Autologous hGMSC-Derived iPS: A New Proposal for Tissue Regeneration.

The high mortality in the global population due to chronic diseases highlights the urgency to identify effective alternative therapies. Regenerative medicine provides promising new approaches for this purpose, particularly in the use of induced pluripotent stem cells (iPSCs). The aim of the work is to establish a new pluripotency cell line obtained for the first time by reprogramming human gingival mesenchymal stem cells (hGMSCs) by a non-integrating method. The hGMSC-derived iPS line characterization is performed through morphological analysis with optical and electron scanning microscopy and through the pluripotency markers expression evaluation in cytofluorimetry, immunofluorescence, and RT-PCR. To confirm the pluripotency of new hGMSC-derived iPS, the formation of embryoid bodies (EBs), as an alternative to the teratoma formation test, is studied in morphological analysis and through three germ layers' markers' expression in immunofluorescence and RT-PCR. At the end, a comparative study between parental hGMSCs and derived iPS cells is performed also for the extracellular vesicles (EVs) and their miRNA content. The new hGMSC-derived iPS line demonstrated to be pluripotent in all aspects, thus representing an innovative dynamic platform for personalized tissue regeneration.

Cytotoxicity of two fluoride-releasing adhesive tapes.

Sodium fluoride-polyvinyl alcohol (NaF-PVA) tape was developed to deliver fluoride to teeth by adding fluoride to polymer tape. Previous studies have demonstrated that tapes are effective and have antimicrobial properties. This study aimed to evaluate the cytotoxicity of two fluoride-releasing adhesive tapes. We investigated two polyvinyl alcohol (PVA) tapes: (i) a fluoride-PVA (F-PVA) tape, and (ii) a pullulan-incorporated F-PVA (PF-PVA) tape. The cytotoxicity test was conducted on human gingival fibroblasts (HGF) and human periodontal ligament (PDL) cells. Using an adhesive tape containing fluoride, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on these cells. Genetic analysis of the cells was performed to conduct a stability test on humans. In the MTT assay, PF-PVA had 66% greater cytotoxicity than control by PDL and 69% by HGF. F-PVA showed less cytotoxicity than PF-PVA by 29% in PDL and 33% in HGF. Gene ontology (GO) analysis and gene set enrichment analysis (GSEA) were performed as gene expression analyses. GO analysis indicated that PF-PVA displayed more expression changes of genes related to cytotoxicity than F-PVA. In addition, GSEA found more inflammatory response associations in PF-PVA than in F-PVA. MTT and genetic testing yielded comparable results.

Dimethyl Fumarate-Loaded Gellan Gum Hydrogels Can Reduce In Vitro Chemokine Expression in Oral Cells.

Dimethyl fumarate (DMF), originally proposed to treat multiple sclerosis, is considered to have a spectrum of anti-inflammatory effects that effectively control periodontitis, mainly when applied with a hydrogel delivery system. Chemokine expression by gingival fibroblasts is a significant driver of periodontitis; thus, hydrogel-based strategies to deliver DMF, which in turn dampen chemokine expression, are of potential clinical relevance. To test this approach, we have established a bioassay where chemokine expression is induced by exposing gingival fibroblast to IL1ß and TNFα, or with saliva. We show herein that DMF effectively reduced the expression of CXCL8, CXCL1, CXCL2, and CCL2-and lowered the phosphorylation of ERK and JNK-without affecting cell viability. This observation was confirmed by immunoassays with CXCL8. Consistently, the forced chemokine expression in HSC2 oral squamous epithelial cells was greatly diminished by DMF. To implement our hydrogel-based delivery system, gingival fibroblasts were cocultured with gellan gum hydrogels enriched for DMF. In support of our strategy, DMF-enriched gellan gum hydrogels significantly reduced the forced chemokine expression in gingival fibroblasts. Our data suggest that DMF exerts its anti-inflammatory activity in periodontal cells when released from gellan gum hydrogels, suggesting a potential clinical relevance to control overshooting chemokine expression under chronic inflammatory conditions.

Mesenchymal Stem/Stromal Cells Derived from Dental Tissues Mediate the Immunoregulation of T Cells through the Purinergic Pathway.

Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to bone marrow-derived mesenchymal stem cells (BM-MSCs) for potential clinical applications because of their accessibility and anti-inflammatory capacity. We previously demonstrated that DT-MSCs from dental pulp (DP-MSCs), periodontal ligaments (PDL-MSCs), and gingival tissue (G-MSCs) show immunosuppressive effects similar to those of BM, but to date, the DT-MSC-mediated immunoregulation of T lymphocytes through the purinergic pathway remains unknown. In the present study, we compared DP-MSCs, PDL-MSCs, and G-MSCs in terms of CD26, CD39, and CD73 expression; their ability to generate adenosine (ADO) from ATP and AMP; and whether the concentrations of ADO that they generate induce an immunomodulatory effect on T lymphocytes. BM-MSCs were included as the gold standard. Our results show that DT-MSCs present similar characteristics among the different sources analyzed in terms of the properties evaluated; however, interestingly, they express more CD39 than BM-MSCs; therefore, they generate more ADO from ATP. In contrast to those produced by BM-MSCs, the concentrations of ADO produced by DT-MSCs from ATP inhibited the proliferation of CD3+ T cells and promoted the generation of CD4+CD25+FoxP3+CD39+CD73+ Tregs and Th17+CD39+ lymphocytes. Our data suggest that DT-MSCs utilize the adenosinergic pathway as an immunomodulatory mechanism and that this mechanism is more efficient than that of BM-MSCs.

Developing a novel calcium silver zeolite for caries management.

OBJECTIVE: To develop a novel calcium silver zeolite (Ca-Ag-Zeo) and assess its biocompatibility, physiochemical properties and antimicrobial effects. METHODS: Ca-Ag-Zeo was synthesized using ion-exchange method with calcium chloride, silver nitrate and Zeolite X (Zeo). Silver zeolite X (Ag-Zeo) and Zeo were set as control. The chemical structure, morphology, crystal structure and elemental composition of Ca-Ag-Zeo was characterized by X-ray diffraction spectrum, scanning electron microscopy, transmission electron microscopy and energy dispersive spectroscopy, respectively. Its biocompatibility on the human gingival fibroblasts was assessed by cell counting kit-8 assay. Its physiochemical properties were determined by the released calcium and silver ion using Inductive Coupled Plasma Emission Spectrometry for up to 12 weeks. The antimicrobial properties on Streptococcus mutans, Lactobacillus acidophilus, Lactobacillus casei, and Candida albicans were assessed by minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) assay. RESULTS: Ca-Ag-Zeo with a hexagonal cage structure was synthesized. As for biocompatibility, the half-maximal inhibitory concentration (± SD in mg/mL) of Ca-Ag-Zeo, Ag-Zeo and Zeo in human gingival fibroblasts were 0.52 ± 0.05, 0.15 ± 0.01 and 3.35 ± 0.58, respectively (Zeo > Ca-Ag-Zeo > Ag-Zeo; p < 0.05). As for physiochemical properties, the accumulated ion release (± SD in mg) of Ca-Ag-Zeo, Ag-Zeo and Zeo were 0.011 ± 0.003, 0 and 0 for calcium ion, respectively (Ca-Ag-Zeo > Ag-Zeo, Zeo; p < 0.001), and 0.213 ± 0.032, 0.209 ± 0.019 and 0 for silver ion, respectively (Ca-Ag-Zeo, Ag-Zeo > Zeo; p < 0.001). As for anti-microbial ability, the MBC/MFC (mg/mL) of Ca-Ag-Zeo, Ag-Zeo and Zeo were 32, 16 and > 256 against Streptococcus mutans; 32, 16, > 256 against Lactobacillus acidophilus; 16, 16, and 256 against Lactobacillus casei; 0.25, 0.125; and 2, 1, > 256 against Candida albicans, respectively. CONCLUSION: A novel Ca-Ag-Zeo was developed. It presented better biocompatibility compared to Ag-Zeo. It released calcium and silver ions sustainably, and it could inhibit the growth of common cariogenic microorganisms.

RNAseq of Gingival Fibroblasts Exposed to PRF Membrane Lysates and PRF Serum.

