Human oral mucosa cell atlas reveals a stromal-neutrophil axis regulating tissue immunity.

The oral mucosa remains an understudied barrier tissue. This is a site of rich exposure to antigens and commensals, and a tissue susceptible to one of the most prevalent human inflammatory diseases, periodontitis. To aid in understanding tissue-specific pathophysiology, we compile a single-cell transcriptome atlas of human oral mucosa in healthy individuals and patients with periodontitis. We uncover the complex cellular landscape of oral mucosal tissues and identify epithelial and stromal cell populations with inflammatory signatures that promote antimicrobial defenses and neutrophil recruitment. Our findings link exaggerated stromal cell responsiveness with enhanced neutrophil and leukocyte infiltration in periodontitis. Our work provides a resource characterizing the role of tissue stroma in regulating mucosal tissue homeostasis and disease pathogenesis.

On-going Mechanical Damage from Mastication Drives Homeostatic Th17 Cell Responses at the Oral Barrier.

Immuno-surveillance networks operating at barrier sites are tuned by local tissue cues to ensure effective immunity. Site-specific commensal bacteria provide key signals ensuring host defense in the skin and gut. However, how the oral microbiome and tissue-specific signals balance immunity and regulation at the gingiva, a key oral barrier, remains minimally explored. In contrast to the skin and gut, we demonstrate that gingiva-resident T helper 17 (Th17) cells developed via a commensal colonization-independent mechanism. Accumulation of Th17 cells at the gingiva was driven in response to the physiological barrier damage that occurs during mastication. Physiological mechanical damage, via induction of interleukin 6 (IL-6) from epithelial cells, tailored effector T cell function, promoting increases in gingival Th17 cell numbers. These data highlight that diverse tissue-specific mechanisms govern education of Th17 cell responses and demonstrate that mechanical damage helps define the immune tone of this important oral barrier.

TRPV4 mediates IL-1-induced Ca2+ signaling, ERK activation and MMP expression.

Ca2+ permeation through TRPV4 in fibroblasts is associated with pathological matrix degradation. In human gingival fibroblasts, IL-1ß binding to its signaling receptor (IL-1R1) induces activation of extracellular regulated kinase (ERK) and MMP1 expression, processes that require Ca2+ flux across the plasma membrane. It is not known how IL-1R1, which does not conduct Ca2+, generates Ca2+ signals in response to IL-1. We examined whether TRPV4 mediates the Ca2+ fluxes required for ERK signaling in IL-1 stimulated gingival fibroblasts. TRPV4 was immunostained in fibroblasts of human gingival connective tissue and in focal adhesions of cultured mouse gingival fibroblasts. Human gingival fibroblasts treated with IL-1ß showed no change of TRPV4 expression but there was increased MMP1 expression. In mouse, gingival fibroblasts expressing TRPV4, IL-1 strongly increased [Ca2+]i. Pre-incubation of cells with IL-1 Receptor Antagonist blocked Ca2+ entry induced by IL-1 or the TRPV4 agonist GSK101. Knockout of TRPV4 or expression of a non-Ca2+-conducting TRPV4 pore-mutant or pre-incubation with the TRPV4 inhibitor RN1734, blocked IL-1-induced Ca2+ transients and expression of the mouse interstitial collagenase, MMP13. Treatment of mouse gingival fibroblasts with GSK101 phenocopied Ca2+ and ERK responses induced by IL-1; these responses were absent in TRPV4-null cells or cells expressing a non-conducting TRPV4 pore-mutant. Immunostained IL-1R1 localized with TRPV4 in adhesions within cell extensions. While TRPV4 immunoprecipitates analyzed by mass spectrometry showed no association with IL-1R1, TRPV4 associated with Src-related proteins and Src co-immunoprecipitated with TRPV4. Src inhibition reduced IL-1-induced Ca2+ responses. The functional linkage of TRPV4 with IL-1R1 expands its repertoire of innate immune signaling processes by mediating IL-1-driven Ca2+ responses that drive matrix remodeling in fibroblasts. Thus, inhibiting TRPV4 activity may provide a new pharmacological approach for blunting matrix degradation in inflammatory diseases.

TLR4/7-mediated host-defense responses of gingival epithelial cells.

