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Immune cell pattern-recognition receptors such as Toll-like receptors (TLRs) play important roles in the regulation of host responses to periodontal pathogens. Our previous studies have demonstrated that immune regulatory B cells were activated by TLRs and alleviated periodontitis inflammation and bone loss. The purpose of this study is to determine the role of TLR9 signaling in the activation and IL-10 production of the primed-immune B cells in vitro. Wild-type (WT) and TLR9 knockout (TLR9KO) mice (C57BL/6 background, n = 5) were pre-immunized intraperitoneally with 1 × 108 formalin-fixed P. gingivalis and boosted once with 1 × 107 formalin-fixed P. gingivalis. Isolated splenocytes and purified B cells from each mouse were cultured with 1 × 108 formalin-fixed P. gingivalis for 48 h. Immunocytochemistry was performed to detect CD45+ IL-10+ cells. Levels of IL-10 expression and secretion in splenocytes and B cells were detected using qRT-PCR and ELISA, respectively. After stimulation with fixed P. gingivalis, the percentage of CD45+ IL-10+ B cells and the level of IL-10 expression were significantly increased (p < 0.01) in splenocytes and purified B cells isolated from WT mice. However, these changes were not observed in splenocytes and purified B cells from TLR9KO mice when the cells were treated with fixed P. gingivalis. The percentage of CD45+ IL-10+ B cells was significantly reduced in splenocytes and purified B cells from TLR9KO mice compared to those from WT mice when challenged with P. gingivalis. IL-10 expression in B cells from TLR9KO mice was significantly decreased compared to those from WT mice at both the mRNA and protein levels. Additionally, P. gingivalis-induced up-regulation of TNF-α mRNA expressions were consistently observed in B cells from both WT and TLR9KO mice. P. gingivalis-induced B10 activation and IL-10 production during adaptive responses by primed B cells requires TLR9 signaling and can be achieved independent of T-cell help.

This study probed in vitro the mechanisms of competition/coexistence between Streptococcussanguinis (known for being correlated with health in the oral cavity) and Streptococcusmutans (responsible for aciduric oral environment and formation of caries) by means of quantitative Raman spectroscopy and imaging. In situ Raman assessments of live bacterial culture/coculture focusing on biofilm exopolysaccharides supported the hypothesis that both species engaged in antagonistic interactions. Experiments of simultaneous colonization always resulted in coexistence, but they also revealed fundamental alterations of the biofilm with respect to their water-insoluble glucan structure. Raman spectra (collected at fixed time but different bacterial ratios) showed clear changes in chemical bonds in glucans, which pointed to an action by Streptococcus sanguinis to discontinue the impermeability of the biofilm constructed by Streptococcus mutans. The concurrent effects of glycosidic bond cleavage in water-insoluble α − 1,3–glucan and oxidation at various sites in glucans’ molecular chains supported the hypothesis that secretion of oxygen radicals was the main “chemical weapon” used by Streptococcus sanguinis in coculture.

SARS-CoV-2, the causative agent of the debilitating COVID-19, is mainly transmitted by first infecting nose and lung epithelial cells. The mouth is also believed to be a viral portal site since certain types of oral epithelial cells were shown to express ACE2 receptor. However, it is unclear whether oral epithelial cells are directly infected by SARS-CoV-2. In this study, we addressed whether epithelial cells of the oral gingiva were susceptible to infection. Interestingly, we found that KRT5+ and KRT18+ gingival epithelial cells do not express ACE2 but highly express TMPRSS2 and Furin as well as CD147, which was proposed to be an alternative receptor for SARS-CoV-2. However, using SARS-CoV-2 pseudoviruses containing the spike protein, we observed that gingival epithelial cells were not susceptible to infection due to the lack of ACE2 expression and the inability of CD147 to mediate viral entry. These results strongly suggest that epithelial cells from the gingiva are not susceptible to SARS-CoV-2 and CD147 is not a receptor for the SARS-CoV-2 virus. The susceptibility of oral cells from other oral structures under healthy and pathological conditions still needs to be confirmed to better understand the role of the oral cavity in COVID-19 infection and transmission.

Cleft alveolus, a common birth defect of the maxillary bone, affects one in 700 live births every year. This defect is traditionally restored by autogenous bone grafts or allografts, which may possibly cause complications. Cell-based therapies using the mesenchymal stem cells (MSCs) derived from human gingiva (gingiva-derived mesenchymal stem cells [GMSCs]) is attracting the research interest due to their highly proliferative and multilineage differentiation capacity. Undifferentiated GMSCs expressed high level of MSC-distinctive surface antigens, including CD73, CD105, CD90, and CD166. Importantly, GMSCs induced with osteogenic medium for a week increased the surface markers of osteogenic phenotypes, such as CD10, CD92, and CD140b, indicating their osteogenic potential. The objective of this study was to assess the bone regenerative efficacy of predifferentiated GMSCs (dGMSCs) toward an osteogenic lineage in combination with a self-assembling hydrogel scaffold PuraMatrix™ (PM) and/or bone morphogenetic protein 2 (BMP2), on a rodent model of maxillary alveolar bone defect. A critical size maxillary alveolar defect of 7 mm × 1 mm × 1 mm was surgically created in athymic nude rats. The defect was filled with either PM/BMP2 or PM/dGMSCs or the combination of three (PM/dGMSCs/BMP2) and the bone regeneration was evaluated at 4 and 8 weeks postsurgery. New bone formation was evaluated by microcomputed tomography and histology using Hematoxylin and Eosin staining. The results demonstrated the absence of spontaneous bone healing, either at 4 or 8 weeks postsurgery in the defect group. However, the PM/dGMSCs/BMP2 group showed significant enhancement in bone regeneration at 4 and 8 weeks postsurgery, compared with the transplantation of individual material/cells alone. Apart from developing the smallest critical size defect, results showed that PM/dGMSCs/BMP2 could serve as a promising option for the regeneration of bone in the cranio/maxillofacial region in humans. IMPACT STATEMENT: Gingiva-derived mesenchymal stem cells (GMSCs) are the most appealing cell source as they are readily accessible and capable of multilineage differentiation. They are most suitable for bone regeneration in craniofacial defects, due to their origin from neural crest progenitor cells. In this study, we have demonstrated that combination of predifferentiated osteogenic GMSCs (dGMSCs), self-assembling hydrogel, and low doses of BMP2 accelerated bone regeneration of alveolar bone defect in rat model suggesting that dGMSCs may lead to a novel cell therapy for enhanced bone regeneration in alveolar cleft and other bone defect complications in the craniofacial area.

