Parameters associated with radiographic distal surface caries in the mandibular second molar adjacent to an impacted third molar.

BACKGROUND: To determine the risk factors for the development of radiographic distal surface caries (rDSC) in patients who attend routine dental check-ups during an era of National Institute for Health Care Excellence third molar surgery guidelines. METHODS: Radiographs taken during routine dental examinations involving 1012 patients from Manchester, UK were accessed. Clinical parameters, oral health, patient demographics, and socioeconomic factors were assessed. Risk factors were identified by multivariate logistic regression analysis. RESULTS: The detected rate of rDSC was 63.9% and rDSC was distributed homogenously across all five socioeconomic groups (p = 0.425). Risk factors associated with rDSC (p < 0.001) were identified as partially erupted mesio-angularly impacted mandibular third molars, third molars with compromised molar to molar contact points, loss of lamina dura of ≥ 2 mm, male gender, increasing age, and a higher modified Decayed Missing Filled Tooth score. CONCLUSION: rDSC was significantly associated with the angulation of third molars, the compromised contact position of the adjacent third molar, the periodontal status of the distal aspect of the second molar and the cumulative history of oral health in a population governed by specific third molar guidelines. An active approach to third molar surgical management could reduce rDSC and serve this population, irrespective of patients' socioeconomic or deprivation status.

Locoregional recurrence rate and disease-specific survival following marginal vs segmental resection for oral squamous cell carcinoma with mandibular bone invasion.

BACKGROUND AND OBJECTIVES: To determine locoregional recurrence rate (LRR) and disease-specific survival (DSS) following marginal vs segmental mandibulectomy. METHODS: Included were 210 patients, who had marginal or segmental mandibulectomy between 2000 and 2017. Marginal resection was performed when complete removal of the tumor was deemed feasible on the condition that at least 1 cm bone height of the inferior border of the mandible could be preserved. Segmental resection was performed in case less than 1 cm bone height of the mandible would remain. Clinical and histopathological data were collected from medical records. LRR and DSS were computed using Kaplan-Meier analysis. Cox-regression analysis was used to identify risk factors for LRR and DSS. RESULTS: A total of 59 marginal and 151 segmental resections had been performed. There was no significant difference in 3- and 5-year LRR (P = .904) and no significant difference in 3- and 5-year DSS (P = .362) between the marginal and segmental resection group. Cox-regression analysis showed a trend for surgical margin less than equal to 1 mm, to affect LRR (P = .05) and surgical margin less than equal 1 mm, perineural invasion and lymph node metastasis to affect DSS (P < .05). CONCLUSIONS: There was no difference in outcome between the two types of mandibulectomy.

Strontium, oxygen, and carbon isotope variation in modern human dental enamel.

OBJECTIVES: Isotopic analyses using human dental enamel provide information on the mobility and diet of individuals in forensic and archeological studies. Thus far, no study has systematically examined intraindividual coupled strontium (Sr), oxygen (O), and carbon (C) isotope variation in human enamel or the effect that caries have on the isotopic integrity of the enamel. The inadequate quantification of isotopic variation affects interpretations and may constrain sample selection of elements affected by caries. This study aims to quantify the intraindividual isotopic variation and provides recommendations for enamel sampling methods. MATERIAL AND METHODS: This study presents the first systematic results on intraindividual variation in Sr-O-C isotope composition and Sr concentration in modern human dental enamel of third molars (affected and unaffected by caries). A multiloci sampling approach (n = 6-20) was used to analyze surface and inner enamel, employing thermal ionization mass spectrometry (TIMS) and isotope ratio mass spectrometry (IRMS). Third molars were analyzed from 47 individuals from the Netherlands, Iceland, the United States, the Caribbean, Colombia, Somalia, and South Africa. RESULTS: Intradental isotopic variation in modern Dutch dental elements was recorded for Sr, O, and C and exceeded the variation introduced by the analytical error. Single loci and bulk sampling approaches of third molars established that a single analysis is only representative of the bulk Sr isotope composition in 60% of the elements analyzed. Dental elements affected by caries showed twice the variation seen in unaffected dental elements. Caries did not consistently incorporate the isotopic composition of the geographical environment in which they developed. DISCUSSION: The isotopic variability recorded in unaffected inner enamel indicates that variations greater than 0.000200 for 87 Sr/86 Sr and larger than 2‰ for δ18 O and δ13 C are required to demonstrate changes in modern Dutch human diet or geographic location.