Gingival epithelial cells (GECs) are physical and immunological barriers against outward pathogens while coping with a plethora of non-pathogenic commensal bacteria. GECs express several members of Toll-like receptors (TLRs) and control subsequent innate immune responses. TLR4 senses lipopolysaccharide (LPS) while TLR7/8 recognizes single-strand RNA (ssRNA) playing important roles against viral infection. However, their distinct roles in GECs have not been fully demonstrated. Here, we analyzed biological responses of GECs to  LPS and CL075, a TLR7/8 agonist. GE1, a mouse gingival epithelial cell line, constitutively express TLR4 and TLR7, but not TLR8, like primary skin keratinocytes. Stimulation of GE1 cells with CL075 induced cytokine, chemokine, and antimicrobial peptide  expressions, the pattern of which is rather different from that with LPS: higher mRNA levels of interferon (IFN) ß, CXCL10, and ß-defensin (BD) 14 (mouse homolog of human BD3); lower levels of tumor necrosis factor (TNF), CCL5, CCL11, CCL20, CXCL2, and CX3CL1. As for the intracellular signal transduction of GE1 cells, CL075 rapidly induced significant AKT phosphorylation but failed to activate IKKα/ß-NFκB pathway, whereas LPS induced marked IKKα/ß-NFκB activation without significant AKT phosphorylation. In contrast, both CL075 and LPS induced rapid IKKα/ß-NFκB activation and AKT phosphorylation in a macrophage cell line. Furthermore, specific inhibition of AKT activity abrogated CL075-induced IFNß, CXCL10, and BD14 mRNA expression in GE1 cells. Thus, TLR4/7 ligands appear to induce rather different host-defense responses of GECs through distinct intracellular signaling mechanisms.

Activating the healing process: three-dimensional culture of stem cells in Matrigel for tissue repair.

BACKGROUND: To establish a strategy for stem cell-related tissue regeneration therapy, human gingival mesenchymal stem cells (hGMSCs) were loaded with three-dimensional (3D) bioengineered Matrigel matrix scaffolds in high-cell density microtissues to promote local tissue restoration. METHODS: The biological performance and stemness of hGMSCs under 3D culture conditions were investigated by viability and multidirectional differentiation analyses. A Sprague‒Dawley (SD) rat full-thickness buccal mucosa wound model was established, and hGMSCs/Matrigel were injected into the submucosa of the wound. Autologous stem cell proliferation and wound repair in local tissue were assessed by histomorphometry and immunohistochemical staining. RESULTS: Three-dimensional suspension culture can provide a more natural environment for extensions and contacts between hGMSCs, and the viability and adipogenic differentiation capacity of hGMSCs were significantly enhanced. An animal study showed that hGMSCs/Matrigel significantly accelerated soft tissue repair by promoting autologous stem cell proliferation and enhancing the generation of collagen fibers in local tissue. CONCLUSION: Three-dimensional cell culture with hydrogel scaffolds, such as Matrigel, can effectively improve the biological function and maintain the stemness of stem cells. The therapeutic efficacy of hGMSCs/Matrigel was confirmed, as these cells could effectively stimulate soft tissue repair to promote the healing process by activating the host microenvironment and autologous stem cells.

New insights on collagen structural organization and spatial distribution around dental implants: a comparison between machined and laser-treated surfaces.

BACKGROUND: One of the main factors for the osseointegration of dental implants is the development of an adequate soft tissue barrier, mainly composed by collagen, which protects the implant from bacterial development. The structural features of the peri-implant collagen are influenced by the implant components and, in particular, by the type of the surface. In the clinical practice, healing abutments are characterized by smooth surfaces, named machined. Recently, a new laser technique, Synthegra, has been developed to obtain a topography-controlled surface with micrometric regular pores that seems reducing the risk of peri-implantitis. Based on this background, this study aims investigating the structural organization and spatial distribution of collagen surrounding healing abutments characterized by laser-treated and machined surfaces. METHODS: Gingiva portions surrounding custom-made healing abutments (HA), characterized by alternated laser-treated and machined surfaces, were collected and analyzed by combining Fourier Transform InfraRed Imaging (FTIRI) spectroscopy, a non-invasive and high-resolution bidimensional analytical technique, with histological and multivariate analyses. RESULTS: Masson's trichrome staining, specific for collagen, highlighted a massive presence of collagen in all the analyzed samples, evidencing a surface-related spatial distribution. The nature of collagen, investigated by the FTIRI spectroscopy, appeared more abundant close to the laser-treated surface, with a perpendicular disposition of the bundles respect to the HA; conversely, a parallel distribution was observed around the machined surface. A different secondary structure was also found, with a higher amount of triple helices and a lower quantity of random coils in collagen close to the laser treated surfaces. CONCLUSIONS: FTIRI spectroscopy demonstrates that the use of a laser treated transmucosal surface can improve the morphological organization of the peri-implant collagen, which presents a distribution more similar to that of natural teeth. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov Identifier: (Registration Number: NCT05754970). Registered 06/03/2023, retrospectively registered, https://clinicaltrials.gov/show/NCT05754970 .

