miRNA-148a-containing GMSC-derived EVs modulate Treg/Th17 balance via IKKB/NF-κB pathway and treat a rheumatoid arthritis model.

Mesenchymal stem cells (MSCs) have demonstrated potent immunomodulatory properties that have shown promise in the treatment of autoimmune diseases, including rheumatoid arthritis (RA). However, the inherent heterogeneity of MSCs triggered conflicting therapeutic outcomes, raising safety concerns and limiting their clinical application. This study aimed to investigate the potential of extracellular vesicles derived from human gingival mesenchymal stem cells (GMSC-EVs) as a therapeutic strategy for RA. Through in vivo experiments using an experimental RA model, our results demonstrate that GMSC-EVs selectively homed to inflamed joints and recovered Treg and Th17 cell balance, resulting in the reduction of arthritis progression. Our investigations also uncovered miR-148a-3p as a critical contributor to the Treg/Th17 balance modulation via IKKB/NF-κB signaling orchestrated by GMSC-EVs, which was subsequently validated in a model of human xenograft versus host disease (xGvHD). Furthermore, we successfully developed a humanized animal model by utilizing synovial fibroblasts obtained from patients with RA (RASFs). We found that GMSC-EVs impeded the invasiveness of RASFs and minimized cartilage destruction, indicating their potential therapeutic efficacy in the context of patients with RA. Overall, the unique characteristics - including reduced immunogenicity, simplified administration, and inherent ability to target inflamed tissues - position GMSC-EVs as a viable alternative for RA and other autoimmune diseases.

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.

Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis.

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFß/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFß/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFß-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFß-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.

[A preliminary in vivo and in vitro study of endothelial cell pyroptosis in the periodontal inflammatory environment].

Objective: To observe whether endothelial cells undergo pyroptosis in the inflammatory periodontal environment by using a model in vivo and in vitro, providing an experimental basis for indepth understanding of the underlying pathogenesis of periodontitis. Methods: According to the classification of periodontal diseases of 2018, gingival tissues were collected from periodontally healthy subjects and patients with stage Ⅲ-Ⅳ, grade C periodontitis, who presented Department of Oral and Maxillofacial Surgery and Department of Periodontology, School of Stomatology, The Fourth Military Medical University from April to May 2022. Immunohistochemical staining was performed to detect the expression level and distribution of gasdermin D (GSDMD), a hallmark protein of cell pyroptosis, in gingival tissues. Periodontitis models were established in each group by ligating the maxillary second molar teeth of three mice for 2 weeks (ligation group). The alveolar bone resorption was determined by micro-CT (mice without ligation treatment were used as the control group), and the colocalization of GSDMD and CD31 were quantitatively analyzed by immunofluorescence staining in gingival tissues of healthy and inflammatory mice. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) combined with adenosine triphosphate (ATP) at various concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 mg/L, respectively, and the 0 mg/L group was set as the control group at the same time. Scanning electron microscopy was used to observe the morphology of HUVECs. Western blotting was used to detect the expression of gasdermin D-N terminal domains (GSDMD-N) protein and immunofluorescence cell staining was used to detect the expression and distribution of GSDMD. Cell counting kit-8 (CCK-8) was used to detect the proliferative ability of HUVECs, and propidium iodide (PI) staining was used to detect the integrity of cell membrane of HUVECs. Results: Immunohistochemistry showed that GSDMD in gingival tissues of periodontitis was mainly distributed around blood vessels and its expression level was higher than that in healthy tissues. Micro-CT showed that alveolar bone resorption around the maxillary second molar significantly increased in ligation group mice compared with control subjects (t=8.88, P<0.001). Immunofluorescence staining showed significant colocalization of GSDMD with CD31 in the gingival vascular endothelial cells in mice of ligation group. The results of scanning electron microscopy showed that there were pores of different sizes, the typical morphology of pyroptosis, on HUVECs cell membranes in the inflammatory environment simulated by ATP combined with different concentrations of LPS, and 2.5 mg/L group showed the most dilated and fused pores on cell membranes, with the cells tended to lyse and die. Western blotting showed that the expression of GSDMD-N, the hallmark protein of cell pyroptosis, was significantly higher in 2.5 and 5.0 mg/L groups than that in the control group (F=3.86, P<0.01). Immunofluorescence cell staining showed that the average fluorescence intensity of GSDMD in 2.5 mg/L group elevated the most significantly in comparison with that in the control group (F=35.25, P<0.001). The CCK-8 proliferation assay showed that compared to the control group (1.00±0.02), 0.5 mg/L (0.52±0.07), 1.0 mg/L (0.57±0.10), 2.5 mg/L (0.58±0.04), 5.0 mg/L (0.55±0.04), 10.0 mg/L (0.61±0.03) groups inhibited cell proliferation (F=39.95, P<0.001). PI staining showed that the proportion of positive stained cells was highest [(56.07±3.22)%] in 2.5 mg/L group (F=88.24, P<0.001). Conclusions: Endothelial cells undergo significant pyroptosis in both in vivo and in vitro periodontal inflammatory environments, suggesting that endothelial cell pyroptosis may be an important pathogenic factor contributing to the pathogenesis of periodontitis.

