Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts.

Aim: Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail. Methods: The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs. Results: Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast's cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81. Conclusion: RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs.

Metabolomic signatures distinguish extracellular vesicles from osteoclasts and odontoclasts.

AIMS: Pathological dental root resorption and alveolar bone loss are often detected only after irreversible damage. Biomarkers in the gingival crevicular fluid or saliva could provide a means for early detection; however, such biomarkers have proven elusive. We hypothesize that a multiomic approach might yield reliable diagnostic signatures for root resorption and alveolar bone loss. Previously, we showed that extracellular vesicles (EVs) from osteoclasts and odontoclasts differ in their protein composition. In this study, we investigated the metabolome of EVs from osteoclasts, odontoclasts and clasts (non-resorbing clastic cells). MATERIALS AND METHODS: Mouse haematopoietic precursors were cultured on dentine, bone or plastic, in the presence of recombinant RANKL and CSF-1 to trigger differentiation along the clastic line. On Day 7, the cells were fixed and the differentiation state and resorptive status of the clastic cells were confirmed. EVs were isolated from the conditioned media on Day 7 and characterized by nanoparticle tracking and electron microscopy to ensure quality. Global metabolomic profiling was performed using a Thermo Q-Exactive Orbitrap mass spectrometer with a Dionex UHPLC and autosampler. RESULTS: We identified 978 metabolites in clastic EVs. Of those, 79 are potential biomarkers with Variable Interdependent Parameters scores of 2 or greater. Known metabolites cytidine, isocytosine, thymine, succinate and citrulline were found at statistically higher levels in EVs from odontoclasts compared with osteoclasts. CONCLUSION: We conclude that numerous metabolites found in odontoclast EVs differ from those in osteoclast EVs, and thus represent potential biomarkers for root resorption and periodontal tissue destruction.

Towards resorbable 3D-printed scaffolds for craniofacial bone regeneration.

This review will briefly examine the development of 3D-printed scaffolds for craniofacial bone regeneration. We will, in particular, highlight our work using Poly(L-lactic acid) (PLLA) and collagen-based bio-inks. This paper is a narrative review of the materials used for scaffold fabrication by 3D printing. We have also reviewed two types of scaffolds that we designed and fabricated. Poly(L-lactic acid) (PLLA) scaffolds were printed using fused deposition modelling technology. Collagen-based scaffolds were printed using a bioprinting technique. These scaffolds were tested for their physical properties and biocompatibility. Work in the emerging field of 3D-printed scaffolds for bone repair is briefly reviewed. Our work provides an example of PLLA scaffolds that were successfully 3D-printed with optimal porosity, pore size and fibre thickness. The compressive modulus was similar to, or better than, the trabecular bone of the mandible. PLLA scaffolds generated an electric potential upon cyclic/repeated loading. The crystallinity was reduced during the 3D printing. The hydrolytic degradation was relatively slow. Osteoblast-like cells did not attach to uncoated scaffolds but attached well and proliferated after coating the scaffold with fibrinogen. Collagen-based bio-ink scaffolds were also printed successfully. Osteoclast-like cells adhered, differentiated, and survived well on the scaffold. Efforts are underway to identify means to improve the structural stability of the collagen-based scaffolds, perhaps through mineralization by the polymer-induced liquid precursor process. 3D-printing technology is promising for constructing next-generation bone regeneration scaffolds. We describe our efforts to test PLLA and collagen scaffolds produced by 3D printing. The 3D-printed PLLA scaffolds showed promising properties akin to natural bone. Collagen scaffolds need further work to improve structural integrity. Ideally, such biological scaffolds will be mineralized to produce true bone biomimetics. These scaffolds warrant further investigation for bone regeneration.

Identification of an Electrogenic 2Cl-/H+ Exchanger, ClC5, as a Chloride-Secreting Transporter Candidate in Kidney Cyst Epithelium in Tuberous Sclerosis.