PGRN is involved in macrophage M2 polarization regulation through TNFR2 in periodontitis.

BACKGROUND AND OBJECTIVE: Progranulin (PGRN), a multifunctional growth factor, plays indispensable roles in the regulation of cancer, inflammation, metabolic diseases, and neurodegenerative diseases. Nevertheless, its immune regulatory role in periodontitis is insufficiently understood. This study attempts to explore the regulatory effects of PGRN on macrophage polarization in periodontitis microenvironment. METHODS: Immunohistochemical (IHC) and multiplex immunohistochemical (mIHC) stainings were performed to evaluate the expression of macrophage-related markers and PGRN in gingival samples from periodontally healthy subjects and periodontitis subjects. RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were polarized towards M1 or M2 macrophages by the addition of LPS or IL-4, respectively, and were treated with or without PGRN. Real-time fluorescence quantitative PCR (qRT-PCR), immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), and flow cytometry were used to determine the expressions of M1 and M2 macrophage-related markers. Co-immunoprecipitation was performed to detect the interaction between PGRN and tumor necrosis factor receptor 2 (TNFR2). Neutralizing antibody was used to block TNFR2 to confirm the role of TNFR2 in PGRN-mediated macrophage polarization. RESULTS: The IHC and mIHC staining of human gingival slices showed a significant accumulation of macrophages in the microenvironment of periodontitis, with increased expressions of both M1 and M2 macrophage markers. Meanwhile, PGRN was widely expressed in the gingival tissue of periodontitis and co-expressed mainly with M2 macrophages. In vitro experiments showed that in RAW264.7 cells and BMDMs, M1 markers (CD86, TNF-α, iNOS, and IL-6) substantially decreased and M2 markers (CD206, IL-10, and Arg-1) significantly increased when PGRN was applied to LPS-stimulated macrophages relatively to LPS stimulation alone. Besides, PGRN synergistically promoted IL-4-induced M2 markers expression, such as CD206, IL-10, and Arg1. In addition, the co-immunoprecipitation result showed the direct interaction of PGRN with TNFR2. mIHC staining further revealed the co-localization of PGRN and TNFR2 on M2 macrophages (CD206+). Blocking TNFR2 inhibited the regulation role of PGRN on macrophage M2 polarization. CONCLUSIONS: In summary, PGRN promotes macrophage M2 polarization through binding to TNFR2 in both pro- and anti-inflammatory periodontal microenvironments.

Effects of toothpaste containing inactivated Lacticaseibacillus paracasei Probio-01 on plaque-induced gingivitis and dental plaque microbiota.

Plaque-induced gingivitis is an inflammatory response in gingival tissues resulting from bacterial plaque accumulation at the gingival margin. Postbiotics can promote the proliferation of beneficial bacteria and optimise the state of microbiota in the oral cavity. In this study, we investigated the effect of inactivated Lacticaseibacillus paracasei Probio-01 on plaque-induced gingivitis and the dental plaque microbiota. A total of 32 healthy gingival participants (Group N, using blank toothpaste for 3 months) and 60 patients with plaque-induced gingivitis (30 in Group F, using inactivated Probio-01 toothpaste for 3 months, and 30 in Group B, using blank toothpaste for 3 months, respectively) were recruited. Clinical indices, which included bleeding on probing (BOP), gingival index (GI), and plaque index (PI), were used to assess the severity of gingivitis. Furthermore, 16SrDNA amplicon sequencing was used to explore changes in the gingival state and dental plaque microbiota in patients with plaque-induced gingivitis. The results showed that inactivated Probio-01 significantly reduced clinical indices of gingivitis, including BOP, GI, and PI, in participants with plaque-induced gingivitis and effectively relieved gingival inflammation, compared with that observed in the control group (group B). Inactivated Probio-01 did not significantly influence the diversity of dental plaque microbiota, but increased the relative abundance of dental plaque core bacteria, such as Leptotrichia and Fusobacterium (P < 0.05). Strong correlations were observed between the indices and abundance of dental plaque microbiota. Overall, the inactivated Probio-01 significantly reduced the clinical indices of gingivitis and effectively improved gingival inflammation in patients with plaque-induced gingivitis. The activity of inactivated Probio-01 against plaque-induced gingivitis was possibly mediated by its ability to regulate the dental plaque microbiota, as indicated by the close correlation between the plaque microbiota and clinical indices of gingivitis.