[Inhibitory effect of downregulating G protein-coupled receptor class C group 5 member A expression on lipopolysaccharide-induced inflammatory response in human gingival fibroblasts].

Objective: To clarify the effect and the mechanism of G protein-coupled receptor class C group 5 member A (GPRC5A) on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (GFs), thus to provide a foundation for delving into the role of G protein coupled receptor (GPCR) in periodontitis. Methods: Gingival tissue samples were collected from 3 individuals periodontally healthy (health group) and 3 patients with periodontitis (periodontitis group) in Shandong Stomatological Hospital from December 2022 to February 2023. The expressions of GPRC5A of the two groups were detected by immunohistochemistry staining. GFs used in this study were isolated from a portion of gingiva for the extraction of impacted teeth in School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University from December 2022 to February 2023. GFs were isolated with enzymic digestion and transfected with 30, 50 and 80 µmol/L small interfering RNA-GPRC5A (siGPRC5A) or small interfering RNA-negative control (siNC), regarded as the experimental group and the negative control one, respectively. The silencing efficiency of siGPRC5A was evaluated by real-time fluorescence quantitative PCR (RT-qPCR). Experiments were then conducted using these cells which were divided into four groups of negative control (NC), LPS, siGPRC5A+LPS and siGPRC5A. The mRNA and protein levels of GPRC5A in GFs under 1 mg/L LPS-induced GFs inflammatory state were evaluated by RT-qPCR and Western blotting analysis after GPRC5A knockdown. RT-qPCR was used to detect the gene expression levels of the inflammatory cytokines in GFs induced by LPS, namely, interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α, prostaglandin endoperoxide synthase 2 (PTGS2) after GPRC5A knockdown. Western blotting analysis and immunofluorescence staining were used to further investigate the activation of nuclear factor-kappa B (NF-κB) signaling pathway. Results: Immunohistochemistry staining showed that the expression of GPRC5A in gingival tissues of periodontitis group (0.132±0.006) increased compared with that in periodontally healthy group (0.036±0.019) (t=8.24, P=0.001). Meanwhile, RT-qPCR results showed that the gene expression levels of GPRC5A at different time point (2, 6, 12, 24 h) in LPS-induced GFs (0.026±0.002, 0.042±0.005, 0.004±0.000, 0.016±0.000) were upregulated compared with those in the NC group (0.004±0.000, 0.004±0.000, 0.002±0.000, 0.007±0.000) (all P<0.001), respectively, and peaked at 6 h. The 50 µmol/L group displayed the most significant decrease in siGPRC5A expression (31.16±3.29) compared with that of the siNC group (100.00±4.88) (F=297.98, P<0.001). The results of RT-qPCR and Western blotting analysis showed that siGPRC5A (0.27±0.03, 0.71±0.00) suppressed the expressions of GPRC5A at both gene and protein levels, while LPS (1.30±0.10, 1.43±0.03) was able to promote the expressions of GPRC5A compared with those of the NC group (1.00±0.01, 1.00±0.00)(all P<0.001). The siGPRC5A+LPS group (0.39±0.03, 1.06±0.16) also inhibited the increase of GPRC5A at both gene and protein levels induced by LPS (1.30±0.10, 1.43±0.03) (F=208.38, P<0.001; F=42.04, P<0.001). RT-qPCR results showed that the expressions of IL-8, IL-1ß, IL-6, TNF-α, and PTGS2 at the gene level in LPS group were highly increased compared with those in the NC group (all P<0.001). siGPRC5A significantly suppressed LPS-induced expressions of these inflammatory cytokines in GFs (all P<0.001). Western blotting analysis showed that the levels of p65 and IκBα protein phosphorylation in the LPS group were highly increased compared with those in the NC group, and siGPRC5A could effectively suppressed LPS-induced protein phosphorylation (all P<0.01). Furthermore, immunofluorescence staining showed that NF-κB p65 in the control group was mainly concentrated in the cytoplasm, and partially translocated to the nucleus under the stimulation of LPS. siGPRC5A was able to inhibit LPS-induced intranuclear translocation of p65 to a certain extent. Conclusions: GPRC5A expression was upregulated in periodontitis, and GPRC5A knockdown inhibited LPS-induced inflammation. Moreover, GPRC5A played a role in inflammation regulation by interacting with NF-κB signaling pathway.