Kidney cyst expansion in tuberous sclerosis complex (TSC) or polycystic kidney disease (PKD) requires active secretion of chloride (Cl-) into the cyst lumen. In PKD, Cl- secretion is primarily mediated via the cystic fibrosis transmembrane conductance regulator (CFTR) in principal cells. Kidney cystogenesis in TSC is predominantly composed of type A intercalated cells, which do not exhibit noticeable expression of CFTR. The identity of the Cl--secreting molecule(s) in TSC cyst epithelia remains speculative. RNA-sequencing analysis results were used to examine the expression of FOXi1, the chief regulator of acid base transporters in intercalated cells, along with localization of Cl- channel 5 (ClC5), in various models of TSC. Results from Tsc2+/- mice showed that the expansion of kidney cysts corresponded to the induction of Foxi1 and correlated with the appearance of ClC5 and H+-ATPase on the apical membrane of cyst epithelia. In various mouse models of TSC, Foxi1 was robustly induced in the kidney, and ClC5 and H+-ATPase were expressed on the apical membrane of cyst epithelia. Expression of ClC5 was also detected on the apical membrane of cyst epithelia in humans with TSC but was absent in humans with autosomal dominant PKD or in a mouse model of PKD. These results indicate that ClC5 is expressed on the apical membrane of cyst epithelia and is a likely candidate mediating Cl- secretion into the kidney cyst lumen in TSC.

Cigarette smoke exposed airway epithelial cell-derived EVs promote pro-inflammatory macrophage activation in alpha-1 antitrypsin deficiency.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder most commonly secondary to a single mutation in the SERPINA1 gene (PI*Z) that causes misfolding and accumulation of alpha-1 antitrypsin (AAT) in hepatocytes and mononuclear phagocytes which reduces plasma AAT and creates a toxic gain of function. This toxic gain of function promotes a pro-inflammatory phenotype in macrophages that contributes to lung inflammation and early-onset COPD, especially in individuals who smoke cigarettes. The aim of this study is to determine the role of cigarette exposed AATD macrophages and bronchial epithelial cells in AATD-mediated lung inflammation. METHODS: Peripheral blood mononuclear cells from AATD and healthy individuals were differentiated into alveolar-like macrophages and exposed to air or cigarette smoke while in culture. Macrophage endoplasmic reticulum stress was quantified and secreted cytokines were measured using qPCR and cytokine ELISAs. To determine whether there is "cross talk" between epithelial cells and macrophages, macrophages were exposed to extracellular vesicles released by airway epithelial cells exposed to cigarette smoke and their inflammatory response was determined. RESULTS: AATD macrophages spontaneously produce several-fold more pro-inflammatory cytokines as compared to normal macrophages. AATD macrophages have an enhanced inflammatory response when exposed to cigarette smoke-induced extracellular vesicles (EVs) released from airway epithelial cells. Cigarette smoke-induced EVs induce expression of GM-CSF and IL-8 in AATD macrophages but have no effect on normal macrophages. Release of AAT polymers, potent neutrophil chemo attractants, were also increased from AATD macrophages after exposure to cigarette smoke-induced EVs. CONCLUSIONS: The expression of mutated AAT confers an inflammatory phenotype in AATD macrophages which disposes them to an exaggerated inflammatory response to cigarette smoke-induced EVs, and thus could contribute to progressive lung inflammation and damage in AATD individuals.

Identification of IQGAP1 as a SLC26A4 (Pendrin)-Binding Protein in the Kidney.

Background: Several members of the SLC26A family of transporters, including SLC26A3 (DRA), SLC26A5 (prestin), SLC26A6 (PAT-1; CFEX) and SLC26A9, form multi-protein complexes with a number of molecules (e.g., cytoskeletal proteins, anchoring or adaptor proteins, cystic fibrosis transmembrane conductance regulator, and protein kinases). These interactions provide regulatory signals for these molecules. However, the identity of proteins that interact with the Cl-/HCO3 - exchanger, SLC26A4 (pendrin), have yet to be determined. The purpose of this study is to identify the protein(s) that interact with pendrin. Methods: A yeast two hybrid (Y2H) system was employed to screen a mouse kidney cDNA library using the C-terminal fragment of SLC26A4 as bait. Immunofluorescence microscopic examination of kidney sections, as well as co-immunoprecipitation assays, were performed using affinity purified antibodies and kidney protein extracts to confirm the co-localization and interaction of pendrin and the identified binding partners. Co-expression studies were carried out in cultured cells to examine the effect of binding partners on pendrin trafficking and activity. Results: The Y2H studies identified IQ motif-containing GTPase-activating protein 1 (IQGAP1) as a protein that binds to SLC26A4's C-terminus. Co-immunoprecipitation experiments using affinity purified anti-IQGAP1 antibodies followed by western blot analysis of kidney protein eluates using pendrin-specific antibodies confirmed the interaction of pendrin and IQGAP1. Immunofluorescence microscopy studies demonstrated that IQGAP1 co-localizes with pendrin on the apical membrane of B-intercalated cells, whereas it shows basolateral expression in A-intercalated cells in the cortical collecting duct (CCD). Functional and confocal studies in HEK-293 cells, as well as confocal studies in MDCK cells, demonstrated that the co-transfection of pendrin and IQGAP1 shows strong co-localization of the two molecules on the plasma membrane along with enhanced Cl-/HCO3 - exchanger activity. Conclusion: IQGAP1 was identified as a protein that binds to the C-terminus of pendrin in B-intercalated cells. IQGAP1 co-localized with pendrin on the apical membrane of B-intercalated cells. Co-expression of IQGAP1 with pendrin resulted in strong co-localization of the two molecules and increased the activity of pendrin in the plasma membrane in cultured cells. We propose that pendrin's interaction with IQGAP1 may play a critical role in the regulation of CCD function and physiology, and that disruption of this interaction could contribute to altered pendrin trafficking and/or activity in pathophysiologic states.