Site-specific autonomic vasomotor responses and their interactions in rat gingiva.

Blood flow in the gingiva, comprising the interdental papilla as well as attached and marginal gingiva, is important for maintaining of gingival function and is modulated by risk factors such as stress that may lead to periodontal disease. Marked blood flow changes mediated by the autonomic (parasympathetic and sympathetic) nervous system may be essential for gingival hemodynamics. However, differences in autonomic vasomotor responses and their functional significance in different parts of the gingiva are unclear. We examined the differences in autonomic vasomotor responses and their interactions in the gingiva of anesthetized rats. Parasympathetic vasodilation evoked by the trigeminal (lingual nerve)-mediated reflex elicited frequency-dependent blood flow increases in gingivae, with the increases being greatest in the interdental papilla. Parasympathetic blood flow increases were significantly reduced by intravenous administration of the atropine and VIP antagonist. The blood flow increase evoked by acetylcholine administration was higher in the interdental papilla than in the attached gingiva, whereas that evoked by VIP agonist administration was greater in the attached gingiva than in the interdental papilla. Activation of the cervical sympathetic nerves decreased gingival blood flow and inhibited parasympathetically induced blood flow increases. Our results suggest that trigeminal-parasympathetic reflex vasodilation 1) is more involved in the regulation of blood flow in the interdental papilla than in the other parts of the gingiva, 2) is mediated by cholinergic (interdental papilla) and VIPergic systems (attached gingiva), and 3) is inhibited by excess sympathetic activity. These results suggest a role in the etiology of periodontal diseases during mental stress.

Reproducibility of Laser Doppler Flowmetry in gingival microcirculation. A study on six different protocols.

OBJECTIVES: Laser Doppler Flowmetry (LDF) is a non-invasive technique for the assessment of tissue blood flow, but increased reproducibility would facilitate longitudinal studies. The aim of the study was to assess the interday reproducibility of Laser Doppler Flowmetry (LDF) at rest, at elevated local temperatures, and with the use of the vasodilator Methyl Nicotinate (MN) in six interconnected protocols for the measurement of the blood supply to the microvascular bed of the gingiva. METHODS: Ten healthy volunteers were included. Interweek LDF measurements with custom-made acrylic splints were performed. Six protocols were applied in separate regions of interest (ROI): 1; basal LDF, 2; LDF with thermoprobe 42 °C, 3; LDF with thermoprobe 45 °C, 4; LDF with thermoprobe 42 °C and MN, 5; LDF with thermoprobe 45 °C and MN and 6; LDF with MN. RESULTS: Intra-individual reproducibility was assessed by the within-subject coefficient of variation (wCV) and the intraclass correlation coefficient (ICC). Basal LDF measurements demonstrated high reproducibility with wCV 11.1 in 2 min and 10.3 in 5 min. ICC was 0.9 and 0.92. wCV after heat and MN was 4.9-10.3 and ICC 0.82-0.93. The topically applied MN yielded increased blood flow. CONCLUSION: This is the first study evaluating the reproducibility of basal LDF compared to single or multiple vasodilatory stimuli in gingiva. Multiple collector fibers probe and stabilizing acrylic splints are recommended. Vasodilatory stimulation showed a tendency toward higher reproducibility. Furthermore, MN yields vasodilation in gingiva.

Vulvar-vaginal-gingival-otic syndrome.

In order to retrospectively analyse the multi-site involvement pattern of erosive lichen planus patients, we retrospectively reported the clinical and medical data of three patients with erosive lichen planus which involving their vulva, vagina, gingiva, and ear canal. We confirmed the existence of otic lichen planus, and found that it is more common in patients with vulvovaginal-gingival syndrome of erosive lichen planus. Therefore, we propose 'vulvovaginal-gingival-otic syndrome' to further describe this rare compound pattern of lichen planus.

Galectin-3 inhibition alleviated LPS-induced periodontal inflammation in gingival fibroblasts and experimental periodontitis mice.