The effect of Activin-A on periodontal ligament fibroblasts-mediated osteoclast formation in healthy donors and in patients with fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disease characterized by heterotopic ossification (HO). The disease is caused by a mutation in the activin receptor type 1 (ACVR1) gene that enhances this receptor's responsiveness to Activin-A. Binding of Activin-A to the mutated ACVR1 receptor induces osteogenic differentiation. Whether Activin-A also affects osteoclast formation in FOP is not known. Therefore we investigated its effect on the osteoclastogenesis-inducing potential of periodontal ligament fibroblasts (PLF) from teeth of healthy controls and patients with FOP. We used western blot analysis of phosphorylated SMAD3 (pSMAD3) and quantitative polymerase chain reaction to assess the effect of Activin-A on the PLF. PLF-induced osteoclast formation and gene expression were studied by coculturing control and FOP PLF with CD14-positive osteoclast precursor cells from healthy donors. Osteoclast formation was also assessed in control CD14 cultures stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANK-L). Although Activin-A increased activation of the pSMAD3 pathway in both control and FOP PLF, it increased ACVR1, FK binding protein 12 (FKBP12), an inhibitor of DNA binding 1 protein (ID-1) expression only in FOP PLF. Activin-A inhibited PLF mediated osteoclast formation albeit only significantly when induced by FOP PLF. In these cocultures, it reduced M-CSF and dendritic cell-specific transmembrane protein (DC-STAMP) expression. Activin-A also inhibited osteoclast formation in M-CSF and RANK-L mediated monocultures of CD14+ cells by inhibiting their proliferation. This study brings new insight on the role of Activin A in osteoclast formation, which may further add to understanding FOP pathophysiology; in addition to the known Activin-A-mediated HO, this study shows that Activin-A may also inhibit osteoclast formation, thereby further promoting HO formation.

Gender-Affirming Hormone Treatment Induces Facial Feminization in Transwomen and Masculinization in Transmen: Quantification by 3D Scanning and Patient-Reported Outcome Measures.

INTRODUCTION: Hormone treatment induces feminization of the body in transwomen and masculinization in transmen. However, the effect of hormone treatment on facial characteristics is still unknown. AIM: We aimed to study whether hormone treatment induces facial feminization and masculinization and how this potential change affects satisfaction and self-esteem. METHODS: In this single-center cohort study, we included 27 transwomen and 15 transmen who received standardized hormone treatment in the Center of Expertise on Gender Dysphoria, VU University Medical Center Amsterdam. Facial 3-dimensional images were obtained at baseline and at 3 and 12 months. At each image, 22 facial landmarks were placed. Furthermore, the FACE-Q Satisfaction with Facial Appearance Overall and the Rosenberg self-esteem scale were obtained at the same measurement points. MAIN OUTCOME MEASURES: The main outcome measures included the relative local shift of skin in millimeters in the 22 landmarks in the transverse (x-axis), coronal (y-axis), and sagittal (z-axis) anatomic axes, the color maps, and the outcomes of the questionnaires. RESULTS: After 12 months, cheek tissue in transwomen increased, with 0.50 mm (95% CI 0.04-0.96) in the x-axis and 1.08 mm (95% CI 0.31-1.85) in the z-axis. Tissue in the jaws decreased with -0.60 mm (95% CI -1.28-0.08) in the x-axis and -0.18 mm (95% CI -0.03-0.33) in the y-axis. Cheek tissue in transmen decreased with -0.45 mm (95% CI -1.00-0.11) in the x-axis and -0.84 mm (95% CI -1.92-0.25) in the z-axis. These changes already started after 3 months. An increase in satisfaction with the facial appearance was found in both transwomen and transmen. There were no changes in reported self-esteem. CLINICAL IMPLICATION: These results could lead to more realistic expectations of facial changes. Furthermore, our results suggest that the face continues to change for at least a year, which could suggest that performing facial feminization surgery after 1 year of hormone treatment might be too early. STRENGTH & LIMITATIONS: This study is the first that provides insight into the facial changes in transgender individuals receiving hormone treatment, and it introduces an objective method to examine (small) facial changes. Our study is limited by the poor reliability of the landmarks, the difficulty of facial fixation, and the lack of gender-specific questions in the questionnaires. CONCLUSIONS: Hormone treatment in transwomen induces an increase in cheek tissue and a decrease in jaw tissue. In transmen a tendency of decrease in cheek tissue and an increase in jaw tissue was found. These changes are in the direction of the desired gender. Tebbens M, Nota NM, Liberton NPTJ, et al. Gender-Affirming Hormone Treatment Induces Facial Feminization in Transwomen and Masculinization in Transmen: Quantification by 3D Scanning and Patient-Reported Outcome Measures. J Sex Med 2019;16:746-754.

