific targeting of neuroimmune and neuroskeletal regulation can offer promising therapeutic targets for periodontitis supplementing conventional treatments.

Delivery of Alkaline Phosphatase Promotes Periodontal Regeneration in Mice.

Factors regulating the ratio of pyrophosphate (PPi) to phosphate (Pi) modulate biomineralization. Tissue-nonspecific alkaline phosphatase (TNAP) is a key promineralization enzyme that hydrolyzes the potent mineralization inhibitor PPi. The goal of this study was to determine whether TNAP could promote periodontal regeneration in bone sialoprotein knockout mice (Ibsp-/- mice), which are known to have a periodontal disease phenotype. Delivery of TNAP was accomplished either systemically (through a lentiviral construct expressing a mineral-targeted TNAP-D10 protein) or locally (through addition of recombinant human TNAP to a fenestration defect model). Systemic TNAP-D10 delivered by intramuscular injection at 5 d postnatal (dpn) increased circulating alkaline phosphatase (ALP) levels in Ibsp-/- mice by 5-fold at 30 dpn, with levels returning to normal by 60 dpn when tissues were evaluated by micro-computed tomography and histology. Local delivery of recombinant human TNAP to fenestration defects in 5-wk-old wild type (WT) and Ibsp-/- mice did not alter long-term circulating ALP levels, and tissues were evaluated by micro-computed tomography and histology at postoperative day 45. Systemic and local delivery of TNAP significantly increased alveolar bone volume (20% and 37%, respectively) and cementum thickness (3- and 42-fold) in Ibsp-/- mice, with evidence for periodontal ligament attachment and bone/cementum marker localization. Local delivery significantly increased regenerated cementum and bone in WT mice. Addition of 100-µg/mL bovine intestinal ALP to culture media to increase ALP in vitro increased media Pi concentration, mineralization, and Spp1 and Dmp1 marker gene expression in WT and Ibsp-/- OCCM.30 cementoblasts. Use of phosphonoformic acid, a nonspecific inhibitor of sodium Pi cotransport, indicated that effects of bovine intestinal ALP on mineralization and marker gene expression were in part through Pi transport. These findings show for the first time through multiple in vivo and in vitro approaches that pharmacologic modulation of Pi/PPi metabolism can overcome periodontal breakdown and accomplish regeneration.

Reduced Orthodontic Tooth Movement in Enpp1 Mutant Mice with Hypercementosis.

Previous studies revealed that cementum formation is tightly regulated by inorganic pyrophosphate (PPi), a mineralization inhibitor. Local PPi concentrations are determined by regulators, including ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which increases PPi concentrations by adenosine triphosphate hydrolysis. Orthodontic forces stimulate alveolar bone remodelling, leading to orthodontic tooth movement (OTM). To better understand how disturbed mineral metabolism and the resulting altered periodontal structures affect OTM, we employed Enpp1 mutant mice that feature reduced PPi and increased cervical cementum in a model of OTM induced by a stretched closed-coil spring ligated between the maxillary left first molar and maxillary incisors. We analyzed tooth movement, osteoclast/odontoclast response, and tooth root resorption by micro-computed tomography, histology, histomorphometry, and immunohistochemistry. Preoperatively, we noted an altered periodontium in Enpp1 mutant mice, with significantly increased periodontal ligament (PDL) volume and thickness, as well as increased PDL-bone/tooth root surface area, compared to wild-type (WT) controls. After 11 d of orthodontic treatment, Enpp1 mutant mice displayed 38% reduced tooth movement versus WT mice. Molar roots in Enpp1 mutant mice exhibited less change in PDL width in compression and tension zones compared to WT mice. Root resorption was noted in both groups with no difference in average depths, but resorption lacunae in Enpp1 mutant mice were almost entirely limited to cementum, with 150% increased cementum resorption and 92% decreased dentin resorption. Osteoclast/odontoclast cells were reduced by 64% in Enpp1 mutant mice, with a predominance of tartrate-resistant acid phosphatase (TRAP)-positive cells on root surfaces, compared to WT mice. Increased numbers of TRAP-positive cells on root surfaces were associated with robust immunolocalization of osteopontin (OPN) and receptor-activator of NF-κB ligand (RANKL). Collectively, reduced response to orthodontic forces, decreased tooth movement, and altered osteoclast/odontoclast distribution suggests Enpp1 loss of function has direct effects on clastic function/recruitment and/or indirect effects on periodontal remodeling via altered periodontal structure or tissue mineralization.

Periodontitis in Chédiak-Higashi Syndrome: An Altered Immunoinflammatory Response.