OBJECTIVES: Clinical studies have confirmed that galectin-3 (Gal-3) levels are significantly elevated in periodontitis patients. The present study aimed to explore the effects of Gal-3 inhibition on periodontal inflammation in vitro and in vivo. METHODS: Human gingival fibroblasts (HGFs) with or without Gal-3 knockdown were stimulated by lipopolysaccharide (LPS), and a ligation-induced mouse periodontitis model treated with a Gal-3 inhibitor was established. Hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining were used to evaluate Gal-3 levels in gingival tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect Gal-3, interleukin (IL)-6, IL-8, and C-C motif ligand 2 (CCL2) expression. Immunofluorescence and western blotting were used to detect NF-κB and ERK signaling pathway activation. Micro-computed tomography was used to analyse the degree of bone loss. RESULTS: Gal-3 was significantly up-regulated in inflamed gingival tissues and LPS-induced HGFs. Gal-3 knockdown markedly decreased LPS-induced IL-6, IL-8, and CCL2 expression and blocked NF-κB and ERK signaling pathway activation in HGFs. In the mouse periodontitis model, Gal-3 inhibition significantly alleviated IL-1ß and IL-6 infiltration in gingival tissue and mitigated periodontal bone loss. CONCLUSIONS: Gal-3 inhibition notably alleviated periodontal inflammation partly through blocking NF-κB and ERK signaling pathway activation.

Macrophage-enriched novel functional long noncoding RNAs LRRC75A-AS1 and GAPLINC regulate polarization and innate immune responses.

INTRODUCTION: Macrophages (Mφs) are functionally dynamic immune cells that bridge innate and adaptive immune responses; however, the underlying epigenetic mechanisms that control Mφ plasticity and innate immune functions are not well elucidated. OBJECTIVE: To identify novel functions of macrophage-enriched lncRNAs in regulating polarization and innate immune responses. METHODS: Total RNA isolated from differentiating monocyte-derived M1 and M2 Mφs was profiled for lncRNAs expression using RNAseq. Impact of LRRC75A-AS1, GAPLINC and AL139099.5 knockdown was examined on macrophage differentiation, polarization markers, phagocytosis, and antigen processing by flow cytometry and florescence microscopy. Cytokine profiles were examined by multiplex bead array and cytoskeletal signaling pathway genes were quantified by PCR-based array. Gingival biopsies were collected from periodontally healthy and diseased subjects to examine lncRNAs, M1/M2 marker expression. RESULTS: Transcriptome profiling of M1 and M2 Mφs identified thousands of differentially expressed known and novel lncRNAs. We characterized three Mφ-enriched lncRNAs LRRC75A-AS1, GAPLINC and AL139099.5 in polarization and innate immunity. Knockdown of LRRC75A-AS1 and GAPLINC downregulated the Mφ differentiation markers and skewed Mφ polarization by decreasing M1 markers without a significant impact on M2 markers. LRRC75A-AS1 and GAPLINC knockdown also attenuated bacterial phagocytosis, antigen processing and inflammatory cytokine secretion in Mφs, supporting their functional role in potentiating innate immune functions. Mechanistically, LRRC75A-AS1 and GAPLINC knockdown impaired Mφ migration by downregulating the expression of multiple cytoskeletal signaling pathways suggesting their critical role in regulating Mφ migration. Finally, we showed that LRRC75A-AS1 and GAPLINC were upregulated in periodontitis and their expression correlates with higher M1 markers suggesting their role in macrophage polarization in vivo. CONCLUSION: Our results show that polarized Mφs acquire a unique lncRNA repertoire and identified many previously unknown lncRNA sequences. LRRC75A-AS1 and GAPLINC, which are induced in periodontitis, regulate Mφ polarization and innate immune functions supporting their critical role in inflammation.