Sjögren's syndrome is not a risk factor for periodontal disease: a systematic review.

OBJECTIVES: Sjögren's syndrome (SS) is an autoimmune disorder causing irreversible damage to the exocrine glands. Evidence whether SS patients are at a higher risk to develop periodontal disease is conflicting. Therefore, we systematically reviewed the literature on the prevalence of periodontal disease in patients with SS. METHODS: Searches were performed in MEDLINE and CENTRAL databases on prevalence of periodontal diseases in SS. Meta-analyses were performed for gingival index (GI), plaque index (PI), probing pocket depth (PPD), clinical attachment level (CAL), DMFT and DMFS (Decayed Missing Filled Teeth, respectively, Surfaces). RESULTS: Out of 512 studies, 10 studies were eligible for quantitative synthesis. Meta-analyses of the data indicated that in SS patients CAL, GI, PPD and PI are comparable to controls. DMFT and DMFS values were higher in SS patients than controls. CONCLUSIONS: No significant differences in the GI, PI, CAL, and PPD were observed in patients with SS compared to controls. These results indicate that there is no evidence of a higher risk for periodontal disease in patients with SS, while SS patients are more susceptible to caries compared to non-SS patients.

T Cell Proliferation Is Induced by Chronically TLR2-Stimulated Gingival Fibroblasts or Monocytes.

During inflammation of the gums, resident cells of the periodontium, gingival fibroblasts (GFs), interact with heterogeneous cell populations of the innate and adaptive immune system that play a crucial role in protecting the host from pathogenic infectious agents. We investigated the effects of chronic inflammation, by exposing peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocyte (PBL) cultures, and GF-PBMC cocultures to Toll-like receptor 2 (TLR2) and TLR4 activators for 21 days and assessed whether this influenced leukocyte retention, survival, and proliferation. Chronic stimulation of PBMC-GF cocultures with TLR2 and TLR4 agonists induced a reduction of NK (CD56+CD3-), T (CD3+), and B (CD19+) cells, whereas the number of TLR-expressing monocytes were unaffected. TLR2 agonists doubled the T cell proliferation, likely of a selective population, given the net decrease of T cells. Subsequent chronic exposure experiments without GF, using PBMC and PBL cultures, showed a significantly (p < 0.0001) increased proinflammatory cytokine production of TNF-α and IL-1ß up to 21 days only in TLR2-activated PBMC with concomitant T cell proliferation, suggesting a role for monocytes. In conclusion, chronic TLR activation mediates the shift in cell populations during infection. Particularly, TLR2 activators play an important role in T cell proliferation and proinflammatory cytokine production by monocytes, suggesting that TLR2 activation represents a bridge between innate and adaptive immunity.

Osteogenic and osteoclastogenic potential of jaw bone-derived cells-A case study.