Chédiak-Higashi syndrome (CHS), a rare autosomal recessive disorder caused by mutations in the lysosomal trafficking regulator gene (LYST), is associated with aggressive periodontitis. It is suggested that LYST mutations affect the toll-like receptor (TLR)-mediated immunoinflammatory response, leading to frequent infections. This study sought to determine the periodontal status of patients with classic (severe) and atypical (milder) forms of CHS and the immunoregulatory functions of gingival fibroblasts in CHS patients. In contrast to aged-matched healthy controls, atypical (n = 4) and classic (n = 3) CHS patients presented with mild chronic periodontitis with no evidence of gingival ulceration, severe tooth mobility, or premature exfoliation of teeth. As a standard of care, all classic CHS patients had undergone bone marrow transplantation (BMT). Primary gingival fibroblasts obtained from atypical and BMT classic CHS patients displayed higher protein expression of TLR-2 (1.81-fold and 1.56-fold, respectively) and decreased expression of TLR-4 (-2.5-fold and -3.85-fold, respectively) at baseline when compared with healthy control gingival fibroblasts. When challenged with whole bacterial extract of Fusobacterium nucleatum, both atypical and classic CHS gingival fibroblasts failed to up-regulate TLR-2 and TLR-4 expression when compared with their respective untreated groups and control cells. Cytokine multiplex analysis following F. nucleatum challenge showed that atypical CHS gingival fibroblasts featured significantly increased cytokine expression (interleukin [IL]-2, IL-4, IL-5, IL-6, IL-10, IL-12, interferon-γ, tumor necrosis factor-α), whereas classic CHS cells featured similar/decreased cytokine expression when compared with treated control cells. Collectively, these results suggest that LYST mutations in CHS patients affect TLR-2 and TLR-4 expression/function, leading to dysregulated immunoinflammatory response, which in turn may influence the periodontal phenotype noted in CHS patients. Furthermore, our results suggest that atypical CHS patients and classic CHS patients who undergo BMT early in life are less susceptible to aggressive periodontitis and that hematopoietic cells play a critical role in mitigating the risk of aggressive periodontitis in CHS. Knowledge Transfer Statement: Results from this study can be used to create awareness among clinicians and researchers that not all CHS patients exhibit historically reported aggressive periodontitis, especially if they have atypical CHS disease or have received bone marrow transplantation. LYST mutations in CHS patients may affect TLR-2 and TLR-4 expression/function leading to dysregulated immunoinflammatory response, which in turn may influence the periodontal phenotype noted in CHS patients.

Hypercementosis Associated with ENPP1 Mutations and GACI.

Mineralization of bones and teeth is tightly regulated by levels of extracellular inorganic phosphate (Pi) and pyrophosphate (PPi). Three regulators that control pericellular concentrations of Pi and PPi include tissue-nonspecific alkaline phosphatase (TNAP), progressive ankylosis protein (ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Inactivation of these factors results in mineralization disorders affecting teeth and their supporting structures. This study for the first time analyzed the effect of decreased PPi on dental development in individuals with generalized arterial calcification of infancy (GACI) due to loss-of-function mutations in the ENPP1 gene. Four of the 5 subjects reported a history of infraocclusion, overretained primary teeth, ankylosis, and/or slow orthodontic tooth movement, suggesting altered mineral metabolism contributing to disrupted tooth movement and exfoliation. All subjects had radiographic evidence of unusually protruding cervical root morphology in primary and/or secondary dentitions. High-resolution micro-computed tomography (micro-CT) analyses of extracted primary teeth from 3 GACI subjects revealed 4-fold increased cervical cementum thickness ( P = 0.00007) and a 23% increase in cementum density ( P = 0.009) compared to age-matched healthy control teeth. There were no differences in enamel and dentin densities between GACI and control teeth. Histology revealed dramatically expanded cervical cementum in GACI teeth, including cementocyte-like cells and unusual patterns of cementum resorption and repair. Micro-CT analysis of Enpp1 mutant mouse molars revealed 4-fold increased acellular cementum thickness ( P = 0.002) and 5-fold increased cementum volume ( P = 0.002), with no changes in enamel or dentin. Immunohistochemistry identified elevated ENPP1 expression in cementoblasts of human and mouse control teeth. Collectively, these findings reveal a novel dental phenotype in GACI and identify ENPP1 genetic mutations associated with hypercementosis. The sensitivity of cementum to reduced PPi levels in both human and mouse teeth establishes this as a well-conserved and fundamental biological process directing cementogenesis across species (ClinicalTrials.gov NCT00369421).

PTH and Vitamin D Repress DMP1 in Cementoblasts.

A complex feedback mechanism between parathyroid hormone (PTH), 1,25(OH)2D3 (1,25D), and fibroblast growth factor 23 (FGF-23) maintains mineral homeostasis, in part by regulating calcium and phosphate absorption/reabsorption. Previously, we showed that 1,25D regulates mineral homeostasis by repressing dentin matrix protein 1 (DMP1) via the vitamin D receptor pathway. Similar to 1,25D, PTH may modulate DMP1, but the underlying mechanism remains unknown. Immortalized murine cementoblasts (OCCM.30), similar to osteoblasts and known to express DMP1, were treated with PTH (1-34). Real-time quantitative polymerase chain reaction (PCR) and Western blot revealed that PTH decreased DMP1 gene transcription (85%) and protein expression (30%), respectively. PTH mediated the downregulation of DMP1 via the cAMP/protein kinase A (PKA) pathway. Immunohistochemistry confirmed the decreased localization of DMP1 in vivo in cellular cementum and alveolar bone of mice treated with a single dose (50 µg/kg) of PTH (1-34). RNA-seq was employed to further identify patterns of gene expression shared by PTH and 1,25D in regulating DMP1, as well as other factors involved in mineral homeostasis. PTH and 1,25D mutually upregulated 36 genes and mutually downregulated 27 genes by ≥2-fold expression (P ≤ 0.05). Many identified genes were linked with the regulation of bone/tooth homeostasis, cell growth and differentiation, calcium signaling, and DMP1 transcription. Validation of RNA-seq results via PCR array confirmed a similar gene expression pattern in response to PTH and 1,25D treatment. Collectively, these results suggest that PTH and 1,25D share complementary effects in maintaining mineral homeostasis by mutual regulation of genes/proteins associated with calcium and phosphate metabolism while also exerting distinct roles on factors modulating mineral metabolism. Furthermore, PTH may modulate phosphate homeostasis by downregulating DMP1 expression via the cAMP/PKA pathway. Targeting genes/proteins mutually governed by PTH and 1,25D may be a viable approach for designing new therapies for preserving mineralized tissue health.