Platelet-rich fibrin (PRF) is prepared by spontaneous coagulation of fractionated blood. When squeezed between two plates, PRF is separated into solid PRF membranes and a liquid exudate, the PRF serum. The question arises regarding how much the overall activity remains in the PRF membranes and what is discarded into the PRF serum. To this end, we have exposed gingival fibroblasts to lysates prepared from PRF membranes and PRF serum, followed by bulk RNA sequencing. A total of 268 up- and 136 down-regulated genes in gingival fibroblasts exposed to PRF membrane lysates were significantly regulated under the premise of a minimum log2 with 2.5-fold change and a minus log10 significance level of two, respectively. PRF serum only caused 62 up- and 32 down-regulated genes under these conditions. Among the 46 commonly up-regulated genes were CXCL1, CXCL5, CXCL6, CXCL8, IL33, IL6, and PTGS2/COX2, stanniocalcin-1-all linked to an inflammatory response. PRF membrane lysates further increased chemokines CCL2, CCL7, CXCL2, CXCL3, and IL1R1, IL1RL1, and IL1RN, as well as the paracrine factors IL11, LIF, IGF1, BMP2, BMP6, FGF2, and CCN2/CTGF, and all hyaluronan synthases. On the other hand, PRF serum increased DKK1. The genes commonly down-regulated by PRF membrane lysates and PRF serum included interferon-induced protein with tetratricopeptide repeats (IFIT1, IFIT2, IFIT3) and odd-skipped-related transcription factors (OSR1 and OSR2), as well as FGF18 and GDF15, respectively. Taken together, PRF membrane lysates, compared to PRF serum, cause a more complex response in gingival fibroblasts, but each increased chemokine expression in gingival fibroblasts.

Combining sandblasting and pink anodisation of Ti implants as a promising method for improving fibroblast adhesion and immune response.

This study examined the effect of combining the sandblasting and anodising of titanium alloys used in implants on the cell response and protein adsorption patterns. The titanium samples were divided into four groups depending on the surface treatment: machining (MC), pink anodisation (PA), sandblasting (MC04) and a combination of the last two (MC04 + PA). Their physicochemical properties were analysed by SEM/EDX, Raman, contact angle measurements and profilometry. In vitro responses were examined using human gingival fibroblastic (HGF) cells and THP-1 macrophages. Cytokine secretion, macrophage adhesion and gene expression were measured by ELISA, confocal microscopy and RT-PCR. Cell adhesion and collagen secretion were evaluated in HGF cultures. The adsorption of immune and regenerative proteins onto the surfaces was assessed employing nLC-MS/MS. MC04 + PA surfaces exhibited a change in the roughness, chemical composition and hydrophilicity of the material, showing more elongated HGF cells and a considerable increase in the area of cells exposed to the MC04 + PA surfaces. Moreover, cells cultured on MC04 + PA generally showed a reduction in the expression of proinflammatory genes (TNF-α, MCP-1, C5, NF-kB and ICAM-1) and an increase in the secretion of anti-inflammatory cytokines, such as IL-4. These results correlated with the proteomic data; we found preferential adsorption of proteins favouring cell adhesion, such as DSC1 and PCOC1. A considerable reduction in the adsorption of immunoglobulins and proteins associated with acute inflammatory response (including SAA4) was also observed. The study highlights the potential advantages of MC04 + PA surface treatment to modify dental implant abutments; it enhances their compatibility with soft tissues and reduces the inflammatory response.

Osteogenic Differentiation of Human Gingival Fibroblasts Inhibits Osteoclast Formation.

Gingival fibroblasts (GFs) can differentiate into osteoblast-like cells and induce osteoclast precursors to differentiate into osteoclasts. As it is unclear whether these two processes influence each other, we investigated how osteogenic differentiation of GFs affects their osteoclast-inducing capacity. To establish step-wise mineralization, GFs were cultured in four groups for 3 weeks, without or with osteogenic medium for the final 1, 2, or all 3 weeks. The mineralization was assessed by ALP activity, calcium concentration, scanning electron microscopy (SEM), Alizarin Red staining, and quantitative PCR (qPCR). To induce osteoclast differentiation, these cultures were then co-cultured for a further 3 weeks with peripheral blood mononuclear cells (PBMCs) containing osteoclast precursors. Osteoclast formation was assessed at different timepoints with qPCR, enzyme-linked immunosorbent assay (ELISA), TRAcP activity, and staining. ALP activity and calcium concentration increased significantly over time. As confirmed with the Alizarin Red staining, SEM images showed that the mineralization process occurred over time. Osteoclast numbers decreased in the GF cultures that had undergone osteogenesis. TNF-α secretion, a costimulatory molecule for osteoclast differentiation, was highest in the control group. GFs can differentiate into osteoblast-like cells and their degree of differentiation reduces their osteoclast-inducing capacity, indicating that, with appropriate stimulation, GFs could be used in regenerative periodontal treatments.