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.

Ingenuity pathway analysis of gingival epithelial cells stimulated with estradiol and progesterone.

OBJECTIVE: Periodontal disease is a risk factor for preterm delivery, and elevated female hormone levels during pregnancy promote hormone-dependent periodontopathogenic bacterial growth and gingivitis. Although the saliva of pregnant women contains female hormones at elevated levels, their effects on the gingiva are poorly understood. Therefore, in this study, we investigated the effects of estradiol and progesterone stimulation on gingival epithelial cells via ingenuity pathway analysis. METHODS: Human gingival epithelial progenitors were cultured in a CnT-Prime medium; 17ß-estradiol (E2) and progesterone (P4) were used as the reagents. Cells treated with dimethyl sulfoxide alone were used as the control group. Cells in the control and experimental groups were incubated for 12 h. RNA was extracted from the cultured cells, RNA-Seq was performed, and pathway analysis was conducted. RESULTS: Differentially expressed genes were detected for 699 (over 2-fold increase) and 348 (decrease) genes in group E2 and for 1448 (increase) and 924 (decrease) genes in group P4 compared with those in the control group (FDR <0.05, n = 4). The z-scores of the pathways suggest that E2 and P4 increased the activity of the wound healing signaling pathway. The activation of this pathway was higher in the E2 and P4 groups than that in the control group. CONCLUSIONS: The results of this study suggest that estradiol and progesterone may affect gingival homeostasis and wound healing.

Passaging of gingival fibroblasts from periodontally healthy and diseased sites upregulates osteogenesis-related genes.

To investigate biological processes of the periodontium, in vitro primary cell models have been established. To study the biology of the gingiva, primary gingival fibroblast cell models are widely used. For such experiments, cells need to be expanded and passaged. A key assumption is that primary cells maintain most of their original characteristics they have in situ. The aim of this research is to explore the impact of early passaging on selected gene expression of human gingival fibroblast cells. For this purpose, gene expression from the outgrowth of the resected tissues until the fourth passage was followed for nine tissue samples, from both healthy and diseased sites. Micrographs were taken from the cultures, RNA was extracted from the samples of each passage and quantitative PCR was performed for selected genes representing various biological processes. Epithelial cells were present during the first outgrowth, but were no longer present in the second passage. Our results indicate that the morphology of the gingival fibroblast cells does not change with passaging and that passages 2-4 contain only gingival fibroblasts. Gene expression of M-CSF, TNF-α, TLR4, POSTN and FAPα was unchanged by passaging, the expression of IL-6, IL-1ß and TLR2 decreased due to passaging and the expression of in particular the selected osteogenesis genes (ALP, RUNX2, Osteonectin, COL1A), OPG and MKI67 increased with passaging. Worldwide, use of the same passage in laboratory experiments using primary cell cultures is the standard. Our results support this, since for certain genes, in particular osteogenesis genes, expression may alter solely due to passaging.

