Adipose-derived mesenchymal stem cells promote salivary duct regeneration via a paracrine effect

ObjectivesThe self-regeneration of exocrine tissues, including salivary glands, is limited and their regeneration mechanism has not yet been fully elucidated. Here we identify the role of adipose-derived mesenchymal stem cells (AMSCs) in salivary gland regeneration.MethodsAMSCs expressing mesenchymal stem cell markers were applied to a submandibular gland injury model and the mechanism of salivary gland repair and regeneration was analyzed.ResultsTransplanted green fluorescent protein (GFP)-labeled AMSCs grew tightly together and promoted ductal regeneration in the regenerative nodule, with slight infiltration of nonspecific immune cells. A comprehensive gene analysis through RNA-sequencing revealed increased expression of bone morphogenetic protein (BMP), transforming growth factor (TGF), and Wnt in AMSC-transplanted regenerative nodules. The factors released from AMSCs scavenge hydrogen peroxidase-induced reactive oxygen species (ROS) through Wnt promoter activity in vitro. Furthermore, AMSC-conditioned medium recovered the growth of the hydrogen peroxidase-damaged primordium of the submandibular gland culture ex vivo.ConclusionsThese results suggest that AMSC-released factors scavenge ROS and maintain salivary gland repair and regeneration via paracrine effects. Thus, AMSCs could be a practical and applicable tool for use in salivary gland regeneration.     

Hemagglutinating properties of a Streptococcus gordonii strain expressing sialic acid-binding adhesin homolog with low binding site similarity to that of strain DL1

ObjectivesThe Hsa adhesin of Streptococcus gordonii strain DL1 was previously identified as a hemagglutinin that binds specifically to sialoglycoconjugates. We recently found that among oral streptococcal species, S. gordonii strains most frequently express Hsa homologs on the bacterial cell surface. However, the effect of amino acid sequence diversity of nonrepetitive region 2 (NR2), a putative binding site of Hsa, on antigenicity and hemagglutinating (HA) properties is unclear due to difficulties in DNA sequencing the NR2 coding region. The aim of this study was to elucidate the similarity of the low NR2 antigenicity Hsa homolog of strain NDU1118 to that of strain DL1 and the association of the homolog with HA properties of the strain.MethodsThe hsa homolog of NDU1118 was sequenced using a long-read next-generation sequencer, and the Hsa homolog was assessed by alignment analysis of the deduced amino acid sequences. The hsa mutant of NDU1118 was generated by insertion of the erythromycin resistance gene. The HA properties of the wild type and the hsa mutant were assessed with human erythrocytes.ResultsThe NR2 amino acid sequence of the NDU1118 Hsa homolog was almost identical to that of the S. gordonii M99 Hsa homolog, also known as GspB, and less similar to that of DL1 Hsa. The hsa mutation of NDU1118 induced reduction of HA activity in untreated erythrocytes, but surprisingly increased lactose-inhibitable HA activity in neuraminidase-treated erythrocytes.ConclusionsThe results suggest the existence of an adhesin other than the Hsa homolog on the cell surface of NDU1118.     

Systematic review and meta-analysis of randomized controlled studies comparing oscillating-rotating and other powered toothbrushes

BackgroundThe aim of this study was to systematically review and analyze the difference in efficacy of oscillating-rotating toothbrushes compared with other powered toothbrushes.MethodsThe authors performed a systematic search of the literature according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The authors used the population, intervention, comparison, and outcome format to develop a search strategy to answer the study question. The authors searched PubMed-MEDLINE databases. Inclusion criteria were randomized controlled clinical trials comparing oscillating-rotating (OR) toothbrushes with other powered toothbrushes published from January 1, 2009 through March 1, 2019.ResultsThe authors’ search resulted in 454 articles; 21 articles were downloaded for review, 15 articles were included in the report, and 12 could be used for meta-analysis. All of the studies were randomized controlled clinical trials that assessed plaque removal and gingival inflammation indexes. Results of the meta-analysis of the randomized controlled clinical trials showed that OR toothbrushes had superior, statistically significant outcomes for whole-mouth plaque reduction, assessed using the Rustogi Modified Navy Plaque Index (P < .01), and gingivitis, assessed by using number of bleeding sites (P < .001), but not for the modified gingival index (P > .05) or gingival bleeding index (P > .05).Practical ImplicationsThere is some evidence to suggest that OR powered toothbrushes might remove more plaque and reduce the number of bleedings sites better than other powered toothbrushes, specifically, sonic action toothbrushes.     