Though the stem cell properties of tooth-derived periodontal ligament and gingival cells have been widely documented, surprisingly little is known about both the osteogenic and osteoclastogenic differentiation capacities of the more clinically relevant jaw bone-derived cells. These cells could be considered being recruited during bone healing such as after tooth extraction, after placing an implant, or after surgical or traumatic injury. Here, we compared the osteoblast and osteoclastogenesis features of four consecutive bone outgrowths with periodontal ligament and gingiva cells. For osteogenesis assay, cells were cultured in osteogenic medium, whereas in osteoclastogenesis assays, cells were cultured in the presence of human peripheral blood mononuclear cells (PBMCs) as a source of osteoclast precursors. After osteogenic stimulus, all six cell types responded by an increased expression of osteoblast markers RUNX2 and DMP1. Periodontal ligament cells expressed significantly higher levels of RUNX2 compared to all bone outgrowths. Alkaline phosphatase enzyme levels in periodontal ligament cells reached earlier and higher peak expression. Mineral deposits were highest in periodontal ligament, gingiva and the first bone outgrowth. Osteoclastogenesis revealed a stepwise increase of secreted pro-osteoclastogenesis proteins M-CSF, IL-1ß, and TNF-α in the last three consecutive bone cultures. OPG mRNA showed the opposite: high expression in periodontal and gingiva cells and the first outgrowth. Osteoclast numbers were similar between the six cultures, both on bone and on plastic. This first study reveals that jaw bone outgrowths contain bone remodelling features that are slightly different from tooth-associated cells.

Sialendoscopy enhances salivary gland function in Sjögren's syndrome: a 6-month follow-up, randomised and controlled, single blind study.

OBJECTIVES: To assess the effect of sialendoscopy of the major salivary glands on salivary flow and xerostomia in patients with Sjögren's syndrome (SS). METHODS: Forty-nine patients with SS were randomly assigned to a control group (n=15) and two intervention groups: irrigation of the major glands with saline (n=16) or with saline followed by triamcinolone acetonide (TA) in saline (n=18). Unstimulated whole saliva flow (UWS), chewing-stimulated whole saliva flow (SWS), citric acid-stimulated parotid flow (SPF), Clinical Oral Dryness Score (CODS), Xerostomia Inventory (XI) score and the European League Against Rheumatism (EULAR) SS Patient-Reported Index (ESSPRI) were obtained 1 week (T0) before, and 1 (T1), 8 (T8), 16 (T16) and 24 (T24) weeks after sialendoscopy. RESULTS: Median baseline UWS, SWS and SPF scores were 0.14, 0.46 and 0.22 mL/min, respectively. After intervention, significant increases in UWS and SWS were observed in the saline group (at T8 (P=0.013) and T24 (P=0.004)) and the saline/TA group (at T24 (P=0.03) and T=16 (P=0.035)). SPF was increased significantly in the saline/TA group at T24 (P=0.03). XI scores declined after sialendoscopy in both intervention groups. Compared with the control group, CODS, XI and ESSPRI improved in the intervention groups. UWS, SWS and SPF were higher in the intervention groups compared with the control group, but these differences were not significant except for SPF in the saline/TA group at T24 (P=0.005). CONCLUSIONS: Irrigation of the major salivary glands in patients with SS enhances salivary flow and reduces xerostomia up to 6 months after sialendoscopy.

Sialendoscopy of Salivary Glands Affected by Sjögren Syndrome: A Randomized Controlled Pilot Study.

PURPOSE: Sialendoscopy of the major salivary glands could alleviate the oral symptoms of Sjögren syndrome (SS) and restore salivary function. The aim of this pilot study was to evaluate the effect of sialendoscopy of the major salivary glands on salivary flow, saliva composition, and mouthfeel in patients with SS and to collect data for sample size analysis for a larger clinical trial. MATERIALS AND METHODS: Twenty patients diagnosed with SS were randomly assigned to a nonintervention control group or a sialendoscopy group. Unstimulated whole saliva flow, stimulated whole saliva flow, Clinical Oral Dryness Score, Xerostomia Inventory score, and EULAR Sjögren's Syndrome Patient Reported Index score were obtained 1 week before (T0), 1 week after (T2), and 8 weeks after (T3) sialendoscopy. Unstimulated whole saliva was analyzed for amylase concentration, activity, and mucin 5B concentration. Amylase and mucin 5B output were calculated. RESULTS: In the sialendoscopy group, unstimulated and stimulated whole saliva flows were numerically higher at T2 and T3 compared with T0. Xerostomia Inventory score was significantly lower in the sialendoscopy group at T2 compared with T0 (P = .03). Unstimulated and stimulated whole saliva flows were higher in the sialendoscopy group compared with the control group at T2 and T3 (not meaningful). Significant differences were found between groups for the EULAR Sjögren's Syndrome Patient Reported Index score at T2 (P = .03) and T3 (P = .001). Xerostomia Inventory score and Clinical Oral Dryness Score in the sialendoscopy group were lower compared with the control group at T2 (P = .02) and at T3 (P = .04), indicating less oral dryness. CONCLUSION: This pilot study indicates a positive effect of sialendoscopy on some parameters, but it cannot yet be concluded that it has a positive effect on salivary flow in patients with SS. These preliminary results need to be verified in a randomized controlled trial with a larger sample and longer follow-up period.