Aging is associated with increased prevalence and severity of pathogenic outcomes of periodontal disease, including soft tissue degeneration and bone loss around the teeth. Although lipopolysaccharide (LPS) derived from the key periodontal pathogen Porphyromonas gingivalis (Pg) plays an important role in the promotion of inflammation and osteoclastogenesis via toll-like receptor (TLR)4 signaling, its pathophysiological role in age-associated periodontitis remains unclear. This study investigated the possible effects of Pg-LPS on RANKL-primed osteoclastogenesis and ligature-induced periodontitis in relation to aging using young (2 months old) and aged (24 months old) mice. To the best of our knowledge, our results indicated that expression of TLR4 was significantly diminished on the surface of osteoclast precursors isolated from aged mice compared with that of young mice. Furthermore, our data demonstrated that the TLR4 antagonist (TAK242) dramatically decreased the numbers of tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts differentiated from RANKL-primed young osteoclast precursors (OCPs) compared with those isolated from aged mice in response to Pg-LPS. In addition, using a ligature-induced periodontitis mouse model, we demonstrated that Pg-LPS elevated (1) secretion of senescence-associated secretory phenotype (SASP) markers, including the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, as well as osteoclastogenic RANKL, and (2) the number of OCPs and TRAP+ osteoclasts in the periodontal lesion induced in young mice. In contrast, Pg-LPS had little, or no, effect on the promotion of periodontitis inflammation induced in aged mice. Altogether, these results indicated that periodontal disease in older mice occurs in a manner independent of canonical signaling elicited by the Pg-LPS/TLR4 axis.

Background Periodontal disease results from the pathogenic interactions between the tissue, immune system, and microbiota; however, standard therapy fails to address the cellular mechanism underlying the chronic inflammation. Dendritic cells (DC) are key regulators of T cell fate, and biomaterials that recruit and program DC locally can direct T cell effector responses. We hypothesized that a biomaterial that recruited and programmed DC toward a tolerogenic phenotype could enrich regulatory T cells within periodontal tissue, with the eventual goal of attenuating T cell mediated pathology. Methods The interaction of previously identified factors that could induce tolerance, granulocyte-macrophage colony stimulating factor (GM-CSF) and thymic stromal lymphopoietin (TSLP), with the periodontitis network was confirmed in silico. The effect of the cytokines on DC migration was explored in vitro using time-lapse imaging. Finally, regulatory T cell enrichment in the dermis and periodontal tissue in response to alginate hydrogels delivering TSLP and GM-CSF was examinedin vivo in mice using immunohistochemistry and live-animal imaging. Results The GM-CSF and TSLP interactome connects to the periodontitis network. GM-CSF enhances DC migration in vitro. An intradermal injection of an alginate hydrogel releasing GM-CSF enhanced DC numbers and the addition of TSLP enriched FOXP3+ regulatory T cells locally. Injection of a hydrogel with GM-CSF and TSLP into the periodontal tissue in mice increased DC and FOXP3+ cell numbers in the tissue, FOXP3+ cells in the lymph node, and IL-10 in the tissue. Conclusion Local biomaterial-mediated delivery of GM-CSF and TSLP can enrich DC and FOXP3+ cells and holds promise for treating the pathologic inflammation of periodontal disease.

Periodontitis is a bacterially-induced inflammatory disease that leads to tooth loss. It results from the damaging effects of a dysregulated immune response, mediated largely by neutrophils, macrophages, T cells and B cells, on the tooth-supporting tissues including the alveolar bone. Specifically, infiltrating B cells at inflamed gingival sites with an ability to secrete RANKL and inflammatory cytokines are thought to play roles in alveolar bone resorption. However, the direct contribution of B cells in alveolar bone resorption has not been fully appreciated. In this study we sought to define the contribution of RANKL expressing B cells in periodontitis by employing a mouse model of pathogen-induced periodontitis that used conditional knockout mice with B cell-targeted RANKL deletion. Briefly, alveolar bone loss was assessed in the wild-type, B-cell deficient (Jh), or B-cell-RANKL deleted (RANKLΔB) mice orally infected with the periodontal pathogen Tannerella forsythia. The RANKLΔB mice were obtained by crossing Cd19-Cre knock-in mice with mice homozygous for conditional RANKL-flox allele (RANKLflox/flox). The alveolar bone resorption was determined by morphometric analysis and osteoclastic activity of the jaw bone. In addition, the bone resorptive potential of the activated effector B cells was assessed ex vivo. The data showed that the RANKL producing B cells increased significantly in the T. forsythia-infected wild-type mice compared to the sham-infected mice. Moreover, T. forsythia-infection induced higher alveolar bone loss in the wild-type and RANKLflox/flox mice compared to infection either in the B cell deficient (Jh) or the B-cell specific RANKL deletion (RANKLΔB) mice. These data established that the oral-pathogen activated B cells contribute significantly to alveolar bone resorption via RANKL production.