[Study on the protection of gingival epithelial barrier by interleukin-22 through regulating microbiota and E-cadherin expression].

Objective: To investigate the regulatory effect and mechanism of interleukin-22 (IL-22) on the gingival epithelial barrier in the context of periodontal inflammation. Methods: IL-22 knockout (IL-22 KO) mice were constructed, and periodontitis mice models were established through oral gavage with polymicrobial inoculation. DNAs were extracted from the oral plaques of IL-22 KO periodontitis mice group (n=7) and their wild-type littermates periodontitis group (n=7) to establish a periodontitis-related oral microbiota database"PD-RiskMicroDB", determining the relationship between changes in oral microbiota and microbial function in two groups using 16S rRNA sequencing results. Gingival epithelial cells (GEC) were cultured by modified trypsinization method, and were stimulated with 100 µg/L IL-22, Porphyromonas gingivalis (Pg) (multiplicity of infection:100), separately or together for 3 and 12 hours. The experimental groups were as follows: control group (no stimulation), IL-22 group, Pg group and Pg+IL-22 group. The expression of barrier protein E-cadherin in each group at 3 h was detected by immunofluorescence, real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. Fluorescein isothiocyanate-dextran-mediated epithelial cell permeability experiment was conducted to clarify the changes in permeability of GEC in each group at 3 and 12 h. The mRNA expressions of E-cadherin in the gingival epithelium of wild-type littermates periodontitis group and IL-22 KO periodontitis group were detected by RT-qPCR. Fifteen C57BL/6 wild-type mice were randomly divided into control group (n=5), periodontitis group (n=5) and periodontitis+IL-22 treatment group (n=5). RT-qPCR and immunohistochemistry (IHC) staining were used to detect the expression level of E-cadherin in the gingival epithelium of each group. Results: 16S rRNA sequencing results showed that the composition of oral microbiota changed in IL-22 KO periodontitis group, of which the abundance of bacterial genera related to periodontal tissue invasion was significantly increased (linear discriminant analysis score: 2.22, P=0.009), compared with wild-type littermates periodontitis group. In vitro cell experiments showed that after Pg infection for 3 hours, the cell connections of GEC in Pg group were interrupted, and the fluorescence intensity of E-cadherin was reduced in Pg group compared with the control group. Meanwhile, the mRNA and protein expression levels of E-cadherin (mRNA: 0.69±0.12; protein: 0.60±0.12) were downregulated compared with the control group [mRNA: 1.00±0.00 (P=0.043); protein: 1.04±0.08 (P=0.003)], respectively. The fluorescence intensity of E-cadherin in the Pg+IL-22 group was enhanced compared with Pg group, and expression levels of E-cadherin mRNA (1.16±0.10) and protein (0.98±0.07) in Pg+IL-22 group showed a significant increase compared with Pg group [mRNA: 0.69±0.12 (P=0.005); protein: 0.60±0.12 (P=0.007)]. The result of epithelial permeability test showed that there was no statistical difference in epithelial permeability among control group, Pg group, IL-22 group and Pg+IL-22 group with treatment for 3 hours (F=0.20, P=0.893). While when the treatment time turned to be 12 hours, the epithelial barrier permeability showed a significant increase in Pg group (1.39±0.15) compared with control group (1.00±0.00, P=0.027), and a decrease in Pg+IL-22 group (1.02±0.18) compared with Pg group (1.39±0.15, P=0.034). In vivo, the mRNA expression of E-cadherin in the gingival epithelium of IL-22 KO periodontitis group decreased significantly (0.32±0.21) compared with wild-type littermates periodontitis group (1.01±0.01) (t=5.70, P=0.005). Moreover, RT-qPCR and IHC staining results showed that the mRNA expression level of E-cadherin (0.40±0.07) and absorbance value of E-cadherin positive expression (0.02±0.00) in gingival epithelial tissue of periodontitis group were both significantly down-regulated compared with control group [mRNA: 1.00±0.00 (P=0.005); absorbance value of E-cadherin positive expression: 0.04±0.01 (P=0.006)]. Meanwhile, the mRNA expression level of E-cadherin (1.06±0.24) and the absorbance value of E-cadherin positive expression (0.03±0.01) were both observed increase in periodontitis+IL-22 treatment group compared with periodontitis group (P=0.003, P=0.039). Conclusions: IL-22 may exert a protective effect on the gingival epithelial barrier in an inflammatory environment by regulating the invasiveness of oral microbiota and the expression of host barrier protein.