Non-Invasive Physical Plasma Reduces the Inflammatory Response in Microbially Prestimulated Human Gingival Fibroblasts.

Non-invasive physical plasma (NIPP), an electrically conductive gas, is playing an increasingly important role in medicine due to its antimicrobial and regenerative properties. However, NIPP is not yet well established in dentistry, although it has promising potential, especially for periodontological applications. The aim of the present study was to investigate the effect of NIPP on a commercially available human gingival fibroblast (HGF) cell line and primary HGFs in the presence of periodontitis-associated bacteria. First, primary HGFs from eight patients were characterised by immunofluorescence, and cell numbers were examined by an automatic cell counter over 5 days. Then, HGFs that were preincubated with Fusobacterium nucleatum (F.n.) were treated with NIPP. Afterwards, the IL-6 and IL-8 levels in the cell supernatants were determined by ELISA. In HGFs, F.n. caused a significant increase in IL-6 and IL-8, and this F.n.-induced upregulation of both cytokines was counteracted by NIPP, suggesting a beneficial effect of physical plasma on periodontal cells in a microbial environment. The application of NIPP in periodontal therapy could therefore represent a novel and promising strategy and deserves further investigation.

miRNAs from Inflamed Gingiva Link Gene Signaling to Increased MET Expression.

Several array-based microRNA (miRNA) expression studies independently showed increased expression of miRNAs hsa-miR-130a-3p, -142-3p, -144-3p, -144-5p, -223-3p, -17-5p, and -30e-5p in gingiva affected by periodontal inflammation. We aimed to determine direct target genes and signaling pathways regulated by these miRNAs to identify processes relevant to gingival inflammatory responses and tissue homeostasis. We transfected miRNA mimics (mirVana) for each of the 7 miRNAs separately into human primary gingival fibroblasts cultured from 3 different donors. Following RNA sequencing, differential gene expression and second-generation gene set enrichment analyses were performed. miRNA inhibition and upregulation was validated at the transcript and protein levels using quantitative reverse transcriptase polymerase chain reaction, Western blotting, and reporter gene assays. All 7 miRNAs significantly increased expression of the gene MET proto-oncogene, receptor tyrosine kinase (MET). Expression of known periodontitis risk genes CPEB1, ABCA1, and ATP6V1C1 was significantly repressed by hsa-miR-130a-3p, -144-3p, and -144-5p, respectively. The genes WASL, ENPP5, ARL6IP1, and IDH1 showed the most significant and strongest downregulation after hsa-miR-142-3p, -17-5p, -223-3p, and -30e-5p transfection, respectively. The most significantly regulated gene set of each miRNA related to cell cycle (hsa-miRNA-144-3p and -5p [Padj = 4 × 10-40 and Padj = 4 × 10-6], -miR-17-5p [Padj = 9.5 × 10-23], -miR-30e-5p [Padj = 8.2 × 10-18], -miR-130a-3p [Padj = 5 × 10-15]), integrin cell surface interaction (-miR-223-3p [Padj = 2.4 × 10-7]), and interferon signaling (-miR-142-3p [Padj = 5 × 10-11]). At the end of acute inflammation, gingival miRNAs bring together complex regulatory networks that lead to increased expression of the gene MET. This underscores the importance of mesenchymal cell migration and invasion during gingival tissue remodeling and proliferation in restoring periodontal tissue homeostasis after active inflammation. MET, a receptor of the mitogenic hepatocyte growth factor fibroblast secreted, is a core gene of this process.

Pomegranate Peel Extract Differently Modulates Gene Expression in Gingiva-Derived Mesenchymal Stromal Cells under Physiological and Inflammatory Conditions.