Calcium ions and vitamin B12 are growth factors for Porphyromonas gingivalis

BackgroundPorphyromonas gingivalis is a causative agent of chronic periodontitis. Standard strains of P. gingivalis, such as W83 and ATCC 33277, proliferate in minimal medium when protein is added as the energy source and hemin and menadione are added as growth factors. Nevertheless, minimal medium containing bovine serum albumin sometimes fails to support growth.HighlightsThe proliferation of two W83 strains and seven ATCC 33277 strains in various minimal media was investigated. Previously, we determined that calcium chloride (CaCl₂) was a growth factor for W83NM, a W83 strain. In this study, we found that vitamin B₁₂ enhanced the proliferation of W83NM in a minimal medium with cultures from the fourth passage but not from the first to the third passage. Therefore, using fourth-passage cultures, we assessed the proliferation of two W83 and seven ATCC 33277 strains in minimal media and the effects of CaCl₂ and vitamin B₁₂. Surprisingly, the nine P. gingivalis strains all differed with respect to their proliferation in minimal media, and protein products used as energy sources showed product-to-product and lot-to-lot heterogeneity. Even though strains or protein products were different, we found CaCl₂-dependent growth in nine strains and vitamin B₁₂-dependent growth in seven strains.ConclusionThese results suggest that calcium ions and vitamin B₁₂ are novel growth factors for P. gingivalis.     

A Ser252Trp substitution in mouse FGFR2 results in hyperplasia of embryonic salivary gland parenchyma

ObjectivesMutations in the fibroblast growth factor receptor 2 (FGFR2) gene are responsible for several severe forms of craniosynostotic disorders, such as Apert and Crouzon syndromes. Patients with craniosynostotic disorders caused by a mutation in Fgfr2 present with several clinical symptoms, including hypersalivation. Here we used a transgenic mouse model of Apert syndrome (Fgfr2^+/S252W mice) to evaluate the morphology of the submandibular glands at embryonic day 15.5 (E15.5), the time point reported to mark the start of lumen formation.MethodsFgfr2^+/S252W mice were generated by crossing ACTB-Cre^+/+ and Fgfr2^+/Neo-S252W mice. After measuring body weight, the submandibular glands were collected at E15.5. H&E staining, immunostaining, and RT-qPCR were performed to investigate the development of the submandibular gland.ResultsThe number of ducts and acini in Fgfr2^+/S252W mice was significantly higher than in control littermates; however, lumen formation was not affected. The mRNA expression of Fgf1, Fgfr1, Mmp2, Bmp4, Bmp7, Dusp6, and Etv5 in Fgfr2^+/S252W mice was significantly higher compared to control littermates. Immunoreactivity for FGF3, FGF1, BMP4, and F4/80 was detected in the parenchyma of Fgfr2^+/S252W mice. The area of apoptotic cells stained with TUNEL in Fgfr2^+/S252W mice was significantly larger than that of the control littermates.ConclusionsThese results suggested that increased FGFR1 signaling and apoptosis in the submandibular glands of Fgfr2^+/S252W mice occurred at E15.5, leading to parenchymal hyperplasia. This study demonstrated that a Ser252Trp substitution in mouse FGFR2 resulted in hyperplasia of the submandibular gland parenchyma during development.     