Progressive generation of total joint implant‐derived wear particles is one of the major risk factors in development of peri‐prosthetic osteolysis especially in the aging society. It is commonly accepted that macrophages predominantly drive the inflammatory response to wear debris particles. Among various surface receptors that activate the macrophages to phagocytize particles, it is believed that the Toll‐like receptor 4 (TLR4) and the scavenger macrophage receptor with collagenous structure (MARCO) play key roles in recognition of wear debris particles. However, a strong body of evidence indicates an age‐dependent diminished function of human TLRs. Thus, we hypothesized that the MARCO receptor may be more engaged than TLRs in the phagocytosis of wear debris particles which in turn up‐regulate production of pro‐inflammatory cytokines from aged macrophages. We demonstrated that peritoneal macrophages isolated from aged mice show elevated expression of MARCO receptor compared to that from young mice. In contrast the expression of TLR4 was significantly decreased on the surface of aged macrophages. Furthermore, using anti‐MARCO and anti‐TLR4 neutralizing mAbs, we demonstrated the age‐dependent pathogenic role of MARCO, but not TLR4, receptor in promoting poly‐methyl methacrylate (PMMA) bone cement particles phagocytosis by macrophages leading to the release of pro‐inflammatory cytokines migration inhibitory factor and tumour necrosis factor in vitro. These data also suggest that the approach to neutralize MARCO may lead to the development of therapeutic regimen for the prevention of particle‐induced osteolysis in aged patients.

This study targets on-site/real-time taxonomic identification and metabolic profiling of seven different Candida auris clades/subclades by means of Raman spectroscopy and imaging. Representative Raman spectra from different Candida auris samples were systematically deconvoluted by means of a customized machine-learning algorithm linked to a Raman database in order to decode structural differences at the molecular scale. Raman analyses of metabolites revealed clear differences in cell walls and membrane structure among clades/subclades. Such differences are key in maintaining the integrity and physical strength of the cell walls in the dynamic response to external stress and drugs. It was found that Candida cells use the glucan structure of the extracellular matrix, the degree of α-chitin crystallinity, and the concentration of hydrogen bonds between its antiparallel chains to tailor cell walls’ flexibility. Besides being an effective ploy in survivorship by providing stiff shields in the α–1,3–glucan polymorph, the α–1,3–glycosidic linkages are also water-insoluble, thus forming a rigid and hydrophobic scaffold surrounded by a matrix of pliable and hydrated β–glucans. Raman analysis revealed a variety of strategies by different clades to balance stiffness, hydrophobicity, and impermeability in their cell walls. The selected strategies lead to differences in resistance toward specific environmental stresses of cationic/osmotic, oxidative, and nitrosative origins. A statistical validation based on principal component analysis was found only partially capable of distinguishing among Raman spectra of clades and subclades. Raman barcoding based on an algorithm converting spectrally deconvoluted Raman sub-bands into barcodes allowed for circumventing any speciation deficiency. Empowered by barcoding bioinformatics, Raman analyses, which are fast and require no sample preparation, allow on-site speciation and real-time selection of appropriate treatments.

Diabetes mellitus (DM) may affect the physical and mechanical properties of dentin, which could potentially have an impact on root canal procedures. This study aimed to compare the amount of dentin removed by an endodontic rotary file, comparing dentin from diabetic patients with dentin from control patients under laboratory conditions.The amount of dentin removed was tested using new F3 ProTaper (Dentsply Maillefer, Ballaigues, Switzerland) files applied against the surface of prepared dentin discs for 3 different groups: diabetic type 1 (D1), diabetic type 2 (D2), and nondiabetic (normal). The dentin removed was determined by measuring the depth of penetration of the file using a digital caliper and by measuring the weight loss. Data were analyzed using Kolmogorov-Smirnov, analysis of variance, post hoc Tukey, and Pearson correlation tests (P.05).Significantly more dentin was removed, and the penetration of the F3 instrument was significantly higher (P.05) in DM specimens. The statistical analysis revealed significant differences between the D1, D2, and normal groups (P.05) for the weight loss of the specimen as well as the penetration depth at point B (P.05). Both the weight loss and depth of penetration showed a very high positive correlation (P.05).The dentin of patients suffering from both D1 and D2 exhibited an increased amount of dentin removed compared with the nondiabetic dentin specimens. This can be observed by the increased penetration of the rotary instruments into dentin. Under certain circumstances, this may impact instrumentation, increasing procedural accidents and leading to subsequent weakening of root canal-treated teeth in diabetic patients.

This study was performed to evaluate the effect of type 1 diabetes mellitus (T1DM) on the microhardness of tooth enamel and dentine in mice.Seventy male C57BL/6 J mice were used in this study. Thirty-five mice were rendered diabetic by administration of streptozotocin (STZ), and the remaining animals received citrate buffer (normal/non-diabetic). In each group, specimens were divided into 7 subgroups of 5 mice based on the time points 0, 1, 4, 8, 12, 20, and 28 weeks. The microhardness value (MHV) of the second molars' enamel and root dentine were tested with a Vickers microhardness tester. Five specimens from each subgroup were evaluated for dentinal tubular density by scanning electron microscope (SEM) and color dot map analysis to determine the color intensity of strontium (Sr) and magnesium (Mg) by using ImageJ software.The MHV of enamel was significantly reduced in STZ specimens in time points of 12 weeks (STZ: 274.39 ± 15.42, normal: 291.22 ± 15.28), 20 weeks (STZ: 247.28 ± 19.65, normal: 290.68 ± 11.52), and 28 weeks (STZ: 232.87 ± 15.07, normal: 282.76 ± 10.36) (P 0.05). When comparing the MHV of dentine in subgroups of the normal group, after 20 weeks (169.1 ± 7.5) and 28 weeks (168.6 ± 7.81), the MHV increased significantly (P 0.05). However, in the STZ group, a significant reduction of MHV was noticed between 28 weeks (131.69 ± 6.2) specimens with other subgroups (P 0.05).T1DM negatively affected enamel and dentine microhardness, and enamel was influenced much more negatively and rapidly compared with dentine in diabetic groups.