Pomegranate has shown a favorable effect on gingivitis/periodontitis, but the mechanisms involved are poorly understood. The aim of this study was to test the effect of pomegranate peel extract (PoPEx) on gingiva-derived mesenchymal stromal cells (GMSCs) under physiological and inflammatory conditions. GMSC lines from healthy (H) and periodontitis (P) gingiva (n = 3 of each) were established. The lines were treated with two non-toxic concentrations of PoPEX (low-10; high-40 µg/mL), with or without additional lipopolysaccharide (LPS) stimulation. Twenty-four genes in GMSCs involved in different functions were examined using real-time polymerase chain reaction (RT-PCR). PoPEx (mostly at higher concentrations) inhibited the basal expression of IL-6, MCP-1, GRO-α, RANTES, IP-10, HIF-1α, SDF-1, and HGF but increased the expression of IL-8, TLR3, TGF-ß, TGF-ß/LAP ratio, IDO-1, and IGFB4 genes in H-GMSCs. PoPEx increased IL-6, RANTES, MMP3, and BMP2 but inhibited TLR2 and GRO-α gene expression in P-GMSCs. LPS upregulated genes for proinflammatory cytokines and chemokines, tissue regeneration/repair (MMP3, IGFBP4, HGF), and immunomodulation (IP-10, RANTES, IDO-1, TLR3, COX-2), more strongly in P-GMSCs. PoPEx also potentiated most genes' expression in LPS-stimulated P-GMSCs, including upregulation of osteoblastic genes (RUNX2, BMP2, COL1A1, and OPG), simultaneously inhibiting cell proliferation. In conclusion, the modulatory effects of PoPEx on gene expression in GMSCs are complex and dependent on applied concentrations, GMSC type, and LPS stimulation. Generally, the effect is more pronounced in inflammation-simulating conditions.

Free titanium particles and P. gingivalis lipopolysaccharide create a potentially synergistical effect in a periimplantitis model.

OBJECTIVE: Our aim was to examine the effect of titanium particles and lipopolysaccharide (LPS) from P. gingivalis on the inflammatory profile expression of human gingival fibroblasts (hGFs), cultured on rough titanium discs, in an in vitro peri-implantitis simulation. DESIGN: Human gingival fibroblasts cultured on SLA and TCP surfaces were challenged with LPS, titanium particles or both. At 24, 48 and 72 h after treatment, MTT assay was performed to assess cell proliferation. FDA/PI staining was performed for the same time periods, in order to evaluate cell viability/apoptosis. At 5 and 7 days after the treatment, qPCR was performed to assess gene expressions of IL-6, IL-8 and COL1A1, as well as SEM on titanium discs. RESULTS: All groups presented a significant increase of their population between the time periods of examination. Regarding the interleukin gene expression, the combination of LPS and particles significantly increased the levels of Interleukin-8. Treatment with LPS and particles also induced a significant increase of Interleukin-6 and collagen. FDA/PI microscopy has revealed several apoptotic cells in the treatment groups. SEM micrographs have shown the difficulty of hGFs to adhere on rough surfaces. CONCLUSIONS: The combination of titanium particles and LPS significantly upregulated the expression of IL-6, IL-8 and Col-1a. It appears that particles may arouse similar reactions to the endotoxin, while synergistically intensifying it.

P38α contributes to TNF-α-induced IL-8 production in human gingival cells.

Tumor necrosis factor-alpha (TNF-α) is a major inflammatory cytokine that induces interleukin (IL)-8 production. Although some studies have reported the involvement of the p38 MAPK signaling pathway in TNF-α-induced IL-8 production, its specific regulatory mechanisms in gingival epithelial cells (GECs) are still poorly understood. In the present study, Ca9-22 cells were used as representative GECs to investigate the effect of p38 signaling on TNF-α-induced IL-8 production. We found that TNF-α enhanced IL-8 production in Ca9-22 cells by activating the p38 signaling pathway, and one of its isoforms, p38α, played a key role. P38α deletion markedly inhibited TNF-α-induced IL-8 expression in Ca9-22 cells, while p38α gene rescue could reverse this effect. Further studies revealed that TNF-α-induced IL-8 production was markedly reduced when the threonine 180 and tyrosine 182 p38α phosphorylation sites were targeted for mutagenesis to alanine and phenylalanine, respectively, suggesting their critical role in the process. In conclusion, p38α plays an important role in TNF-α-induced IL-8 production, providing a potential therapeutic target to prevent and treat periodontal disease.

Human ß-defensins are correlated with the immune infiltration and regulated by vitamin D3 in periodontitis.