Roughness affects the response of human fibroblasts and macrophages to sandblasted abutments.

BACKGROUND: A strong seal of soft-tissue around dental implants is essential to block pathogens from entering the peri-implant interface and prevent infections. Therefore, the integration of soft-tissue poses a challenge in implant-prosthetic procedures, prompting a focus on the interface between peri-implant soft-tissues and the transmucosal component. The aim of this study was to analyse the effects of sandblasted roughness levels on in vitro soft-tissue healing around dental implant abutments. In parallel, proteomic techniques were applied to study the interaction of these surfaces with human serum proteins to evaluate their potential to promote soft-tissue regeneration. RESULTS: Grade-5 machined titanium discs (MC) underwent sandblasting with alumina particles of two sizes (4 and 8 µm), resulting in two different surface types: MC04 and MC08. Surface morphology and roughness were characterised employing scanning electron microscopy and optical profilometry. Cell adhesion and collagen synthesis, as well as immune responses, were assessed using human gingival fibroblasts (hGF) and macrophages (THP-1), respectively. The profiles of protein adsorption to the surfaces were characterised using proteomics; samples were incubated with human serum, and the adsorbed proteins analysed employing nLC-MS/MS. hGFs exposed to MC04 showed decreased cell area compared to MC, while no differences were found for MC08. hGF collagen synthesis increased after 7 days for MC08. THP-1 macrophages cultured on MC04 and MC08 showed a reduced TNF-α and increased IL-4 secretion. Thus, the sandblasted topography led a reduction in the immune/inflammatory response. One hundred seventy-six distinct proteins adsorbed on the surfaces were identified. Differentially adsorbed proteins were associated with immune response, blood coagulation, angiogenesis, fibrinolysis and tissue regeneration. CONCLUSIONS: Increased roughness through MC08 treatment resulted in increased collagen synthesis in hGF and resulted in a reduction in the surface immune response in human macrophages. These results correlate with the changes in protein adsorption on the surfaces observed through proteomics.

Antiproliferative effect of low-level laser/ photobiomodulation on gingival fibroblasts derived from calcium channel blocker-induced gingival overgrowth.

The aim of this study was to evaluate the antiproliferative properties of low-level laser therapy (LLLT) on gingival fibroblasts obtained from calcium channel blocker-induced gingival overgrowth (GO). Gingival fibroblasts of patients with GO were compared to healthy gingival fibroblasts (H). Both cells were exposed to LLLT (685 nm wavelength, 25mW power, diode laser) and compared to those not treated with LLLT. Cell proliferation and viability were measured with MTT assay at baseline and after 24 and 72 h. TGF-ß1, CTGF, and collagen Type 1 levels were evaluated with Enzyme-Linked Immunosorbent Assay (ELISA). LLLT significantly decreased the proliferation of GO fibroblasts (p < 0.05) while leading to a significantly higher proliferation in H fibroblasts compared to the untreated cells (p < 0.05). GO cells showed significantly higher CTGF, TGF-ß, and collagen Type 1 expression than the H cells (p < 0.05). LLLT significantly reduced CTGF levels in GO cells compared to the control group (p < 0.05). In H cells, CTGF and TGF-ß levels were also significantly decreased in response to LLLT compared to the control group (p < 0.05). While LLLT significantly reduced collagen expression in the H group (p < 0.05), it did not significantly impact the GO cells. LLLT significantly reduced the synthesis of the growth factors and collagen in both groups with an antiproliferative effect on the gingival fibroblasts from calcium channel blocker-induced GO, suggesting that it can offer a therapeutic approach in the clinical management of drug-induced GO, reversing the fibrotic changes.