Application of hiPSCs in tooth regeneration via cellular modulation

BackgroundInduced pluripotent stem cell (iPSC)-based technology provides limitless resources for customized development of organs without any ethical concerns. In theory, iPSCs generated from terminally differentiated cells can be induced to further differentiate into all types of organs that are derived from the embryonic germ layers. Since iPSC reprogramming technology is relatively new, extensive efforts by the researchers have been put together to optimize the protocols to establish in vitro differentiation of human iPSCs (hiPSCs) into various desirable cell types/organs.HighlightsIn the present study, we review the potential application of iPSCs as an efficient alternative to primary cells for modulating signal molecules. Furthermore, an efficient culture system that promotes the differentiation of cell lineages and tissue formation has been reviewed. We also summarize the recent studies wherein tissue engineering of the three germ layers has been explored. Particularly, we focus on the current research strategies for iPSC-based tooth regeneration via molecular modulation.ConclusionIn recent decades, robust knowledge regarding the hiPSC-based regenerative therapy has been accumulated, especially focusing on cellular modulation. This review provides the optimization of the procedures designed to regenerate specific organs.     

Role of interleukin-1 and inflammasomes in oral disease

BackgroundInflammation promotes immune cell infiltration into tissues and induces production of pro-inflammatory cytokines that mediate innate immune responses. Acute or temporary inflammation results in the required repair of the inflamed tissues. However, chronic inflammation leads to pathogenesis of inflammatory conditions such as periodontal disease. In periodontal tissues, pro-inflammatory cytokines mediate inflammatory responses and accelerate the bone-resorbing activity of osteoclasts, resulting in destruction of alveolar bone. Levels of interleukin-1 (IL-1), a major pro-inflammatory cytokine that strongly promotes osteoclastic activity, are elevated in oral tissues of patients with periodontitis. Therefore, elucidation of the mechanisms underlying IL-1 production will enhance our understanding of the pathogenesis of periodontal disease.HighlightIL-1 has two isoforms: IL-1α and IL-1β. Both isoforms bind to the same IL-1 receptor and have identical biological activity. Unlike that of IL-1α, the IL-1β precursor is not bioactive. To induce its bioactivity, the IL-1β precursor is cleaved by caspase-1, whose activation is mediated by multiprotein complexes termed inflammasomes. Thus, IL-1β maturation and activity are strictly regulated by inflammasomes. This review highlights the current understanding of the molecular mechanisms underlying IL-1 production and the related inflammasome activity.ConclusionInhibition of IL-1 production or the inflammasomes via their regulatory mechanisms may facilitate prevention or treatment of periodontal disease and other inflammatory diseases.     

LPA6-RhoA signals regulate junctional complexes for polarity and morphology establishment of maturation stage ameloblasts

ObjectivesLysophosphatidic acid (LPA) is a potent bioactive phospholipid that exerts various functions upon binding to six known G protein-coupled receptors (LPA_1–6); however; its role in a tooth remains unclear. This study aimed to explore the impact of the LPA/LPA receptor 6 (LPA₆)/RhoA signaling axis on maturation stage ameloblasts (M-ABs), which are responsible for enamel mineralization.MethodsThe expression of LPA₆ and LPA-producing synthetic enzymes during ameloblast differentiation was explored through immunobiological analysis of mouse incisors and molars. To elucidate the role of LPA₆ in ameloblasts, incisors of LPA₆ KO mice were analyzed. In vitro experiments using ameloblast cell lines were performed to validate the function of LPA-LPA₆-RhoA signaling in ameloblasts.ResultsLPA₆ and LPA-producing enzymes were strongly expressed in M-ABs. In LPA₆ knockout mice, M-ABs exhibited abnormal morphology with the loss of cell polarity, and an abnormal enamel epithelium containing cyst-like structures was formed. Moreover, the expression of E-cadherin and zonula occludens-1 (ZO-1) significantly decreased in M-ABs. In vitro experiments demonstrated that LPA upregulated the expression of E-cadherin, ZO-1, and filamentous actin (F-actin) at the cellular membrane, whereas LPA₆ knockdown decreased their expression and changed cell morphology. Furthermore, we showed that RhoA signaling mediates LPA-LPA₆-induced junctional complexes.ConclusionsThis study demonstrated that LPA-LPA₆-RhoA signaling is essential for establishing proper cell morphology and polarity, via cell–cell junction and actin cytoskeleton expression and stability, of M-ABs. These results highlight the biological significance of bioactive lipids in a tooth, providing a novel molecular regulatory mechanism of ameloblasts.     