OBJECTIVE: Exploring the correlation between human ß-defensins (HBDs) and immune infiltration in periodontitis, and whether it is regulated by vitamin D3 . BACKGROUND: The human body produces essential antimicrobial peptides called HBDs, which are associated with periodontitis. There is a strong link between periodontal tissue destruction and the immune cell infiltration. Moreover, vitamin D3 has been reported to regulate the expression of immune cell chemokines. However, the relationship between vitamin D3 , HBDs, and immune infiltration in periodontitis remains to be investigated. METHODS: The Gene Expression Omnibus database was accessed to obtain transcriptomic information of gingival samples taken from periodontitis patients. The expression value of HBD-2 and HBD-3 was calculated. Additionally, using the online program ImmuCellAl, 10 immune cells were scored for immune infiltration in the high-HBDs-expression group and the low-HBDs-expression group, separately. After that, transcriptome sequencing was done based on human gingival fibroblasts that had received vitamin D3 treatment. Furthermore, hGFs were treated by vitamin D3 , tumor necrosis factor-α (TNF-α), and Porphyromonas gingivalis lipopolysaccharide (Pg-LPS). The expressions of HBD-2, HBD-3, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) were detected. To seek the potential mechanism, CYP27A1 siRNA was employed to reduce the expression of CYP27A1, and nuclear factor-gene binding protein 65 (NF-κB p65) was examined. RESULTS: In GSE10334, the expressions of HBD-2 and HBD-3 were down-regulated in periodontitis group. Meanwhile, monocyte, macrophage, and CD4_T cell were less infiltrated in low-HBD-2-expression group, while less Gamma-delta T-cell infiltration was found in low-HBD-3-expression group. Transcriptome sequencing found that 21 genes were significantly expressed, of which the function was enriched in response to bacterial origin and TNF signal pathway. Vitamin D3 could significantly up-regulate the expression of HBD-2 and HBD-3, which could be controlled by knocking down CYP27A1 mRNA expression. With prolonged vitamin D3 stimulation, the expression of HBD-2 and HBD-3 increased. TNF-α/Pg-LPS could significantly increase the expression of HBD-2, HBD-3, IL-8, MCP-1, and p65, all of which were reduced by vitamin D3 . CONCLUSION: HBDs are correlated with immune infiltration in periodontitis. Vitamin D3 inhibits the expression of HBDs and chemokines induced by TNF-α/Pg-LPS, possibly through NF-κB pathway, in human gingival fibroblasts.

Influence of clinical zirconia surface treatments on microscopic characteristics and adhesion-proliferation behavior of human gingival fibroblasts.

Zirconia is favored in dental implant applications due to its biocompatibility, mechanical properties, and esthetic appeal, particularly in its interaction with soft oral tissues such as the gingiva. To optimize zirconia for clinical use, surface treatments like sanding and polishing are essential. The aim of this study was to investigate the effects of clinical surface treatments on the microscopic characteristics of zirconia and the adhesion and proliferation of human gingival fibroblasts (HGFs). Scanning electron microscopy (SEM) and fluorescence microscopy were utilized to examine the microscopic morphology and roughness resulting from various clinical surface treatment procedures on zirconia and to assess their impact on the microscopic appearance and behavior of HGFs. The results showed that the application of surface treatment procedures, particularly polishing treatments, resulted in the formation of a regular shallow groove morphology and a significant reduction in roughness in zirconia. This was accompanied by improved cell proliferation, cell adhesion, and the expression of integrin ß1 in HGFs. The results suggest that smoother zirconia surfaces promote better cell-material interactions, potentially improving the clinical success of dental implants. This research contributes to our understanding of the optimal surface roughness for soft tissue adhesion and the effect of different micro-morphologies on HGF attachment.

Macrophages induce gingival destruction via Piezo1-mediated MMPs-degrading collagens in periodontitis.