Effects of Alendronate and Dexamethasone on Osteoclast Gene Expression and Bone Resorption in Mouse Marrow Cultures.

Osteoclasts are cells whose main function is the resorption of bone matrix. However, several factors, including medications, can interfere with the resorption process. Alendronate (ALN), a nitrogen-containing type of bisphosphonate, and dexamethasone (DEX), a glucocorticoid, are drugs that may affect the resorption activity. The aim of this study is to investigate the effects of ALN, and/or DEX on osteoclast gene expression and resorption activity in primary mouse marrow cultures stimulated with 1,25-dihydroxyvitamin D3, a model for the bone microenvironment. Cultures were treated only with ALN (10-5 M), DEX (10-6 M), and with a combination of both agents. Viability assays performed at days 5, 7, and 9 showed the highest number of viable cells at day 7. All the following assays were then performed at day 7 of cell culture: tartrate resistant acid phosphatase (TRAP) histochemistry, receptor activator of nuclear factor kappa B ligand (RANKL) immunofluorescence, osteoprotegerin (OPG), and RANKL gene expression by qPCR and resorption analysis by scanning electron microscopy. Treatment with ALN, DEX, and the combination of both did not promote significant changes in the number of TRAP+ cells, although larger giant cells were detected in groups treated with DEX. DEX treatment increased the gene expression of RANKL and reduced OPG. The treatment with ALN reduced the depth of the resorption pits, but their inhibitory effect was less effective when administered with DEX.

Genome-wide Association Study Identified Chromosome 8 Locus Associated with Medication-Related Osteonecrosis of the Jaw.

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious drug-related adverse event. To identify pharmacogenomic markers of MRONJ associated with bisphosphonate therapy, we conducted a genomewide association study (GWAS) meta-analysis followed by functional analysis of 5,008 individuals of European ancestry treated with bisphosphonates, which includes the largest number of MRONJ cases to date (444 cases and 4,564 controls). Discovery GWAS was performed in randomly selected 70% of the patients with cancer and replication GWAS was performed in the remaining 30% of the patients with cancer treated with intravenous bisphosphonates followed by meta-analysis of all 3,639 patients with cancer. GWAS was also performed in 1,369 patients with osteoporosis treated with oral bisphosphonates. The lead single-nucleotide polymorphism (SNP), rs2736308 on chromosome 8, was associated with an increased risk of MRONJ with an odds ratio (OR) of 2.71 and 95% confidence interval (CI) of 1.90-3.86 (P = 3.57*10-8 ) in the meta-analysis of patients with cancer. This SNP was validated in the MRONJ GWAS in patients with osteoporosis (OR: 2.82, 95% CI: 1.55-4.09, P = 6.84*10-4 ). The meta-analysis combining patients with cancer and patients with osteoporosis yielded the same lead SNP rs2736308 on chromosome 8 as the top SNP (OR: 2.74, 95% CI: 2.09-3.39, P = 9.65*10-11 ). This locus is associated with regulation of the BLK, CTSB, and FDFT1 genes, which had been associated with bone mineral density. FDFT1 encodes a membrane-associated enzyme, which is implicated in the bisphosphonate pathway. This study provides insights into the potential mechanism of MRONJ.

Orthodontic retention: what's on the horizon?

Orthodontic retention remains one of the great challenges in orthodontics. In this article, we discuss what is on the horizon to help address this challenge, including biological approaches to reduce relapse, treating patients without using retainers, technological developments, personalised medicine and the impact of COVID-19 on approaches to orthodontic retention.