FOXO1 regulates wound-healing responses in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Forkhead box-O 1 (FOXO1) is a transcription factor actively involved in oral wound healing at the epithelial barrier. However, less is known regarding the role of FOXO1 during the tissue repair response in the connective tissue compartment. This study explored the involvement of FOXO1 in the modulation of fibroblast activity related to wound healing. METHODS: Primary cultures of human gingival fibroblasts were obtained from four healthy young donors. Myofibroblastic differentiation, collagen gel contraction, cell migration, cell spreading, and integrin activation were evaluated in the presence or absence of a FOXO1 inhibitor (AS1842856). Variations in mRNA and proteins of interest were evaluated through qRT-PCR and western blot, respectively. Distribution of actin, α-smooth muscle actin, and ß1 integrin was evaluated using immunofluorescence. FOXO1 and TGF-ß1 expression in gingival wound healing was assessed by immunohistochemistry in gingival wounds performed in C57BL/6 mice. Images were analyzed using ImageJ/Fiji. ANOVA or Kruskal-Wallis test followed by Tukey's or Dunn's post-hoc test was performed. All data are expressed as mean ± SD. p < .05 was considered statistically significant. RESULTS: FOXO1 inhibition caused a decrease in the expression of the myofibroblastic marker α-SMA along with a reduction in fibronectin, type I collagen, TGF-ß1, and ß1 integrin mRNA level. The FOXO1 inhibitor also caused decreases in cell migration, cell spreading, collagen gel contraction, and ß1 integrin activation. FOXO1 and TGF-ß1 were prominently expressed in gingival wounds in fibroblastic cells located at the wound bed. CONCLUSION: The present study indicates that FOXO1 plays an important role in the modulation of several wound-healing functions in gingival fibroblast. Moreover, our findings reveal an important regulatory role for FOXO1 on the differentiation of gingival myofibroblasts, the regulation of cell migration, and collagen contraction, all these functions being critical during tissue repair and fibrosis.

ZNF862 induces cytostasis and apoptosis via the p21-RB1 and Bcl-xL-Caspase 3 signaling pathways in human gingival fibroblasts.

OBJECTIVE: This study investigates the effects of ZNF862 on the proliferation and apoptosis of human gingival fibroblasts and their related mechanisms. BACKGROUND: As a major transcription factor family, zinc finger proteins (ZFPs) regulate cell differentiation, growth, and apoptosis through their conserved zinc finger motifs, which allow high flexibility and specificity in gene regulation. In our previous study, ZNF862 mutation was associated with hereditary gingival fibromatosis. Nevertheless, little is known about the biological function of ZNF862. Therefore, this study was aimed to reveal intracellular localization of ZNF862, the influence of ZNF862 on the growth and apoptosis of human gingival fibroblasts (HGFs) and its potential related mechanisms. METHODS: Immunohistochemistry, immunofluorescence staining, and western blotting were performed to determine the intracellular localization of ZNF862 in HGFs. HGFs were divided into three groups: ZNF862 overexpression group, ZNF862 interference group, and the empty vector control group. Then, the effects of ZNF862 on cell proliferation, migration, cell cycle, and apoptosis were evaluated. qRT-PCR and western blotting were performed to further explore the mechanism related to the proliferation and apoptosis of HGFs. RESULTS: ZNF862 was found to be localized in the cytoplasm of HGFs. In vitro experiments revealed that ZNF862 overexpression inhibited HGFs proliferation and migration, induced cell cycle arrest at the G0/G1-phase and apoptosis. Whereas, ZNF862 knockdown promoted HGFs proliferation and migration, accelerated the transition from the G0/G1 phase into the S and G2/M phase and inhibited cell apoptosis. Mechanistically, the effects of ZNF862 on HGFs proliferation and apoptosis were noted to be dependent on inhibiting the cyclin-dependent kinase inhibitor 1A (p21)-retinoblastoma 1 (RB1) signaling pathway and enhancing the B-cell lymphoma-extra-large (Bcl-xL)-Caspase 3 signaling pathway. CONCLUSION: Our results for the first time reveal that ZNF862 is localized in the cytoplasm of HGFs. ZNF862 can inhibit the proliferation of HGFs by inhibiting the p21-RB1 signaling pathway, and it also promotes the apoptosis of HGFs by enhancing the Bcl-xL-Caspase 3 signaling pathway.

Use of ultrasound imaging for assessment of the periodontium: A systematic review.

The objective of this study was to systematically review the literature regarding diagnostic applications of ultrasound imaging for evaluation of the periodontium in humans. The search was conducted on Medline, EMBASE, Web of Science, Scopus, Cochrane, and PubMed up to April 3, 2023. The studies included were exclusively human studies that assessed the periodontium with ultrasound (US) imaging (b-mode). Outcomes measured included alveolar bone level, alveolar bone thickness, gingival thickness, and blood flow quantification. References were imported to Covidence. Two reviewers conducted phases 1 and 2. The JBI risk assessment tool for cross-sectional studies was used. Extracted data included the transducer and measurements used and the study's outcomes. The search yielded 4892 studies after removing duplicates. From these, 25 studies were included and selected for extraction. Included studies retrieved outcomes from US examinations of the periodontal tissues. From the selected studies, 15 used US on natural teeth, 4 used US on implants, 2 used US on edentulous ridges, and 4 used color flow/power in US to evaluate the blood flow. The results of the present systematic review suggest that US might be a feasible and valuable diagnostic tool for the periodontium, with the potential to complement shortfalls of current radiographic technologies.