Macrophages are an integral part of the innate immune response in periodontal tissue and play a crucial role in the progression of periodontitis. Here we reported that macrophages also provoke periodontitis-induced gingival destruction through Piezol-mediated collagen degradation. We discovered that the PIEZO1 expression was markedly elevated in patients with periodontitis through transcriptomic profiling. Moreover, Piezo1 promoted macrophage polarization toward the M1 type in response to lipopolysaccharide (LPS) and induced production of proinflammatory cytokines, which in turn stimulated production of matrix metalloproteinases (MMPs) leading to collagen degradation. Our study suggests that Piezol might be a potential therapeutic target for treating periodontitis-induced gingival destruction.

Interleukin-22 Inhibits Apoptosis of Gingival Epithelial Cells Through TGF-ß Signaling Pathway During Periodontitis.

Periodontitis is a chronic inflammatory disease characterized by the destruction of tooth-supporting tissues. The gingival epithelium is the first barrier of periodontal tissue against oral pathogens and harmful substances. The structure and function of epithelial lining are essential for maintaining the integrity of the epithelial barrier. Abnormal apoptosis can lead to the decrease of functional keratinocytes and break homeostasis in gingival epithelium. Interleukin-22 is a cytokine that plays an important role in epithelial homeostasis in intestinal epithelium, inducing proliferation and inhibiting apoptosis, but its role in gingival epithelium is poorly understood. In this study, we investigated the effect of interleukin-22 on apoptosis of gingival epithelial cells during periodontitis. Interleukin-22 topical injection and Il22 gene knockout were performed in experimental periodontitis mice. Human gingival epithelial cells were co-cultured with Porphyromonas gingivalis with interleukin-22 treatment. We found that interleukin-22 inhibited apoptosis of gingival epithelial cells during periodontitis in vivo and in vitro, decreasing Bax expression and increasing Bcl-xL expression. As for the underlying mechanisms, we found that interleukin-22 reduced the expression of TGF-ß receptor type II and inhibited the phosphorylation of Smad2 in gingival epithelial cells during periodontitis. Blockage of TGF-ß receptors attenuated apoptosis induced by Porphyromonas gingivalis and increased Bcl-xL expression stimulated by interleukin-22. These results confirmed the inhibitory effect of interleukin-22 on apoptosis of gingival epithelial cells and revealed the involvement of TGF-ß signaling pathway in gingival epithelial cell apoptosis during periodontitis.

Stem-cell based soft tissue substitutes: Engineering of crosslinked polylysine-hyaluronic acid microspheres ladened with gingival mesenchymal stem cells for collagen tissue regeneration and angiogenesis.

BACKGROUND: The aim of this study was to construct crosslinked polylysine-hyaluronic acid microspheres (pl-HAM) ladened with gingival mesenchymal stem cells (GMSCs) and explore its biologic behavior in soft tissue regeneration. METHODS: The effects of the crosslinked pl-HAM on the biocompatibility and the recruitment of L-929 cells and GMSCs were detected in vitro. Moreover, the regeneration of subcutaneous collagen tissue, angiogenesis and the endogenous stem cells recruitment were investigated in vivo. We also detected the cell developing capability of pl-HAMs. RESULTS: The crosslinked pl-HAMs appeared to be completely spherical-shaped particles and had good biocompatibility. L-929 cells and GMSCs grew around the pl-HAMs and increased gradually. Cell migration experiments showed that pl-HAMs combined with GMSCs could promote the migration of vascular endothelial cells significantly. Meanwhile, the green fluorescent protein-GMSCs in the pl-HAM group still remain in the soft tissue regeneration area 2 weeks after surgery. The results of in vivo studies showed that denser collagen deposition and more angiogenesis-related indicator CD31 expression in the pl-HAMs+ GMSCs + GeL group compared with the pl-HAMs + GeL group. Immunofluorescence showed that CD44, CD90, CD73 co-staining positive cells surrounded the microspheres in both pl-HAMs + GeL group and pl-HAM + GMSCs + GeL group. CONCLUSIONS: The crosslinked pl-HAM ladened with GMSCs system could provide a suitable microenvironment for collagen tissue regeneration, angiogenesis and endogenous stem cells recruitment, which may be an alternative to autogenous soft tissue grafts for minimally invasive treatments for periodontal soft tissue defects in the future.