RANKL and RANK in extracellular vesicles: surprising new players in bone remodeling.

Receptor activator of nuclear factor kappa B-ligand (RANKL), its receptor RANK, and osteoprotegerin which binds RANKL and acts as a soluble decoy receptor, are essential controllers of bone remodeling. They also play important roles in establishing immune tolerance and in the development of the lymphatic system and mammary glands. In bone, RANKL stimulates osteoclast formation by binding RANK on osteoclast precursors and osteoclasts. This is required for bone resorption. Recently, RANKL and RANK have been shown to be functional components of extracellular vesicles (EVs). Data linking RANKL and RANK in EVs to biological regulatory roles are reviewed, and crucial unanswered questions are examined. RANKL and RANK are transmembrane proteins and their presence in EVs allows them to act at a distance from their cell of origin. Because RANKL-bearing osteocytes and osteoblasts are often spatially distant from RANK-containing osteoclasts in vivo, this may be crucial for the stimulation of osteoclast formation and bone resorption. RANK in EVs from osteoclasts has the capacity to stimulate a RANKL reverse signaling pathway in osteoblasts that promotes bone formation. This serves to couple bone resorption with bone formation and has inspired novel bifunctional therapeutic agents. RANKL- and RANK- containing EVs in serum may serve as biomarkers for bone and immune pathologies. In summary, EVs containing RANKL and RANK have been identified as intercellular regulators in bone biology. They add complexity to the central signaling network responsible for maintaining bone. RANKL- and RANK-containing EVs are attractive as drug targets and as biomarkers.

Activation of (pro)renin by (pro)renin receptor in extracellular vesicles from osteoclasts.

The (pro)renin receptor (PRR) is a multifunctional integral membrane protein that serves as a component of the vacuolar H+-ATPase (V-ATPase) and also activates (pro)renin. We recently showed that full-length PRR, found as part of a V-ATPase sub-complex, is abundant in extracellular vesicles shed by osteoclasts. Here, we tested whether these extracellular vesicles stimulate (pro)renin. Extracellular vesicles isolated from the conditioned media of RAW 264.7 osteoclast-like cells or primary osteoclasts were characterized and counted by nanoparticle tracking. Immunoblotting confirmed that full-length PRR was present. Extracellular vesicles from osteoclasts dose-dependently stimulated (pro)renin activity, while extracellular vesicles from 4T1 cancer cells, in which we did not detect PRR, did not activate (pro)renin. To confirm that the ability of extracellular vesicles from osteoclasts to stimulate (pro)renin activity was due to the PRR, the "handle region peptide" from the PRR, a competitive inhibitor of PRR activity, was tested. It dose-dependently blocked the ability of extracellular vesicles to stimulate the enzymatic activity of (pro)renin. In summary, the PRR, an abundant component of extracellular vesicles shed by osteoclasts, stimulates (pro)renin activity. This represents a novel mechanism by which extracellular vesicles can function in intercellular regulation, with direct implications for bone biology.

SIRT1 Gene SNP rs932658 Is Associated With Medication-Related Osteonecrosis of the Jaw.

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious adverse drug reaction. Our previous whole-exome sequencing study found SIRT1 intronic region single-nucleotide polymorphism (SNP) rs7896005 to be associated with MRONJ in cancer patients treated with intravenous (iv) bisphosphonates (BPs). This study aimed to identify causal variants for this association. In silico analyses identified three SNPs (rs3758391, rs932658, and rs2394443) in the SIRT1 promoter region that are in high linkage disequilibrium (r2 > 0.8) with rs7896005. To validate the association between these SNPs and MRONJ, we genotyped these three SNPs on the germline DNA from 104 cancer patients of European ancestry treated with iv BPs (46 cases and 58 controls). Multivariable logistic regression analysis showed the minor alleles of these three SNPs were associated with lower odds for MRONJ. The odds ratios (95% confidence interval) and p values were 0.351 (0.164-0.751; p = 0.007) for rs3758391, 0.351 (0.164-0.751; p = 0.007) for rs932658, and 0.331 (0.157-0.697; p = 0.0036) for rs2394443, respectively. In the reporter gene assays, constructs containing rs932658 with variant allele A had higher luciferase activity than the reference allele, whereas constructs containing SNP rs3758391 and/or rs2394443 did not significantly affect activity. These results indicate that the promoter SNP rs932658 regulates the expression of SIRT1 and presumably lowers the risk of MRONJ by increasing SIRT1 expression. © 2020 American Society for Bone and Mineral Research (ASBMR).