Made available in DSpace on 2020-12-10T20:01:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-05 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR, USA) Maranhao State Research Foundation (FAPEMA, Brazil) Background Desipramine is a tricyclic antidepressant with immune-modulatory activity, whose effects on ligature-induced periodontitis are yet to be investigated. Hence, its actions on alveolar bone resorption, gingival collagen content and key inflammatory mediators were herewith analyzed. Methods A total of 60 male Wistar rats were randomly assigned into three groups: 1) control: rats without ligature treated with vehicle (saline); 2) ligature: rats with ligature-induced periodontitis treated with vehicle; 3) ligature + desipramine: rats with ligature-induced periodontitis treated with desipramine (20 mg/kg/d in vehicle). Mandibles and gingival tissues were collected 3 or 15 days after ligature insertion (or no ligature insertion for controls) and treatments. Alveolar bone resorption and gingival collagen fibers were histologically analyzed using either HE or picrosirius red staining. Gingival mRNA expressions of interleukin (IL)-1 beta, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 were obtained through reverse transcription polymerase chain reaction. MMP-9 activity was analyzed by zymography. Results Alveolar bone loss was significantly reduced in the ligature + desipramine group (P < 0.05), whereas gingival collagen degradation was like the ligature group (P > 0.05). Desipramine administration downregulated mRNA expressions of IL-1 beta, iNOS, COX-2, and TIMP-1 when compared to vehicle alone in the ligature group (P < 0.05). MMP-9 expression and MMP-9/TIMP-1 ratio were similar among rats with ligature-induced periodontitis (P > 0.05); however, MMP-9 activity was lower in the group treated with desipramine (P < 0.05). Conclusion Desipramine administration reduced alveolar bone loss as histologically observed, and modulated key bone remodeling and inflammatory mediators in rats with ligature-induced periodontitis. Univ Fed Maranhao, Post Grad Program Dent, Sao Luis, Maranhao, Brazil Univ Estadual Ponta Grossa, Dept Gen Biol, Ponta Grossa, Parana, Brazil Fac Sci Tocantins, Araguaina, Tocantins, Brazil Univ Ceuma, Post Grad Program Dent, Sao Luis, Maranhao, Brazil Univ Fed Maranhao, Sch Med, Sao Luis, Maranhao, Brazil Univ Taubate, Nucleus Periodontal Res, Taubate, SP, Brazil Sao Paulo State Univ, Dept Biosci & Oral Diag, Sao Jose Dos Campos, SP, Brazil Nova Southeastern Univ, Coll Dent Med, Ft Lauderdale, FL 33314 USA Univ Fed Alfenas, Biol Sci Grad Program, Rua Gabriel Monteiroda Silva 700,Predio E Sala, BR-37130001 Alfenas, MG, Brazil Sao Paulo State Univ, Dept Biosci & Oral Diag, Sao Jose Dos Campos, SP, Brazil FAPESP: 2008/00566-6 CAPES: 4073/08-8 National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR, USA): R15 DE027851 Maranhao State Research Foundation (FAPEMA, Brazil): 1818/2012

To evaluate the effect(s) of mineral trioxide aggregate (MTA) on in vitro RANKL-mediated osteoclast-dependent bone resorption events and the influence of CaTwo types of osteoclast precursors, RAW 264.7 (RAW) cell line or bone marrow cells (obtained from BALB/c mice and stimulated with recombinant (r) macrophage colony stimulation factor (M-CSF), were stimulated with or without recombinant (r) activator of nuclear kappa B ligand (RANKL), in the presence or absence of MTA for 6 to 8 days. White Angelus MTA and Bios MTA (Angelus, Londrina, Paraná, Brazil) were prepared and inserted into capillary tubes (direct contact surface = 0.50 mmIn bone marrow and RAW cells, the number of TRAP-positive mature osteoclast cells induced by rRANKL was significantly inhibited by the presence of MTA compared with control rRANKL stimulation without MTA (p 0.05), along with the reduction of TRAP enzyme activity (p 0.05) and the low expression of cathepsin K gene (p 0.05). In contrast, to control mature osteoclasts, the resorption area on dentin was significantly decreased for mature osteoclasts incubated with MTA (p 0.05). rRANKL-stimulated RAW cells treated with CaMTA significantly suppressed RANKL-mediated osteoclastogenesis and osteoclast activity and, therefore, appears able to suppress bone resorption events in periapical lesions. This process might be related to CaMTA is an important worldwidely acknowleged biomaterial. The knowledge about its molecular activities on osteoclasts might contribute to improving the understanding of its clinical efficacy.

Background: Porphyromonas gingivalis (Pg) infection causes periodontal disease and is involved in the exacerbation of rheumatoid arthritis (RA). Gut microbiota dysbiosis has shown strong associations with systemic diseases, including RA, diabetes mellitus, and inflammatory bowel disease, and inoculation of periodontopathogenic bacteria (i.e. Pg) can alter gut microbiota composition. Therefore, this study investigated dysbiosis-mediated arthritis exacerbation by Pg oral inoculation in an experimental arthritis model mouse.Methods: Pg inoculation in the oral cavity twice a week for 6 weeks was performed to induce periodontitis in SKG mice. Concomitantly, a single intraperitoneal (i.p.) injection of laminarin (LA) was administered to induce experimental arthritis (Pg-LA mouse). Citrullinated protein (CP) and IL-6 levels in periodontal, intestinal, and joint tissues, and serum were measured by ELISA. Gut microbiota composition was determined by sequencing the bacterial 16S rRNA after DNA purification of mouse feces. Fecal microbiota transplantation (FMT) was performed by transferring Pg-RA-derived feces to normal SKG mice. The effect of the Pg peptidylarginine deaminase (PgPAD) on the level of citrullinated proteins and arthritis progression were determined using PgPAD knockout mutant.Results: Periodontal alveolar bone loss and IL-6 in gingival tissue were induced by Pg oral infection, as well as severe joint destruction, increased arthritis scores (AS), and IL-6 and CP production in serum, joint, and intestinal tissues. Distribution of Deferribacteres and S24-7 was decreased, while CP was significantly increased in gingival, joint, and intestinal tissues of Pg-inoculated experimental arthritis mice compared to experimental arthritis mice without Pg inoculation. Further, FMT from Pg-inoculated experimental arthritis mice showed the reproduction of donor gut microbiota and resulted in severe joint destruction with increased IL-6 and CP production in joint and intestinal tissues. The avarage AS of FMT from Pg-inoculated experimental arthritis was much higher than that of donor mouse. However, inoculation of the PgPAD knockout mutant inhibited the elevation of arthritis scores, ACPA level in serum and reduced CP amount in gingival, joint, and intestinal tissues compared to Pg wild type inoculation .Conclusion: Pg oral infection affected gut microbiota dysbiosis and joint destruction via increased CP generation.