[An in vitro experiment on the stability and irritant of hypochlorous acid in oral cavity].

PURPOSE: To study the stability of physicochemical properties and sterilizing effect about two commercially available hypochlorous acid (HClO) products under simulated clinical conditions, and to evaluate the compatibility of HClO on soft and hard tissues and cells in oral cavity. METHODS: Samples of HClO solution with different production processes were prepared, to detect the changes of physicochemical indexes of each sample over time under simulated clinical conditions (shielded from light at 20-25 ℃, open the cover for 5 minutes every day), including free available chlorine, oxidation-reduction potential and pH. Through suspension quantitative germicidal test, the antibiosis-concentration curve of HClO solution was made, so as to calibrate the change of antibacterial ability of disinfectant with the decrease of available chlorine content during storage. Pulp, tongue and dentine were immersed in PBS, 100 ppm HClO, 200 ppm HClO and 3% NaClO. The influence on soft and hard tissues was evaluated by weighing method and microhardness test. The toxic effects of HClO, NaClO and their 10-fold diluent on human gingival fibroblasts were determined by CCK-8 cytotoxicity assay. GraphPad PRIS 8.0 software was used to analyze the data. RESULTS: Under simulated conditions, the free available chlorine (FAC) of HClO solution decayed with time, and the attenuation degree was less than 20 ppm within 1 month. The bactericidal effect of each HClO sample was still higher than 5log after concentration decay. There was no obvious dissolution and destruction to soft and hard tissues for HClO(P＞0.05). The cell viability of HClO to human gingival fibroblast cells (HGFC) was greater than 80%, which was much higher than 3% NaClO (P＜0.001). CONCLUSIONS: The bactericidal effect and stability of HClO solution can meet clinical needs, which has low cytotoxicity and good histocompatibility. It is expected to become a safe and efficient disinfection product in the field of living pulp preservation and dental pulp regeneration.

Investigating adhesion of primary human gingival fibroblasts and osteoblasts to orthodontic mini-implants by scanning electron microscopy.

Miniscrews offer controlled anchorage and thus optimize tooth movement in orthodontic treatment. Nevertheless, failures such as soft tissue problems, instability due to loosening, partial osseointegration, or even device fracture can occur. While clinical technique can play a role in some of these problems, the manufacturer's design and material choice influence how the implant interacts with the surrounding tissue. In some cases, the design and material may trigger unwanted bone and soft tissue responses. This in vitro study investigates how the implant surface affects cell adhesion and growth of human primary fibroblasts and osteoblasts on commercially available orthodontic TiAl6V4 miniscrews from three producers: tomas-pin SD N 08 (Dentaurum), OrthoEasy Pin (Forestadent), and Dual Top G2 (Promedia, Jeil Medical). Cell-implant interaction at the top, neck, and drilling part of the screws was assessed qualitatively by scanning electron microscopy. While both cell types adhered to and grew on all products, subtle differences in cell shape and spreading were detected, depending on the microstructure of the implant surface. This indicates that cell adhesion to implant surfaces can be controlled by manipulating the machining conditions.

Anti-proliferative impact of resveratrol on gingival fibroblasts from juvenile hyaline fibromatosis.

AIM: Resveratrol is a natural polyphenolic compound with biological activities such as anti-inflammation and antioxidation. Its anti-fibrotic effect has been experimentally demonstrated in the pancreas and liver. This study aims to determine the anti-proliferative effect of resveratrol on fibroblasts obtained from hyperplastic gingival tissues from a patient diagnosed with Juvenile Hyaline Fibromatosis (JHF). MATERIALS AND METHODS: Primary gingival fibroblast cell lines were obtained from gingival growth tissues by the gingivectomy of a patient with JHF. Gingival fibroblasts were treated with or without 3 different doses of resveratrol (50, 100, 200 µM). Cytotoxicity and cell proliferation were evaluated after 24, 48, and 72 h. Collagen, TGF, and CTGF were analyzed by ELISA in the 48-hour supernatants. RESULTS: All three doses of resveratrol suppressed the proliferation of JHF gingival fibroblasts at 24 and 48 h without showing any cytotoxic effect compared to the control group (p < 0.0001). At 72 h, 100 and 200 µM resveratrol showed significantly less proliferation (p < 0.0001), less collagen, CTGF, and TGF- ß (p < 0.001) than the control group. CONCLUSION: Resveratrol had a profound anti-proliferative effect on gingival fibroblasts obtained from gingival enlargements with JHF, suggesting that it can be used as a therapeutic to prevent excessive cell growth by suppressing collagen, CTGF, and TGF- ß synthesis in the pathogenesis of hyperplasia.