Dipotassium glycyrrhizate and hinokitiol enhance macrophage efferocytosis by regulating recognition, uptake, and metabolism of apoptotic cells in vitro.

BACKGROUND AND OBJECTIVE: Efferocytosis is a process whereby macrophages remove apoptotic cells, such as neutrophils, that have accumulated in tissues, which is required for resolution of inflammation. Efferocytosis is impaired in individuals with increasing age and in those with various systemic diseases. Recently, efferocytosis has been reported to be related to the pathogenesis and progression of periodontitis, and enhancement of efferocytosis, especially in the subjects with impaired efferocytosis, was suggested to lead to periodontitis prevention and care. Various anti-inflammatory ingredients are used in oral care products, but their effect on efferocytosis is unclear. Here, we aimed to identify ingredients contained in oral care products that are effective for efferocytosis regulation. METHODS: The ability of dead cells to induce inflammation in human gingival fibroblast (HGF) cells were evaluated by measuring IL-6 secretion. Six ingredients in oral care products used as anti-inflammatory agents were evaluated for their effect on efferocytosis using flow cytometry. The expression of various efferocytosis-related molecules, such as MERTK and LRP1 involved in recognition, and LXRα and ABCA1 that function in metabolism, were measured in RAW264.7 cells with or without ingredient treatment. Rac1 activity, which is related to the uptake of dead cells, was measured using the G-LISA kit. RESULTS: Dead cells elicited IL-6 secretion in HGF cells. Among the six ingredients, GK2 and hinokitiol enhanced efferocytosis activity. GK2 and hinokitiol significantly increased the expression of MERTK and LRP1, and also enhanced LXRα and ABCA1 expression after efferocytosis. Furthermore, they increased Rac1 activity in the presence of dead cells. CONCLUSION: Among the six ingredients tested, GK2 and hinokitiol promoted efferocytosis by regulating apoptotic cell recognition, uptake, and metabolism-related molecules. Efferocytosis upregulation may be one of the mechanisms of GK2 and hinokitiol in the treatment of inflammatory diseases, such as periodontitis.

Phytocannabinoids and gingival inflammation: Preclinical findings and a placebo-controlled double-blind randomized clinical trial with cannabidiol.

OBJECTIVE: The aim of this study was to: (1) evaluate the anti-inflammatory effects of cannabidiol (CBD) on primary cultures of human gingival fibroblasts (HGFs) and (2) to clinically monitor the effect of CBD in subjects with periodontitis. BACKGROUND: The use of phytocannabinoids is a new approach in the treatment of widely prevalent periodontal disease. MATERIALS AND METHODS: Cannabinoid receptors were analyzed by western blot and interleukin production detected using enzyme immunoassay. Activation of the Nrf2 pathway was studied via monitoring the mRNA level of heme oxygenase-1. Antimicrobial effects were determined by standard microdilution and 16S rRNA screening. In the clinical part, a placebo-control double-blind randomized study was conducted (56 days) in three groups (n = 90) using dental gel without CBD (group A) and with 1% (w/w) CBD (group B) and corresponding toothpaste (group A - no CBD, group B - with CBD) for home use to maintain oral health. Group C used dental gel containing 1% chlorhexidine digluconate (active comparator) and toothpaste without CBD. RESULTS: Human gingival fibroblasts were confirmed to express the cannabinoid receptor CB2. Lipopolysaccharide-induced cells exhibited increased production of pro-inflammatory IL-6 and IL-8, with deceasing levels upon exposure to CBD. CBD also exhibited antimicrobial activities against Porphyromonas gingivalis, with an MIC of 1.5 µg/mL. Activation of the Nrf2 pathway was also demonstrated. In the clinical part, statistically significant improvement was found for the gingival, gingival bleeding, and modified gingival indices between placebo group A and CBD group B after 56 days. CONCLUSIONS: Cannabidiol reduced inflammation and the growth of selected periodontal pathogenic bacteria. The clinical trial demonstrated a statistically significant improvement after CBD application. No adverse effects of CBD were reported by patients or observed upon clinical examination during the study. The results are a promising basis for a more comprehensive investigation of the application of non-psychotropic cannabinoids in dentistry.

Analysis of inflammation and bone remodeling of atmospheric plasma therapy in experimental periodontitis.