To evaluate the selenium (Se) behavior when used as an endodontic dressing in teeth with pulp necrosis. Additionally, its effects was also compared with the calcium hydroxide (C.H.), which is used globally as a root canal dressing, and the combination of the C.H. with Se (C.H. + Se). The sample consisted of 60 patients requiring endodontic treatment who were divided into groups, i.e., without intracanal medication (empty) and with medications as follows: selenium (Se), calcium hydroxide (C.H.), and calcium hydroxide + selenium (C.H. + Se) (n = 15). After the coronary opening, three absorbent paper points were placed in the RCS and maintained for 2 min for microbial evaluation. Following the cleaning and shaping procedures, new paper points were introduced into the root canal system, passing passively through the root apex (2 mm) into the periapical tissues for 2 min, for immune evaluation. The collections were performed again 15 days later. Real-time PCR quantified the expression of the prokaryotic 16S ribosomal RNA. The 16S mRNA was evaluated before the cleaning and shaping procedures and 15 days later in the groups treated with or without medication. A significant reduction in the microbial load was observed only in the groups that received endodontic dressing (p

Objectives Interrelations between bone and the immune system are involved in both homeostatic and pathogenic bone remodeling processes. Although emerging evidence supports that mechanosensitive Ca2+-channel Piezo1 plays a pivotal role in osteoblast-mediated bone regeneration, possible engagement of Piezo1 in the differentiation of osteoclasts from monocytes and resulting bone resorption is largely unknown. In the present study, we sought to establish the role of Piezo1 in RANKL-stimulated monocytes for their differentiation to osteoclasts in vitro. Methods Mouse RAW 264.7 monocyte cell line or bone marrow derived mononuclear cells (BMMCs) were stimulated with M-CSF and/or RANKL, in the presence or absence of Yoda1 (Piezo1 activator) or with or without hydrostatic pressure (HP). Ca2+ influx assay was employed to confirm Piezo1 channel activity. To evaluate the level of osteoclastogenesis and bone-resorptive function by mature osteoclasts, TRAP staining and pit formation assays were performed. To monitor the expressions of mRNAs for Piezo1 as well as OC-STAMP, TRAP (ACP5) and MMP9, qPCR were employed. Results Yoda1-mediated Ca2+ influx was significantly elevated in RANKL BMMCs and RAW264.7 cells, while significantly suppressing the expressions of osteoclastogenesis-related genes, OC-STAMP, MMP9 and ACP5. Moreover, the number and size of multi-nucleated TRAP-positive cells as well as formation of resorption pits were significantly down-regulated by the treatment with Yoda1 as well as HP. Conclusions The present study suggests that mechanosensitive Piezo1 channel expressed on monocytes may play a gatekeeper role in down-modulating over-activation of osteoclastogenesis and function to resorb bone. Supported by NIH NIDCR grants, DE-027851, DE-028715 and DE-331851

Objectives Anti-citrullinated-protein auto-antibodies (ACPA), especially that react to citrullinated vimentin (CV), are implicated in the pathogenesis of Rheumatoid arthritis (RA). P. gingivalis (Pg), a key pathogen of periodontitis, can citrullinate the host proteins by production of Pg-derived peptidylarginine deiminase (PPAD). This study evaluated the relationship among antibodies (Abs) that react cyclic citrullinated protein (CCP), CV and periodontal pathogens in the serum of RA patients or control subjects. Methods Blood serum of healthy subjects (n=10) and RA patients (n=26) were obtained from Precision for Medicine. Six distinctive peptide sequences of Vimentin (#1–#6) and corresponding CV (#1’–#6’) peptides were synthesized. ELISA was employed to detect IgG Ab titers for CCP, intact vimentin and CV peptides. Fixed periodontal pathogens, Pg 33277, F. nucleatum, P. intermedia, A. actinomycetemcomitans (Aa) Y4, S. mitis, S. gordonii, A. odontolyticus, and C. gingivalis were also used as IgG ELISA antigens. Results The levels of anti-CCP Ab were higher in RA patients with anti-Pg Ab than those without anti-Pg Ab. Since elevated anti-Pg Ab is hallmark of periodontitis, it is conceivable that RA patients with anti-Pg Ab would have periodontitis. However, there was no association between anti-CCP Ab and the Abs reactive to the other bacteria. Very interestingly, RA patients with anti-Pg Ab, but not those without anti-Pg Ab, showed significantly elevated Ab response to #6’ CV peptide compared to intact #6 vimentin peptide, among six peptide sequences tested. Conclusions Our findings indicated that periodontitis may be associated with the induction of ACPA via PPAD-mediated citrullination of host proteins. Supported by NIH NIDCR grants, DE-027851, DE-028715 and DE-331851