A multifunctional gingival retraction cord with antibacterial and hemostasis properties based on Chitosan/Propolis/Tranexamic acid for dental treatment.

A novel gingival retraction cord named P/TA@CSy was prepared using chitosan yarns (CSy) loaded with tranexamic acid (TA) and Propolis (P). P/TA@CSy has good toughness with a breaking strength of 41.3 Pa, benefiting from the twisting structure and Propolis coating. A short coagulation time of 456 s was achieved for P/TA@CSy because of the potent blood absorption ability from the effective attachment of tranexamic acid. Moreover, excellent antibacterial ability was obtained with the antibacterial rates against E. coli of 94.73 %, S. aureus of 99.99 % and S. mutans of 99.99 %, contributing to Propolis's antibacterial ability. In addition, suppression of the expression of pro-inflammatory cytokines (IL-6 and TNF-α) was found, which could prevent wound infection. P/TA@CSy displayed excellent cytocompatibility with the cell activity of 100 % after 24 h. Therefore, P/TA@CSy could rapidly respond to gingival hemostasis and infection prevention, showing excellent potential in dental treatment.

A mechanistic study on the toluidine blue/ photodynamic therapy inhibition of lipopolysaccharide-induced inflammatory response in rat gingival fibroblasts.

The purpose of this research was to investigate the effect of toluidine blue (TB) mediated photodynamic therapy (PDT) on the inhibition of lipopolysaccharide (LPS)-induced inflammation in rat gingival fibroblasts through in vitro experiments. Rat gingival fibroblasts were divided into five groups: (1) control, (2) LPS treatment, (3) laser treatment, (4) TB treatment (1.0 µg/mL), and (5) PDT treatment (TB plus laser irradiation at 320 mW/cm2 for 240 s). After 24 h, cell growth activity was measured using MTT assay. The levels of receptor activator for nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in the cell culture supernatant were measured using enzyme-linked immunosorbent assay (ELISA). Nuclear proteins were extracted and the phosphorylation levels of phosphorylated nuclear factor-κB/p65 (p-p65) and phosphorylated inhibitor of nuclear factor-κB (p-IκBα) were determined using Western Blot. MTT results showed no significant difference in cell viability between the groups (P > 0.05). After LPS induction, OPG expression decreased, RANKL expression increased, and the OPG/RANKL ratio decreased, which was different from the control group (P < 0.05). After PDT treatment, OPG expression increased, RANKL expression decreased (P < 0.05), and the OPG/RANKL ratio increased (P < 0.05). Compared to the control group, there was no significant difference in OPG and RANKL expression or the OPG/RANKL ratio (P > 0.05). The activation of NF-κB was closely related to the phosphorylation levels of p-p65 and p-IκBα. LPS significantly up-regulated p-p65 and p-IκBα expression (P < 0.05), while PDT treatment decreased their phosphorylation levels (P < 0.05). TB-PDT treatment can inhibit NF-κB signaling pathway activation, decrease RANKL and OPG expression, and reduce the OPG/RANKL ratio, thereby reducing inflammation and playing a role in periodontitis treatment.

Streptococcus mitis enhances metal-induced apoptosis in reconstructed human gingiva but not skin.

BACKGROUND: Commensal bacteria colonizing oral mucosa and skin play an essential role in maintaining host-microbiome homeostasis. It is unknown whether cytotoxicity resulting from metal ions leaching from medical devices may be influenced by commensal microbes. OBJECTIVE: Determine whether the extent of apoptosis triggered by nickel or titanium ions is influenced by Streptococcus mitis and whether apoptosis occurs via the intrinsic or extrinsic apoptosis pathway. METHODS: Reconstructed Human Gingiva (RHG) and Skin (RHS) were topically exposed to titanium or nickel salts in the presence or absence of S. mitis. Cytotoxicity and apoptosis were assessed by histology, immunohistochemistry, TUNEL assay, and Western Blot. RESULTS: S. mitis alone resulted in negligible cytotoxicity. After metal exposure, localized apoptosis was observed in the epithelium and fibroblasts within the lamina propria hydrogel of both RHG and RHS. S. mitis enhanced metal-mediated apoptosis in gingiva but not in skin. Apoptosis was mediated via the extrinsic pathway caspase 8. Activation of the execution phase of apoptosis occurred via caspases 3 and 7, and PARP-1. CONCLUSION: Our study supports the finding that metals have irritant, cytotoxic properties resulting in apoptosis when leaching into skin or gingiva. Particularly for gingiva, commensal microbes exaggerate this detrimental effect.