Application of Additive Manufacturing in Oral and Maxillofacial Surgery.

Additive manufacturing is the process of joining materials to create objects from digital 3-dimensional (3D) model data, which is a promising technology in oral and maxillofacial surgery. The management of lost craniofacial tissues owing to congenital abnormalities, trauma, or cancer treatment poses a challenge to oral and maxillofacial surgeons. Many strategies have been proposed for the management of such defects, but autogenous bone grafts remain the gold standard for reconstructive bone surgery. Nevertheless, cell-based treatments using adipose stem cells combined with osteoconductive biomaterials or scaffolds have become a promising alternative to autogenous bone grafts. Such treatment protocols often require customized 3D scaffolds that fulfill functional and esthetic requirements, provide adequate blood supply, and meet the load-bearing requirements of the head. Currently, such customized 3D scaffolds are being manufactured using additive manufacturing technology. In this review, 2 of the current and emerging modalities for reconstruction of oral and maxillofacial bone defects are highlighted and discussed, namely human maxillary sinus floor elevation as a valid model to test bone tissue-engineering approaches enabling the application of 1-step surgical procedures and seeding of Good Manufacturing Practice-level adipose stem cells on computer-aided manufactured scaffolds to reconstruct large bone defects in a 2-step surgical procedure, in which cells are expanded ex vivo and seeded on resorbable scaffolds before implantation. Furthermore, imaging-guided tissue-engineering technologies to predetermine the surgical location and to facilitate the manufacturing of custom-made implants that meet the specific patient's demands are discussed. The potential of tissue-engineered constructs designed for the repair of large oral and maxillofacial bone defects in load-bearing situations in a 1-step surgical procedure combining these 2 innovative approaches is particularly emphasized.

A 22-year follow-up of an endodontic implant.

Root fractures in the middle and apical thirds of the root are treated by repositioning and for approximately 6 weeks of immobilization while those in the cervical third are immobilized for 3 months. Even though the results are good, some root-fractured teeth are lost and replaced by dental implants or fixed partial dentures. One historic but effective treatment option for those root fractures with unfavorable crown to root ratios is an endodontic implant in middle and apical third root fractures. This method offers immediate stable fixation of a crown and its coronal root segment to the underlying alveolar bone. This report documents the long-term survival of a tooth treated with an endodontic implant. A 25-year-old male patient presented following a bicycle accident with a dislocated unfavorable root fracture in the middle third. The crown with the coronal root segment was secured to the bone using a commercially available endodontic implant. The apical part of the root was removed. Although the clinical and radiological follow-up results of the endodontic implant demonstrated a good clinical function and little bone loss, the implant ultimately had to be removed after 22 years of service due to pain and increasing mobility.

Proposal for a new staging system for osteoradionecrosis of the mandible.

A new staging system for osteoradionecrosis of the mandible has been retrospectively applied to a group of 31 patients. In this system clinicoradiographic signs and symptoms are incorporated in a simplified manner. For imaging purposes the use of plain radiographs such as periapical films and panoramic radiographs is recommended, mainly because of their readily availability. The presented staging system seems well reproducible, facilitating the comparison of study groups dealing with the various issues of osteoradionecrosis of the mandible. It is yet to be evaluated whether the presently proposed staging system is useful for management purposes.

Radiographic Detection Rate of Distal Surface Caries in the Mandibular Second Molar in Populations with Different Third Molar Management Strategies: A Multicenter Study.