Characterization and evaluation of ascorbic acid-induced cell sheet formation in human periodontal ligament stem cells: An in vitro study

ObjectivesPeriodontal ligament-derived stem cells (PDLSCs) are regarded as a viable option for periodontal regeneration using cell sheet technology. The objective of the present in vitro study was to characterize human PDLSCs based on their phenotypic and biological properties and to evaluate the ascorbic acid (AA or vitamin C)-induced cell sheet by analyzing the molecular markers.MethodsPDLSCs were established from premolars, and their morphology, viability, proliferation, phenotypic marker expression, and ability to differentiate into osteocytes and adipocytes were analyzed. PDLSCs were then induced to form cell sheets using 100 μM AA, and gene expression was examined by real-time polymerase chain reaction.ResultsPDLSCs showed fibroblastic morphology with >95% viability. The cells were highly proliferative and positive for surface antigens CD29, CD73, and CD90 but negative for CD34 and CD45. They were capable of differentiating into osteocytes and adipocytes. Induction with 100 μM AA transformed PDLSCs into two-to three-layered cell sheets. There was no significant upregulation in ALP and RUNX2 expression in the AA-induced cell sheet. However, the expression levels of late osteoblast differentiation marker (bone gamma-carboxy glutamate protein); cementogenic markers (cementum attachment protein and CP23), and genes encoding extracellular matrix (ECM) proteins [collagen type 1 alpha 1 and integrin beta 1) were higher in AA-induced cell sheets by PDLSCs.ConclusionsThe stimulating effect of AA on cell sheet formation by PDLSCs was confirmed by the expression of typical markers involved in osteogenesis/cementogenesis and ECM secretion, which makes this procedure a prospective option for periodontal tissue regeneration applications.     

Comparison of bleaching effects when applied to white-spot lesions before or after resin infiltration: An in vitro study

BackgroundThe purpose of this study was to compare color alterations (ΔE) of white-spot lesions (WSLs) bleached before versus after resin infiltration (RI).MethodsUsing the facial surfaces of bovine maxillary incisors, WSLs were created and the teeth were allocated into 2 groups (n = 45/group): bleach then RI (B-RI group) and RI then bleach (RI-B group). To determine ΔE, Commission Internationale de l’Eclairage L1 a1 b1 (L1 represents lightness, ranging from black to white [0-100]; a1 represents green to red chromaticity [–150-+100]; and b1 represents blue to yellow chromaticity [–100-+150]) measurements were obtained at baseline, after WSL formation, and after RI and bleaching. Representative specimens were evaluated by means of scanning electron microscopy. Statistical analyses included the Mann-Whitney U and Wilcoxon signed rank tests (P ≤ .0016) and repeated measures analysis of variance (P ≤ .05).ResultsNo differences in ΔE were found comparing B-RI with RI-B groups or when the B-RI group was compared with bleached enamel. A statistically significant difference was found when the RI-B group was compared with bleached enamel (ΔE, 0.81; P < .001), but the difference was deemed not clinically significant. Scanning electron microscopy revealed that bleaching after RI increased surface roughness of the resin.ConclusionsThere were no clinically significant differences in ΔE of WSLs when bleach was applied before or after RI; however, applying bleaching agent after RI roughened the surface of the resin material.Practical ImplicationsResults indicate that ΔE were not clinically significantly different between WSLs bleached before versus after RI, although it is best to sequence bleaching before RI therapy, as bleaching after RI roughened the restoration’s surface.     