BACKGROUND AND OBJECTIVE: The biological effects of atmospheric plasma (cold plasma) show its applicability for controlling the etiological factors that involve tissue repair. Thus, the study evaluated the effect of atmospheric plasma therapy in the control of tissue inflammation and bone remodeling in experimental periodontitis. METHODS: Fifty-six rats were subjected to ligation in the cervical region of the first maxillary molars (8 weeks). The animals were divided into two groups (n = 28): periodontitis without treatment group (P group), and periodontitis with atmospheric plasma treatment group (P + AP group). Tissue samples were collected at 2 and 4 weeks after treatment to analyze the inflammation and bone remodeling by biochemical, histomorphometric, and immunohistochemical analyses. RESULTS: Inflammatory infiltration in the gingival and periodontal ligament was lower in the P + AP group than in the P group (p < .05). The MPO and NAG levels were higher in the P + AP group compared to P group (p < .05). At 4 weeks, the TNF-α level was lower and the IL-10 level was higher in the P + AP group compared to P group (p < .05). In the P + AP group, the IL-1ß level increased in the second week and decreased in the fourth week (p < .05), the number of blood vessels was high in the gingival and periodontal ligament in the second and fourth week (p < .05); and the number of fibroblasts in the gingival tissue was low in the fourth week, and higher in the periodontal tissue in both period (p < .05). Regarding bone remodeling, the RANK and RANKL levels decreased in the P + AP group (p < .05). The OPG level did not differ between the P and P + AP groups (p > .05), but decreased from the second to the fourth experimental week in P + AP group (p < .05). CONCLUSIONS: The treatment of experimental periodontitis with atmospheric plasma for 4 weeks modulated the inflammatory response to favor the repair process and decreased the bone resorption biomarkers, indicating a better control of bone remodeling in periodontal disease.

Oxytocin alleviates periodontitis in adult rats.

OBJECTIVE: The objective of the study was to evaluate the expression of oxytocin receptors in normal and inflamed gingiva, as well as the effects of systemic administration of oxytocin in bone loss and gum inflammatory mediators in a rat model of experimental periodontitis. BACKGROUND DATA: Current evidence supports the hypothesis of a disbalance between the oral microbiota and the host's immune response in the pathogenesis of periodontitis. Increased complexity of the microbial biofilm present in the periodontal pocket leads to local production of nitrogen and oxygen-reactive species, cytokines, chemokines, and other proinflammatory mediators which contribute to periodontal tissue destruction and bone loss. Oxytocin has been suggested to participate in the modulation of immune and inflammatory processes. We have previously shown that oxytocin, nitric oxide, and endocannabinoid system interact providing a mechanism of regulation for systemic inflammation. Here, we aimed at investigating not only the presence and levels of expression of oxytocin receptors on healthy and inflamed gingiva, but also the effects of oxytocin treatment on alveolar bone loss, and systemic and gum expression of inflammatory mediators involved in periodontal tissue damage using ligature-induced periodontitis. Therefore, anti-inflammatory strategies oriented at modulating the host's immune response could be valuable adjuvants to the main treatment of periodontal disease. METHODS: We used an animal model of ligature-induced periodontitis involving the placement of a linen thread (Barbour flax 100% linen suture, No. 50; size 2/0) ligature around the neck of first lower molars of adult male rats. The ligature was left in place during the entire experiment (7 days) until euthanasia. Animals with periodontitis received daily treatment with oxytocin (OXT, 1000 µg/kg, sc.) or vehicle and/or atosiban (3 mg/kg, sc.), an antagonist of oxytocin receptors. The distance between the cement-enamel junction and the alveolar bone crest was measured in stained hemimandibles in the long axis of both buccal and lingual surfaces of both inferior first molars using a caliper. TNF-α levels in plasma were determined using specific rat enzyme-linked immunosorbent assays (ELISA). OXT receptors, IL-6, IL-1ß, and TNF-α expression were determined in gingival tissues by semiquantitative or real-time PCR. RESULTS: We show that oxytocin receptors are expressed in normal and inflamed gingival tissues in male rats. We also show that the systemic administration of oxytocin prevents the experimental periodontitis-induced increased gum expression of oxytocin receptors, TNF-α, IL-6, and IL-1ß (p < .05). Furthermore, we observed a reduction in bone loss in rats treated with oxytocin in our model. CONCLUSIONS: Our results demonstrate that oxytocin is a novel and potent modulator of the gingival inflammatory process together with bone loss preventing effects in an experimental model of ligature-induced periodontitis.