Background: Distal surface caries (DSC) has been associated with partially erupted impacted third molars. The purpose of this study was to compare the rates of DSC between populations that had undergone different third molar management strategies. Methods: Radiographs that had been taken during routine examinations of 1012, 251 and 250 patients in Manchester, Bucharest and Amsterdam, respectively, were evaluated. The following parameters were assessed: the state of the distal surface in the second mandibular molar, loss of periodontal support, impaction type of the third molar, contact point localization, and patients' genders, ages and their cumulative history of dental health. Results: The rate of DSC in the second mandibular molar was 63.9%, 19.9% and 26.0% in the Manchester, Bucharest and Amsterdam populations, respectively. A loss of lamina dura of ≥2 mm, increased percentages of decayed, missing or filled teeth and male gender were risk factors in all three populations. All assessed parameters apart from the site of the mandible reached statistical significance in the Manchester sample (p < 0.001). The DSC rate was cumulative with increasing age in the Manchester population, in which third molars were strategically retained. Conclusions: The UK population, treated according to strict guidelines that limit the removal of third molars, had a statistically significant higher DSC prevalence rate (p < 0.001) than the Romanian or Dutch populations. The active surgical management of mandibular third molars seems to have the potential to reduce the DSC rate in the adjacent second molar.

Historical aspects about third molar removal versus retention and distal surface caries in the second mandibular molar adjacent to impacted third molars.

This paper provides an insight into the historical recommendations regarding removal of mandibular third molars, as set out by the Royal College of Surgeons of England and the National Institutes of Health in the USA, as well as regional guidance from the National Institute for Health and Care Excellence and the controversy that surrounds surgical removal of third molars. The influences of third molar management as it developed in the UK, the historical economic evaluations, and the available evidence base on third-molar removal versus retention are described. This article seeks to address the growing concerns regarding the increasing frequency of distal surface caries (DSC) in mandibular second molar teeth when the decay is associated with asymptomatic, partially erupted, mandibular third molars, especially when they are mesially or horizontally impacted. Lastly, we illustrate radiographs of patients affected by DSC and how guidance that has been issued by a guideline institution regarding third molar surgery, even though it is based on insufficient evidence, is perceived as a strictly compulsory clinical strategy, and has been used in clinical practice in the UK for more than 20 years.

Silibinin Attenuates Experimental Periodontitis by Downregulation of Inflammation and Oxidative Stress.

Periodontitis is an oral microbiota-induced inflammatory disease, in which inflammation and oxidative stress play a critical role. Silibinin (SB), a Silybum marianum-derived compound, exhibits strong anti-inflammatory and antioxidative properties. We adopted a rat ligature-induced periodontitis model and a lipopolysaccharide- (LPS-) stimulated human periodontal ligament cells (hPDLCs) model to evaluate the protective effects of SB. In the in vivo model, SB reduced alveolar bone loss and apoptosis of PDLCs in the periodontal tissue. SB also maintained the expression of nuclear factor-E2-related factor 2 (Nrf2), a key regulator of cellular resistance to oxidative stress, and attenuated lipid, protein, and DNA oxidative damages in the periodontal lesion area. Meanwhile, in the in vitro model, SB administration reduced the production of intracellular reactive oxidative species (ROS). Furthermore, SB exerted a strong anti-inflammatory property in both in vivo and in vitro models by inhibiting the expression of inflammatory mediators including nuclear factor-κB (NF-κB) as well as nucleotide binding oligomerization domain- (NOD-) like receptor family pyrin domain-containing 3 (NLRP3) and downregulating the levels of proinflammatory cytokines. This study, for the first time, demonstrates that SB exhibits the anti-inflammatory and antioxidative properties against periodontitis by downregulating the expression of NF-κB and NLRP3 and upregulating Nrf2 expression, suggesting a promising potential clinical application of SB in periodontitis.

Diabetes Medication Metformin Inhibits Osteoclast Formation and Activity in In Vitro Models for Periodontitis.