Effects of the antiosteoblastogenesis factor Semaphorin 4D (Sema4D), expressed by thrombin-activated platelets (TPs), on osteoblastogenesis, as well as osteoclastogenesis, were investigated in vitro. Intact platelets released both Sema4D and IGF-1. However, in response to stimulation with thrombin, platelets upregulated the release of Sema4D, but not IGF-1. Anti-Sema4D-neutralizing monoclonal antibody (mAb) upregulated TP-mediated osteoblastogenesis in MC3T3-E1 osteoblast precursors. MC3T3-E1 cells exposed to TPs induced phosphorylation of Akt and ERK further upregulated by the addition of anti-sema4D-mAb, suggesting the suppressive effects of TP-expressing Sema4D on osteoblastogenesis. On the other hand, TPs promoted RANKL-mediated osteoclastogenesis in the primary culture of bone-marrow-derived mononuclear cells (BMMCs). Among the known three receptors of Sema4D, including Plexin B1, Plexin B2 and CD72, little Plexin B2 was detected, and no Plexin B1 was detected, but a high level of CD72 mRNA was detected in RANKL-stimulated BMMCs by qPCR. Both anti-Sema4D-mAb and anti-CD72-mAb suppressed RANKL-induced osteoclast formation and bone resorptive activity, suggesting that Sema4D released by TPs promotes osteoclastogenesis via ligation to a CD72 receptor. This study demonstrated that Sema4D released by TPs suppresses osteogenic activity and promotes osteoclastogenesis, suggesting the novel property of platelets in bone-remodeling processes.

Ceramidases are a group of enzymes that degrade pro-inflammatory ceramide by cleaving a fatty acid to form anti-inflammatory sphingosine lipid. Thus far, acid, neutral and alkaline ceramidase isozymes have been described. However, the expression patterns of ceramidase isoforms as well as their role in periodontal disease pathogenesis remain unknown. In this study, expression patterns of ceramidase isoforms were quantified by real-time PCR and immunohistochemistry in gingival samples of patients with periodontitis and healthy subjects, as well as in EpiGingivalTM-3D culture and OBA-9 gingival epithelial cells both of which were stimulated with or without the presence of live Porphyromonas gingivalis (ATCC 33277 strain). A significantly lower level of acid ceramidase expression was detected in gingival tissues from periodontal patients compared to those from healthy subjects. In addition, acid-ceramidase expression in EpiGingival™ 3D culture and OBA-9 cells was suppressed by stimulation with P. gingivalis in vitro. No significant fluctuation was detected for neutral or alkaline ceramidases in either gingival samples or cell cultures. Next, to elucidate the role of acid ceramidase in P. gingivalis-induced inflammation in vitro, OBA-9 cells were transduced with adenoviral vector expressing the human acid ceramidase (Ad-ASAH1) gene or control adenoviral vector (Ad-control). In response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9 cells showed significantly lower mRNA expressions of caspase-3 as well as the percentage of Annexin V-positive cells, when compared with OBA-9 cells transduced with Ad-control vector. Furthermore, in response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9 cells produced less TNF-α, IL-6, and IL1β pro-inflammatory cytokines than observed in OBA-9 cells transduced with Ad-control vector. Collectively, our data show the novel discovery of anti-inflammatory and anti-apoptotic effects of acid ceramidase in host cells exposed to periodontal bacteria, and the attenuation of the expression of host-protective acid ceramidase in periodontal lesions.

Background and Objective Peri-implantitis and periodontitis are different entities in immune characteristics even though they share similar features in clinical and radiologic signs. Toll-like receptor 2 (TLR-2), one of the key pathogen-recognition receptors in the innate immune system, plays an important role in the progression of periodontitis. However, the role of TLR-2 in peri-implantitis remains unclear. The objective of this study was to investigate the role of TLR-2 in inflammation and alveolar bone loss in a murine model of ligature-induced peri-implantitis and to compare it with ligature-induced periodontitis. Material and Methods Smooth-surface titanium implants were placed in the alveolar bone of the left maxillary molars of wild-type (WT) and Tlr2 knockout (Tlr2-KO) mice 6 weeks after tooth extraction. Silk ligatures were applied to the left implant fixtures and the right maxillary second molars to induce peri-implantitis and periodontitis 4 weeks after implant placement. Two weeks after ligation, bone loss around the implants and maxillary second molars was analysed by micro-computed tomography (micro-CT), and inflammation around the implants and maxillary second molars was assessed at the same time point using histology and TRAP staining, respectively. Expression of mRNA for proinflammatory cytokines (interleukin-1β [Il1β], tumor necrosis factor-α [Tnfα]), an anti-inflammatory cytokine (interleukin-10 [Il10]) and osteoclastogenesis-related cytokines (Rankl, osteoprotegerin [Opg]) were evaluated, in gingival tissue, using real-time quantitative PCR (RT-qPCR). Results The success rate of implant osseointegration was significantly higher in Tlr2-KO mice (85.71%) compared with WT mice (53.66%) (P = .0125). Micro-CT revealed significantly decreased bone loss in Tlr2-KO mice compared with WT mice (P = .0094) in peri-implantitis. The levels of mRNA for Il1β (P = .0055), Tnfα (P = .01) and Il10 (P = .0019) in gingiva were significantly elevated in the peri-implantitis tissues of WT mice, but not in Tlr2-KO mice, compared with controls. However, the gingival mRNA ratios of Rankl/Opg in peri-implant tissues were significantly upregulated in both WT (P = .0488) and Tlr2-KO (P = .0314) mice. Ligature-induced periodontitis exhibited similar patterns of bone loss and inflammatory cytokine profile in both groups of mice, except that the level of Il10 was elevated (P = .0114) whereas the Rankl/Opg ratio was not elevated (P = .9755) in Tlr2-KO mice compared with control mice. Histological findings showed increased numbers of TRAP-positive cells and infiltrated inflammatory cells in ligature-induced peri-implantitis in both WT (P