Oral commensal bacterial flora is responsible for peripheral differentiation of neutrophils in the oral mucosa in the steady state

ObjectivesCommensal bacteria in the host body play a fundamental role in the differentiation and maintenance of the immune system. Studies on intestinal immunity have revealed that, under steady-state conditions, microflora have an important role in the maintenance of health. However, the role of oral commensal bacteria on the oral immune system is still unclear. Here, we clarify the interactions between commensal bacteria and the oral mucosal immune system under steady-state conditions.MethodsWe used germ-free mice that had never been exposed to bacteria and conventional mice grown with normal bacterial flora. Oral cells were isolated from the oral mucosa, stained with specific antibodies, and analyzed by flow cytometry. For the detection of myeloperoxidase and intracellular cytokines, oral cells were stimulated with N-formyl-methionine-leucyl-phenylalanine and phorbol 12-myristate 13-acetate/ionomycin, respectively.ResultsWe found that the oral mucosa harbored more neutrophils in germ-free mice than in conventional mice. However, the majority of neutrophils in the germ-free oral mucosa exhibited an immature phenotype. Other immune cells, including macrophages, T cells, and B cells, in the oral mucosa of germ-free mice showed similar differentiation to those in conventional mice. These results indicate that in the steady-state oral mucosa, the normal commensal flora promote the peripheral differentiation of neutrophils.ConclusionsThe presence of commensal flora is critical for the development of adequate immune system in the oral mucosa.     

Biochemical and X-ray micro-computed tomographic analyses of critical size bone defects grafted with autogenous bone and mercerized bacterial cellulose membranes salified with alendronate

ObjectivesThis study aimed to evaluate the repair of critical-sized bone defects grafted with autogenous bone and mercerized bacterial cellulose membranes (BCm) salified with alendronate (ALN).MethodsForty-eight male Wistar rats underwent surgery to create a 5 mm-diameter bone defect in the calvarium. The removed bone was particularized, regrafted into the defect, and covered by a BCm according to the group: control group (CG), simply mercerized BCm; group 1 (G1), negatively charged BCm (BCm-CM^-) salified with ALN; and group 2 (G2), positively charged BCm (BCm-DEAE⁺) salified with ALN. Serum samples were collected preoperatively and before euthanasia to analyze osteoprotegerin (OPG), parathyroid hormone (PTH), sclerostin (SOST), and fibroblast growth factor 23 (FGF23) levels. The animals were euthanized after 15 or 60 d. Calvaria were analyzed using quantitative microtomography (μCT).ResultsThere was an increased level of PTH in the CG compared to the G2 group, at day 60 (p = 0.019). When analyzing the same group over time, G1 presented an increased FGF23 level on days 15 and 60 (p < 0.05). CG presented an increase in PTH (p = 0.037) at day 60. The μCT analysis detected increased trabecular separation on day 15 in G2 compared to G1 (p = 0.040).ConclusionsSalification of ionized BCm with ALN had no direct effect on bone repair; however, BCm-CM^- increased the levels of FGF23 over time. BCm-DEAE⁺ decreased PTH levels compared to mercerized BCm. BCm-CM-salified with ALN-induced superior bone quality, with respect to trabecular separation, compared to BCm-DEAE⁺.     