Diabetes and periodontitis are comorbidities and may share common pathways. Several reports indicate that diabetes medication metformin may be beneficial for the periodontal status of periodontitis patients. Further research using appropriate cell systems of the periodontium, the tissue that surrounds teeth may reveal the possible mechanism. Periodontal ligament fibroblasts anchor teeth in bone and play a role in the onset of both alveolar bone formation and degradation, the latter by inducing osteoclast formation from adherent precursor cells. Therefore, a cell model including this type of cells is ideal to study the influence of metformin on both processes. We hypothesize that metformin will enhance bone formation, as described for osteoblasts, whereas the effects of metformin on osteoclast formation is yet undetermined. Periodontal ligament fibroblasts were cultured in the presence of osteogenic medium and 0.2 or 1 mM metformin. The influence of metformin on osteoclast formation was first studied in PDLF cultures supplemented with peripheral blood leukocytes, containing osteoclast precursors. Finally, the effect of metformin on osteoclast precursors was studied in cultures of CD14+ monocytes that were stimulated with M-CSF and receptor activator of Nf-κB ligand (RANKL). No effects of metformin were observed on osteogenesis: not on alkaline phosphatase activity, Alizarin red deposition, nor on the expression of osteogenic markers RUNX-2, Collagen I and Osteonectin. Metformin inhibited osteoclast formation and accordingly downregulated the genes involved in osteoclastogenesis: RANKL, macrophage colony stimulating factor (M-CSF) and osteoclast fusion gene DC-STAMP. Osteoclast formation on both plastic and bone as well as bone resorption was inhibited by metformin in M-CSF and RANKL stimulated monocyte cultures, probably by reduction of RANK expression. The present study unraveling the positive effect of metformin in periodontitis patients at the cellular level, indicates that metformin inhibits osteoclast formation and activity, both when orchestrated by periodontal ligament fibroblasts and in cytokine driven osteoclast formation assays. The results indicate that metformin could have a systemic beneficiary effect on bone by inhibiting osteoclast formation and activity.

Intraoperative visualisation and treatment of salivary glands in Sjögren's syndrome by contrast-enhanced ultrasound sialendoscopy (CEUSS): protocol for a phase I single-centre, single-arm, exploratory study.

INTRODUCTION: We established a promising sialendoscopic treatment for in vivo enhancement of salivation in salivary glands affected by Sjögren's syndrome (SS). In this technique, the ducts of the salivary glands are irrigated with saline and steroids. This allows for dilatation of ductal strictures and removal of debris. Unfortunately, it is not possible to assess the delivery and penetration of saline or medications in the ductal system and parenchyma. To address this problem, we will conduct contrast-enhanced ultrasound sialendoscopy (CEUSS) using sulphur hexafluoride microbubbles. To the best of our knowledge, microbubbles have never been used for the treatment of salivary glands in SS. It is, therefore, imperative to test this application for its safety and feasibility. METHODS AND ANALYSIS: A single-arm phase I study will be performed in 10 SS patients. Under local anaesthesia, ultrasound (US) guided infusion of the parotid and submandibular glands with microbubbles will be performed. Continuous US imaging will be used to visualise the glands, including the location of strictures and occlusions. Main outcomes will be the evaluation of safety and technical feasibility of the experimental treatment. Secondary outcomes will consist of determinations of unstimulated whole mouth saliva flow, stimulated whole mouth saliva flow, stimulated parotid saliva flow, clinical oral dryness, reported pain, xerostomia, disease activity, salivary cytokine profiles and clinical SS symptoms. Finally, salivary gland topographical alterations will be evaluated by US. ETHICS AND DISSEMINATION: Ethical approval for this study was obtained from the Medical Ethics Committee of the Amsterdam University Medical Centre, Amsterdam, The Netherlands (NL68283.029.19). data will be presented at national and international conferences and published in a peer-reviewed journal. The study will be implemented and reported in line with the Standard Protocol Items: Recommendations for Interventional Trials' statement. TRIAL REGISTRATION NUMBERS: The Netherlands Trial Register: NL7731, MREC Trial Register: NL68283.029.19; Pre-results.

Chronic Exposure of Gingival Fibroblasts to TLR2 or TLR4 Agonist Inhibits Osteoclastogenesis but Does Not Affect Osteogenesis.