Among several virulence factors produced by the periodontal pathogen Porphyromonas gingivalis (Pg), a recently identified novel class of dihydroceramide lipids that contains a long acyl-chain has the potential to play a pathogenic role in periodontitis because of its higher level of tissue penetration compared to other lipid classes produced by Pg. However, the possible impact of Pg ceramides on osteoclastogenesis is largely unknown. In the present study, we report that the phosphoglycerol dihydroceramide (PGDHC) isolated from Pg enhanced osteoclastogenesis in vitro and in vivo. Using RAW264.7 cells, in vitro assays indicated that PGDHC can promote RANKL-induced osteoclastogenesis by generating remarkably larger TRAP+ multinuclear osteoclasts compared to Pg LPS in a TLR2/4-independent manner. According to fluorescent confocal microscopy, co-localization of non-muscle myosin II-A (Myh9) and PGDHC was observed in the cytoplasm of osteoclasts, indicating the membrane-permeability of PGDHC. Loss- and gain-of-function assays using RNAi-based Myh9 gene silencing, as well as overexpression of the Myh9 gene, in RAW264.7 cells showed that interaction of PGDHC with Myh9 enhances RANKL-induced osteoclastogenesis. It was also demonstrated that PGDHC can upregulate the expression of dendritic cell-specific transmembrane protein (DC-STAMP), an important osteoclast fusogen, through signaling that involves Rac1, suggesting that interaction of PGDHC with Myh9 can elicit the cell signal that promotes osteoclast cell fusion. Taken together, our data indicated that PGDHC is a Pg-derived, cell-permeable ceramide that possesses a unique property of promoting osteoclastogenesis via interaction with Myh9 which, in turn, activates a Rac1/DC-STAMP pathway for upregulation of osteoclast cell fusion.

Background : Porphyromonas gingivalis (Pg) infection causes periodontal disease and is one of the causative bacteria of rheumatoid arthritis (RA) exacerbation. Gut microbiota dysbiosis has shown strong associations with systemic diseases, including RA, diabetes mellitus, and inflammatory bowel disease, and inoculation of periodontopathogenic bacteria (i.e. Pg) can alter gut microbiota composition. Therefore, this study investigated dysbiosis-mediated arthritis exacerbation by Pg oral inoculation in an experimental arthritis model mouse.Methods : Pg inoculation in the oral cavity twice a week for 6 weeks was performed to induce periodontitis in SKG mice. Concomitantly, a single intraperitoneal (i.p.) injection of laminarin (LA) was administered to induce experimental arthritis (Pg-LA mouse). Citrullinated protein (CP) and IL-6 levels in periodontal, intestinal, and joint tissues, and serum were measured by ELISA. Gut microbiota composition was determined by Miseq after DNA purification of mouse feces. Fecal microbiota transplantation (FMT) was performed by transferring Pg-RA-derived feces to normal mice. The effect of the Pg peptidylarginine deaminase (PgPAD) in arthritis progression was determined using PgPAD knockout mutant.Results : Periodontal alveolar bone loss and IL-6 in gingival tissue were induced by Pg oral infection, as well as severe joint destruction, increased arthritis scores (AS), and IL-6 and CP production in serum, joint, and intestinal tissues. Distribution of Deferribacteres and S24-7 was decreased, while CP was significantly increased in gingival, joint, and intestinal tissues of Pg-inoculated experimental arthritis mice compared to experimental arthritis mice without Pg inoculation. Further, FMT from Pg-inoculated experimental arthritis mice showed the reproduction of donor gut microbiota and resulted in severe joint destruction with increased IL-6 and CP production in joint and intestinal tissues. The maximum AS of FMT from Pg-inoculated experimental arthritis was much higher than that of donor mouse. However, inoculation of the PgPAD knockout mutant inhibited the elevation of arthritis scores, ACPA level in serum and reduced CP amount in gingival, joint, and intestinal tissues compared to Pg wild type inoculation .Conclusion : Pg oral infection exacerbated gut microbiota dysbiosis and joint destruction via increased CP generation.

Additional file 1: Figure S1. Assessment of periodontal tissue and evaluation of joint swelling in Pg-administered SKG mice. To determine the effect of Pg infection on periodontal tissue, Pg W83 (108 CFU/50 mL/mouse in 2% CMC/PBS solution) was administered into the oral cavity of SKG mice (6–8 weeks old, female) twice a week for 42 days. SKG mice also received LA i.p. injection. All mice were sacrificed, and gingival and leg joint tissues were collected. Morphological observation of the mouse upper jaw was analyzed (a). ABL of the upper jaw in each group was measured (b). Joint swelling was quantified using Sakaguchi’s AS every week after oral inoculation of Pg (1.0 × 108 CFU/mouse) and LA i.p. injection (c). Ctrl: PBS inoculation, LA: PBS inoculation + LA i.p. injection (10 μg/mouse), Pg: Pg inoculation, Pg/LA: Pg inoculation + LA i.p. injection. Data represent mean ± SD (b, c) of 6 mice per group. Data represent mean ± SD (b, c) of 6 mice per group. Statistical analyses were performed using the Tukey-Kramer test and Bonferroni corrected Mann-Whitney U test for multiple comparisons (* P