Impact of motivational interviewing on early childhood caries: A systematic review and meta-analysis

BackgroundThe authors aimed to assess the scientific evidence on motivational interviewing for the clinical reduction of early childhood caries compared with traditional dental health education.MethodsSearch terms were selected on the basis of Medical Subject Headings and non–Medical Subject Headings terms. The main key words were motivational interviewing, early childhood caries, and education. Potentially eligible studies involved the clinical assessment of caries rate in children whose parents or caregivers received motivational interviewing as an intervention. The authors assessed the risk of bias using the Cochrane risk-of-bias tool. In March 2019, the authors performed an electronic database search of literature published in English within the following databases: Scopus, Cochrane, PubMed, and Embase.ResultsOf 329 articles retrieved initially, 14 were eligible for inclusion in the systematic review and 3 articles contributed to the meta-analysis. For statistical analysis, the mean difference of continuous data was analyzed at a 95% confidence interval using the random-effects model.ConclusionsOverall, the evidence presented in this review was limited. Although the results of the meta-analysis showed that motivational interviewing is as effective as dental health education in controlling early childhood caries, we need more and better designed and reported interventions to assess its impact on early childhood caries accurately.     

Salivary factors related to caries in pregnancy: A systematic review and meta-analysis

BackgroundThe authors of this meta-analysis aimed to assess saliva-related caries risk factors, including calcium and phosphate, hydrogen ion concentration, buffer capacity, Streptococcus mutans and Lactobacillus counts, flow rate, and decayed, missing and filled teeth index in each trimester during pregnancy.Types of Studies ReviewedThe authors searched electronic databases up to July 1, 2019. Eligible observational studies were included. The authors assessed the quality of the included studies by using the Joanna Briggs Institute scale. To estimate the effects of pregnancy, standardized mean differences with 95% confidence intervals were pooled using the random-effects model. Subgroup analysis and meta-regression were used to explore heterogeneity. Publication bias was assessed using Begg and Egger tests.ResultsTwenty-nine studies were included in the meta-analysis, representing 1,230 pregnant women in the case groups and 715 in the control groups (nonpregnant women). The results showed that salivary calcium concentration decreased in the third trimester, salivary phosphate decreased in the second and third trimesters, saliva hydrogen ion concentration decreased in the first and third trimesters, stimulated saliva flow rate increased in the third trimester, and salivary S mutans count increased in the second and third trimesters. In addition, the results showed that saliva calcium, phosphate, S mutans, and buffer capacity amounts had changed from the first trimester to the third.Conclusions and Practical ImplicationsIn the third trimester, most salivary factors related to caries change and can increase the risk of developing caries in the future. Interventions and screening for caries prevention in pregnancy should start in the first or second trimesters.     

Localization of phospholipid-related signal molecules in salivary glands of rodents: A review

BackgroundIn the 1950s, Hokin conducted initial studies on phosphoinositide turnover/cycle in salivary glandular cells. From these studies, the idea emerged that receptor-mediated changes in intramembranous levels of phosphoinositides represent an early step in the stimulus-response pathway. Based on this idea and the general view that knowledge of the exact localization of a given endogenous molecule in cells in situ is important for understanding its functional significance, we have reviewed available information about the localization of several representative phosphoinositide-signaling molecules in the salivary glands in situ in mice.HighlightWe focused on phosphatidylinositol 4-kinase, phosphatidylinositol 4 phosphate 5-kinase α, β, γ, phospholipase Cβ, muscarinic cholinoceptors 1 and 3, diacylglycerol kinase ζ, phospholipase D1 and 2, ADP-ribosylation factor 6 and its exchange factors for Arf6, and cannabinoid receptors. These molecules individually exhibit differential localization in a spatiotemporal manner in the exocrine glands, making it possible to deduce their functional significance, such as their involvement in secretion and cell differentiation.ConclusionAlthough phosphoinositide-signaling molecules whose in situ localization in glandular cells has been clarified are still limited, the obtained information on their localization suggests that their functional significance is more valuable in glandular ducts than in acini. It thus suggests the necessity of greater attention to the ducts in their physio-pharmacological analyses. The purpose of this review is to encourage more in situ localization studies of phosphoinositide-signaling molecules with an aim to further understand their possible involvement in the pathogenesis of salivary gland diseases.