Zoledronic acid affects the process of Porphyromonas gingivalis infecting oral mucosal epithelial barrier: An in-vivo and in-vitro study.

Zoledronic acid (ZA), one of the commonly used bisphosphonates, is mainly used for bone-metabolic diseases. Studies proved that ZA has adverse effects on oral soft tissues. As the first line of innate immunity, the gingival epithelium could be infected by periodontal pathogens, which is a key process of the initiation of periodontal diseases. Yet, how ZA affects the periodontal pathogens infecting the epithelial barrier remains unclear. This study aimed to investigate the influences of ZA on the process of Porphyromonas gingivalis (P. gingivalis) infecting the gingival epithelial barrier via in-vitro and in-vivo experiments. In the in-vitro experiments, under the condition of different concentrations of ZA (0, 1, 10, and 100 µM), P. gingivalis was used to infect human gingival epithelial cells (HGECs). The infections were detected by transmission electron microscope and confocal laser scanning microscope. Besides, the internalization assay was applied to quantify the P. gingivalis, which infected the HGECs, in the different groups. To evaluate the expression levels of pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-6, and IL-8, by infected HGECs, real-time quantitative reverse transcription-polymerase chain reactions were applied. In the in-vivo experiments, rats were given ZA solution (ZA group) or saline (control group) by tail intravenous injection for 8 weeks. Subsequently, we put ligatures around the maxillary second molars of all the rats and inoculated P. gingivalis to the gingiva every other day from day 1 to day 13. The rats were sacrificed on days 3, 7, and 14 for micro-CT and histological analyses. The in-vitro results manifested that the quantity of P. gingivalis that had infected HGECs increased with the ZA concentrations. Pro-inflammatory cytokines expression by HGECs were significantly increased by 100 µM ZA. In the in-vivo study, compared to the control group, more P. gingivalis was detected in the superficial layer of gingival epithelium in the ZA group. Besides, ZA significantly increased the expression level of IL-1ß on day 14 and IL-6 on days 7 and 14 in gingival tissues. These findings suggest that the oral epithelial tissues of patients who receive high-dose ZA treatment may be more susceptible to periodontal infections, resulting in severe inflammatory conditions.

The neutralising and stimulatory effects of antimicrobial peptide LL-37 in human gingival fibroblasts.

OBJECTIVES: To investigate the effects of LL-37, a broad spectrum antimicrobial peptide expressed in periodontal tissues, on human gingival fibroblast responsiveness to microbial challenge and to explore the direct effects of LL-37 on human gingival fibroblasts. DESIGN: The effect of LL-37 on bacterial lipopolysaccharide-induced expression of Interleukin (IL-6) and chemokine C-X-C motif ligand (CXCL) 8 was determined by enzyme linked immunosorbent assay (ELISA). LL-37's influence on bacterial lipopolysaccharide-induced IκBα degradation was investigated by western blot. DNA microarray analysis initially determined the direct effects of LL-37 on gene expression, these findings were subsequently confirmed by quantitative polymerase chain reaction and ELISA analysis of selected genes. RESULTS: Bacterial lipopolysaccharide-induced IL-6 and CXCL8 production by human gingival fibroblasts was significantly reduced in the presence of LL-37 at concentrations in the range of 1-10 µg/ml. LL-37 led to a reduction in lipopolysaccharide-induced IκBα degradation by Escherichia coli lipopolysaccharide and Porphyromonas gingivalis lipopolysaccharide (10 µg/ml). LL-37 (50 µg/ml) significantly altered the gene expression of 367 genes in human gingival fibroblasts by at least 2-fold. CXCL1, CXCL2, CXCL3, Interleukin-24 (IL-24), CXCL8, Chemokine (C-C motif) Ligand 2, and Suppressor of Cytokine Signalling 3 mRNA were significantly upregulated by LL-37. LL-37 also significantly stimulated expression of CXCL8, hepatocyte growth factor and CXCL1 at the protein level. CONCLUSION: LL-37 plays an important regulatory role in the immunomodulatory activity of gingival fibroblasts by inhibiting lipopolysaccharide -induced expression of inflammatory cytokines and directly stimulating the expression of an array of bioactive molecules involved in inflammation and repair.