Made available in DSpace on 2019-10-04T11:57:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-04-01 Nova Southeastern University President Faculty Research Development Grant National Institutes of Health Krakow Award for Excellence in Research from the Harvard School of Dental Medicine Locally produced osteoclastogenic factor RANKL plays a critical role in the development of bone resorption in periradicular periodontitis. However, because RANKL is also required for healthy bone remodeling, it is plausible that a costimulatory molecule that upregulates RANKL production in inflammatory periradicular periodontitis may be involved in the pathogenic bone loss processes. We hypothesized that macrophage migration inhibitory factor (MIF) would play a role in upregulating the RANKL-mediated osteoclastogenesis in the periradicular lesion. In response to pulp exposure, the bone loss and level of MIF mRNA increased in the periradicular periodontitis, which peaked at 14 d, in conjunction with the upregulated expressions of mRNAs for RANKL, proinflammatory cytokines (TNF-alpha, IL-6, and IL-1 beta), chemokines (MCP-1 and SDF-1), and MIF's cognate receptors CXCR4 and CD74. Furthermore, expressions of those mRNAs were found significantly higher in wild-type mice compared with that of MIF-/- mice. In contrast, bacterial LPS elicited the production of MIF from ligament fibroblasts in vitro, which, in turn, enhanced their productions of RANKL and TNF-alpha. rMIF significantly upregulated the number of TRAP(+) osteoclasts in vitro. Finally, periapical bone loss induced in wild-type mice were significantly diminished in MIF-/- mice. Altogether, the current study demonstrated that MIF appeared to function as a key costimulatory molecule to upregulate RANKL-mediated osteoclastogenesis, leading to the pathogenically augmented bone resorption in periradicular lesions. These data also suggest that the approach to neutralize MIF activity may lead to the development of a therapeutic regimen for the prevention of pathogenic bone loss in periradicular periodontitis. Harvard Univ, Sch Dent Med, 188 Longwood Ave, Boston, MA 02115 USA King Abdulaziz Univ, Fac Dent, Jeddah 21589, Saudi Arabia Forsyth Inst, Cambridge, MA 02142 USA Nova Southeastern Univ, Coll Dent Med, 320X S Univ Dr, Ft Lauderdale, FL 33324 USA Univ Michigan, Sch Dent, Ann Arbor, MI 48109 USA Sao Paulo State Univ, Sch Dent, BR-15015050 Aracatuba, SP, Brazil Ohio State Univ, Dept Microbiol, 484 W 12th Ave, Columbus, OH 43210 USA Tohoku Univ, Grad Sch Dent, Sendai, Miyagi 9808575, Japan Sao Paulo State Univ, Sch Dent, BR-15015050 Aracatuba, SP, Brazil National Institutes of Health: AG-053615 National Institutes of Health: DE-18499 National Institutes of Health: DE -19917 National Institutes of Health: DE -007327 National Institutes of Health: DE -027851

Patients with Sjögren’s syndrome (SS), an autoimmune disease primarily affecting the exocrine glands, exhibit enhanced TNF-α expression in the saliva and salivary glands. However, the precise in vivo role of TNF-α during the initiation and development of SS is not clearly defined. The present study is undertaken to determine the function of endogenously produced TNF-α in the pathogenesis of SS in non-obese diabetic (NOD) mice, a model of this human disease. Administration of a neutralizing anti-TNF-α antibody to female NOD mice during the stage prior to disease onset significantly improved salivary secretion, indicating a remission of clinical symptoms of SS. TNF-α blockade also decreased the number of leukocyte foci and reduced the number of T cells and B cells in the submandibular glands. Moreover, TNF-α blockade reduced T-bet protein level in the submandibular glands, suggesting a decrease in T helper 1- and T cytotoxic 1 cells. These cellular changes induced by TNF-α neutralization were associated with a reduction in T and B cell chemoattractants CXCL9 and -13. In addition, TNF-α blockade markedly increased the expression level of tight junction protein claudin-1 and water channel protein aquaporin-5, two key factors required for normal salivary secretion, in the submandibular glands. Collectively, these findings indicate that endogenous TNF-α plays a pathogenic role in the development of SS in the NOD model of this disease.

Bone marrow-derived multipotent stromal cells (BMSCs) have potent antiinflammatory effects. This study aimed to investigate the antiinflammatory potential of BMSCs using a mouse model of ligature-induced periodontitis. BMSCs were isolated from the femurs and tibiae of mice. Periodontitis was induced by placing a ligature around the right maxillary second molar. After 3 days, the mice were administered BMSC in the gingiva of the mesial interdental papilla around the ligatured molar. The ligatured and non-ligatured mice that were not administered BMSC served as controls. Differences in inflammatory infiltration and bone resorption around the roots of the second molar were assessed and were subsequently quantified using microcomputed tomography (micro-CT), histological analysis, and tartrate-resistant acid phosphatase (TRAP) staining. Micro-CT revealed that alveolar bone loss around the ligatured molars increased in a time-dependent manner; however, the effect was significantly less in BMSC-treated mice compared with ligatured control mice. Tissue histopathology revealed that BMSC administration mitigated inflammatory infiltration in ligatured BMSC mice. In addition, the number of TRAP-positive osteoclasts was markedly elevated in ligatured control mice compared with those in BMSC-treated mice. These findings indicate that local BMSC administration can mitigate inflammation and alveolar bone resorption, suggesting that administering BMSC leads to new therapeutics for periodontitis.

Host immune responses play a key role in promoting bone resorption in periodontitis via receptor activator of NF-κB ligand (RANKL)–dependent osteoclastogenesis. Both membrane-bound RANKL (mRANKL) expressed on lymphocytes and soluble RANKL (sRANKL) are found in periodontal lesions. However, the underlying mechanism and cellular source of sRANKL release and its biological role in periodontitis are unclear. TNF-α–converting enzyme (TACE) is reported to cleave the following: 1) precursor TNF-α with release of mature, soluble TNF-α and 2) mRANKL with release of sRANKL. Both soluble TNF-α and sRANKL are found in the periodontitis lesion, leading to the hypothesis that TACE expressed on lymphocytes is engaged in RANKL shedding and that the resulting sRANKL induces osteoclastogenesis. In the current study, upon stimulating PBLs with mitogens in vitro, RANKL expression, sRANKL secretion, and TACE expression were all upregulated. Among the four putative mRANKL sheddases examined in neutralization assays, TACE was the only functional sheddase able to cleave mRANKL expressed on PBL. Moreover, PBL culture supernatant stimulated with mitogens in the presence of anti-TACE Ab or anti-RANKL Ab showed a marked reduction of osteoclastogenesis from osteoclast precursors, indicating that TACE-mediated sRANKL may possess sufficient osteoclastogenic activity. According to double-color confocal microscopy, B cells expressed a more pronounced level of RANKL and TACE expression than T cells or monocytes in periodontally diseased gingiva. Conditioned medium of patients’ gingival lymphocyte culture increased in vitro osteoclastogenic activity, which was suppressed by the addition of anti-TACE Ab and anti-RANKL Ab. Therefore, TACE-mediated cleavage of sRANKL from activated lymphocytes, especially B cells, can promote osteoclastogenesis in periodontitis.