Application of an Antibacterial Coating Layer via Amine-Terminated Hyperbranched Zirconium-Polysiloxane for Stainless Steel Orthodontic Brackets.

The massive growth of various microorganisms on the orthodontic bracket can form plaques and cause diseases. A novel amine-terminated hyperbranched zirconium-polysiloxane (HPZP) antimicrobial coating was developed for an orthodontic stainless steel tank (SST). After synthesizing HPZP and HPZP-Ag coatings, their structures were characterized by nuclear magnetic resonance spectroscopy, scanning electron microscopy, thickness measurement, contact angle detection, mechanical stability testing, and corrosion testing. The cell toxicity of the two coatings to human gingival fibroblasts (hGFs) and human oral keratinocytes (hOKs) was detected by cell counting kit eight assays, and SST, HPZP@SST, and HPZP-Ag@SST were cocultured with Staphylococcus aureus, Escherichia coli, and Streptococcus mutans for 24 hr to detect the antibacterial properties of the coatings, respectively. The results show that the coatings are about 10 µm, and the water contact angle of HPZP coating is significantly higher than that of HPZP-Ag coating (P < 0.01). Both coatings can be uniformly and densely distributed on SST and have good mechanical stability and corrosion resistance. The cell counting test showed that HPZP coating and HPZP-Ag coating were less toxic to cells compared with SST, and the toxicity of HPZP-Ag coating was greater than that of HPZP coating, with the cell survival rate greater than 80% after 72 hr cocultured with hGFs and hOKs. The antibacterial test showed that the number of bacteria on the surface of different materials was ranked from small to large: HPZP@SST < HPZP-Ag@SST < SST and 800 µg/mL HPZP@SST showed a better bactericidal ability than 400 µg/mL after cocultured with S. aureus, E. coli, and S. mutans, respectively (all P < 0.05). The results showed that HPZP coating had a better effect than HPZP-Ag coating, with effective antibacterial and biocompatible properties, which had the potential to be applied in orthodontic process management.

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.

IFN-γ-Preconditioned Human Gingival-Derived Mesenchymal Stromal Cells Inhibit Plasmacytoid Dendritic Cells via Adenosine.

Plasmacytoid dendritic cells (pDCs) are vital players in antiviral immune responses because of their high levels of IFN-α secretion. However, this attribute has also implicated them as critical factors behind the immunopathogenesis of inflammatory diseases, and no currently available therapy can efficiently inhibit pDCs' aberrant activation. Mesenchymal stromal cells (MSCs) possess stromal immunomodulatory functionality, regulating immune cell activation through several mechanisms, including the adenosinergic (CD39/CD73/adenosine) pathway. The IFN-γ preconditioning of bone marrow MSCs improves their inhibitory properties for therapy applications; however, isolating human gingival tissue-derived MSCs (hGMSCs) is more accessible. These cells have shown better immunomodulatory effects, yet the outcome of IFN-γ preconditioning and its impact on the adenosinergic pathway has not been evaluated. This study first validated the immunoregulatory properties of primary-cultured hGMSCs, and the results showed that IFN-γ preconditioning strengthens CD39/CD73 coexpression, adenosine production, and the regulatory properties of hGMSC, which were confirmed by describing for the first time their ability to reduce pDC activation and their IFN-α secretion and to increase the frequency of CD73+ pDC. In addition, when CD73's enzymatic activity was neutralized in hGMSCs, adenosine production and the IFN-γ preconditioning effect were restrained. This evidence might be applied to design hGMSCs- and adenosine-based immunotherapeutic strategies for treating inflammatory disorders that are associated with pDC overactivation.