Chronic exposure to periodontopathogenic bacteria such as Porphyromonas gingivalis and the products of these bacteria that interact with the cells of the tooth surrounding tissues can ultimately result in periodontitis. This is a disease that is characterized by inflammation-related alveolar bone degradation by the bone-resorbing cells, the osteoclasts. Interactions of bacterial products with Toll-like receptors (TLRs), in particular TLR2 and TLR4, play a significant role in this chronic inflammatory reaction, which possibly affects osteoclastic activity and osteogenic capacity. Little is known about how chronic exposure to specific TLR activators affects these two antagonistic activities. Here, we studied the effect of TLR activation on gingival fibroblasts (GF), cells that are anatomically close to infiltrating bacterial products in the mouth. These were co-cultured with naive osteoclast precursor cells (i.e., monocytes), as part of the peripheral blood mononuclear cells (PBMCs). Activation of GF co-cultures (GF + PBMCs) with TLR2 or TLR4 agonists resulted in a weak reduction of the osteoclastogenic potential of these cultures, predominantly due to TLR2. Interestingly, chronic exposure, especially to TLR2 agonist, resulted in increased release of TNF-α at early time points. This effect, was reversed at later time points, thus suggesting an adaptation to chronic exposure. Monocyte cultures primed with M-CSF + RANKL, led to the formation of bone-resorbing osteoclasts, irrespective of being activated with TLR agonists. Late activation of these co-cultures with TLR2 and with TLR4 agonists led to a slight decrease in bone resorption. Activation of GF with TLR2 and TLR4 agonists did not affect the osteogenic capacity of the GF cells. In conclusion, chronic exposure leads to diverse reactions; inhibitory with naive osteoclast precursors, not effecting already formed (pre-)osteoclasts. We suggest that early encounter of naive monocytes with TLR agonists may result in differentiation toward the macrophage lineage, desirable for clearing bacterial products. Once (pre-)osteoclasts are formed, these cells may be relatively insensitive for direct TLR stimulation. Possibly, TLR activation of periodontal cells indirectly stimulates osteoclasts, by secreting osteoclastogenesis stimulating inflammatory cytokines.

Periodontal ligament fibroblasts as a cell model to study osteogenesis and osteoclastogenesis in fibrodysplasia ossificans progressiva.

Fibrodysplasia Ossificans Progressiva (FOP) is a progressive disease characterized by periods of heterotopic ossification of soft connective tissues, including ligaments. Though progress has been made in recent years in unraveling the underlying mechanism, patient-derived cell models are necessary to test potential treatment options. Periodontal ligament fibroblasts (PLF) from extracted teeth can be used to study deviant bone modeling processes in vitro since these cells are derived from genuine ligaments. They further provide a tool to study the hitherto unknown role of the bone morphogenesis protein receptor type 1 (BMPR-1) Activin A type 1 receptor ACVR1-R206H mutation in osteoclastogenesis. To further validate this potential model, osteogenesis and osteoclastogenesis was studied in the presence of TGF-ß/activin receptor inhibitor GW788388. Control and FOP fibroblasts (n=6 of each) were used in osteogenesis and osteoclastogenesis assays in the absence or presence of TGF-ß/activin receptor inhibitor GW788388. For osteogenesis, alkaline phosphatase (ALP) activity, alizarin red staining for mineralization and qPCR for expression of osteogenic markers was assessed. TRACP staining, multinuclearity and expression of osteoclastogenesis markers were used as a measure of osteoclast formation. FOP fibroblasts cultured in osteogenic medium displayed a trend of higher ALP activity at 7days. Gene expression of ALP from FOP fibroblasts was significantly higher at 3days. Mineralization was similar at 21days for both groups. GW788388 did not influence mineral deposition in both groups. Osteoclast formation was inhibited by GW788388 on plastic for both controls and FOP. On cortical bone slices, however, osteoclast formation was significantly lowered by GW788388, only in FOP cultures. qPCR revealed strong expression of RANKL at 7days and a significant decline at 14 and 21days in both FOP and control cultures. In contrast to the osteoclastogenesis results, the RANKL/OPG ratio was higher in the presence of GW788388, only in FOP cultures. TGF-ß expression was significantly higher at 14 and 21days compared to 7days, possibly signifying a role in later stages of osteoclast formation. Addition of GW788388 strongly decreased TGF-ß expression. Our study shows that periodontal ligament fibroblasts from FOP patients displayed at most slightly enhanced in vitro osteogenesis and osteoclastogenesis. This model could be useful to elucidate molecular mechanisms leading to heterotopic ossification in FOP such as in the presence of specific ACVR1-R206H activators as Activin A.