Alpha-1 antitrypsin deficient individuals have circulating extracellular vesicles with profibrogenic cargo.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD)-mediated liver disease is a toxic "gain-of-function" inflammation in the liver associated with intracellular retention of mutant alpha-1 antitrypsin. The clinical presentation of the disease includes fibrosis, cirrhosis and liver failure. However, the pathogenic mechanism of AATD-mediated liver disease is not well understood. Here, we investigated the role of plasma extracellular vesicles (EVs) in progression of AATD-mediated liver disease. METHODS: EVs were isolated from plasma of AATD individuals with liver disease and healthy controls. Their cytokines and miRNA content were examined by multiplex assay and small RNA sequencing. The bioactivity of EVs was assessed by qPCR, western blot analysis and immunofluorescent experiments using human hepatic stellate cells (HSCs) treated with EVs isolated from control or AATD plasma samples. RESULTS: We have found that AATD individuals have a distinct population of EVs with pathological cytokine and miRNA contents. When HSCs were cultured with AATD plasma derived-EVs, the expression of genes related to the development of fibrosis were significantly amplified compared to those treated with healthy control plasma EVs. CONCLUSION: AATD individuals have a distinct population of EVs with abnormal cytokine and miRNA contents and the capacity to activate HSCs and mediate fibrosis. Better understanding of the components which cause liver inflammation and fibrogenesis, leading to further liver injury, has the potential to lead to the development of new treatments or preventive strategies to prevent AATD-mediated liver disease. Video abstract.

Actin and Actin-Associated Proteins in Extracellular Vesicles Shed by Osteoclasts.

Extracellular vesicles (EVs) are shed by all eukaryotic cells and have emerged as important intercellular regulators. EVs released by osteoclasts were recently identified as important coupling factors in bone remodeling. They are shed as osteoclasts resorb bone and stimulate osteoblasts to form bone to replace the bone resorbed. We reported the proteomic content of osteoclast EVs with data from two-dimensional, high resolution liquid chromatography/mass spectrometry. In this article, we examine in detail the actin and actin-associated proteins found in osteoclast EVs. Like EVs from other cell types, actin and various actin-associated proteins were abundant. These include components of the polymerization machinery, myosin mechanoenzymes, proteins that stabilize or depolymerize microfilaments, and actin-associated proteins that are involved in regulating integrins. The selective incorporation of actin-associated proteins into osteoclast EVs suggests that they have roles in the formation of EVs and/or the regulatory signaling functions of the EVs. Regulating integrins so that they bind extracellular matrix tightly, in order to attach EVs to the extracellular matrix at specific locations in organs and tissues, is one potential active role for actin-associated proteins in EVs.

The proteome of extracellular vesicles released by clastic cells differs based on their substrate.

Extracellular vesicles (EVs) from osteoclasts are important regulators in intercellular communication. Here, we investigated the proteome of EVs from clastic cells plated on plastic (clasts), bone (osteoclasts) and dentin (odontoclasts) by two-dimensional high performance liquid chromatography mass spectrometry seeking differences attributable to distinct mineralized matrices. A total of 1,952 proteins were identified. Of the 500 most abundant proteins in EVs, osteoclast and odontoclast EVs were 83.3% identical, while clasts shared 70.7% of the proteins with osteoclasts and 74.2% of proteins with odontoclasts. For each protein, the differences between the total ion count values were mapped to an expression ratio histogram (Z-score) in order to detect proteins differentially expressed. Stabilin-1 and macrophage mannose receptor-1 were significantly-enriched in EVs from odontoclasts compared with osteoclasts (Z = 2.45, Z = 3.34) and clasts (Z = 13.86, Z = 1.81) and were abundant in odontoclast EVs. Numerous less abundant proteins were differentially-enriched. Subunits of known protein complexes were abundant in clastic EVs, and were present at levels consistent with them being in assembled protein complexes. These included the proteasome, COP1, COP9, the T complex and a novel sub-complex of vacuolar H+-ATPase (V-ATPase), which included the (pro) renin receptor. The (pro) renin receptor was immunoprecipitated using an anti-E-subunit antibody from detergent-solubilized EVs, supporting the idea that the V-ATPase subunits present were in the same protein complex. We conclude that the protein composition of EVs released by clastic cells changes based on the substrate. Clastic EVs are enriched in various protein complexes including a previously undescribed V-ATPase sub-complex.

Clastic cells in orthodontic treatment: Translational challenges and recent advances.

OBJECTIVES: Orthodontic treatment consists of numerous appliance activations that rely on stimulation of osteoclasts at alveolar bone sites. However, the action of osteoclast-like cells on dentin ("odontoclasts") is a pathological side effect of orthodontic treatment. The aim of this article is twofold: (a) To report preliminary results from ongoing cell culture experiments to identify unique markers of dentin resorption, and (b) To discuss our work using nanoparticle tracking analysis (NTA) and exosomes for developing biological fluid-based biopsies to monitor clastic cell activity. SETTING AND SAMPLE POPULATION: Twelve healthy volunteers in permanent dentition. MATERIAL AND METHODS: For the in vitro experiments, murine clastic cell precursors were cultured on dentin or bone slices for 7 days and phage-display biopanning was used to identify molecular surface differences between osteoclasts and odontoclasts. In the human study, gingival crevicular fluid (GCF) samples were collected using different tools and analysed for protein and exosome recovery. RESULTS: Biopanning generated antibody fragments that were uniquely reactive to odontoclasts. Numerous nanoparticles in the size range of exosomes were detected in all of the human GCF samples. CONCLUSIONS: Our results support that there are molecular differences between osteoclasts and odontoclasts. Emerging technologies may allow the use of exosomes in GCF as a clinical tool to detect markers of root resorption.

Extracellular vesicle identification in tooth movement models.

Extracellular vesicles (EVs) are 30-150 nm in diameter vesicles released by cells that serve important intercellular regulatory functions. EVs include exosomes and microvesicles. Exosomes form in multivesicular bodies and are released extracellularly as the multivesicular bodies fuse with the plasma membrane. Microvesicles bud directly from the plasma membrane. Here, we examine methods that are available or emerging to detect and study EVs during orthodontic tooth movement (OTM). EV's involvement in regulating bone remodelling associated with OTM may be demonstrated by adding isolated EVs to an animal model to change the rate of tooth movement. Exosomes in multivesicular bodies might be detected by immunogold labelling of markers in sections from the tooth and jaw and detection by electron microscopy. Gingival crevicular fluid (GCF) is enriched in EVs. Detection and characterization of EVs released by osteoclasts during resorption have been described, and this information could be used to analyse EVs in OTM models. Regulatory EVs may be enriched in the GCF from teeth that are being moved or are undergoing root resorption. Emerging approaches, including nanoparticle tracking, ExoView and micro- and nanofluidics, show promise for studying EVs in the GCF. Techniques that amplify signal, including polymerase chain reaction (PCR), provide the sensitivity necessary to utilize EVs from GCF as biomarkers. Studies of the role of EVs in OTM will provide fresh insight that may identify means for enhancing OTM procedures. EVs in GCF may include biomarkers for bone remodelling during OTM, orthodontic-associated root resorption, and other dental pathologies.

Pharmacogenomics of osteonecrosis of the jaw.

Osteonecrosis of the jaw (ONJ) is a rare but serious drug induced adverse event, mainly associated with the use of antiresorptive medications, such as intravenous (IV) bisphosphonates (BPs) in cancer patients. In this review, we evaluated all the pharmacogenomic association studies for ONJ published up to December 2018. To date, two SNPs (CYP2C8 rs1934951 and RBMS3 rs17024608) were identified to be associated with ONJ by two genome-wide association studies (GWAS). However, all six subsequent candidate gene studies failed to replicate these results. In addition, six discovery candidate gene studies tried to identify the genetic markers in several genes associated with bone remodeling, bone mineral density, or osteoporosis. After evaluating the results of these 6 studies, none of the SNPs was significantly associated with ONJ. Recently, two whole-exome sequencing (WES) analysis (including one from our group) were performed to identify variants associated with ONJ. So far, only our study successfully replicated discovery result indicating SIRT1 SNP rs7896005 to be associated with ONJ. However, this SNP also did not reach genome-wide significance. The major limitations of these studies include lack of replication phases and limited sample sizes. Even though some studies had larger sample sizes, they recruited healthy individuals as controls, not subjects treated with BPs. We conclude that a GWAS with a larger sample size followed by replication phase will be needed to fully investigate the pharmacogenomic markers of ONJ.