Platelet-Rich Fibrin Increases CXCL8 Expression in Gingival Fibroblasts.

Platelet-rich fibrin (PRF), the coagulated plasma of fractionated blood, is widely used to support tissue regeneration in dentistry, and the underlying cellular and molecular mechanisms are increasingly being understood. Periodontal connective tissues steadily express CXCL8, a chemokine that attracts granulocytes and lymphocytes, supporting homeostatic immunity. Even though PRF is considered to dampen inflammation, it should not be ruled out that PRF increases the expression of CXCL8 in gingival fibroblasts. To test this hypothesis, we conducted a bioassay where gingival fibroblasts were exposed to PRF lysates and the respective serum. We show here that PRF lysates and, to a lesser extent, PRF serum increased the expression of CXCL8 by the gingival fibroblasts, as confirmed by immunoassay. SB203580, the inhibitor of p38 mitogen-activated protein kinase, reduced CXCL8 expression. Consistently, PRF lysates and, to a weaker range, the PRF serum also caused phosphorylation of p38 in gingival fibroblasts. Assuming that PRF is a rich source of growth factors, the TGF-ß receptor type I kinase inhibitor SB431542 decreased the PRF-induced expression and translation of CXCL8. The findings suggest that PRF lysates and the respective serum drive CXCL8 expression by activating TGF-ß and p38 signaling in gingival fibroblasts.

18­α­glycyrrhetinic acid induces apoptosis in gingival fibroblasts exposed to phenytoin.

Phenytoin (PHT)-induced gingival overgrowth is caused by the increased proliferation and reduced apoptosis of gingival fibroblasts in inflammatory gingiva. Licorice has long been used as a component of therapeutic preparations. It inhibits cell proliferation, induces cell apoptosis and has anti-inflammatory effects. 18-α-glycyrrhetinic acid (18α-GA), the active compound in licorice, promotes apoptosis in various types of cells. The present study determined whether 18α-GA affects apoptosis in gingival fibroblasts exposed to PHT. The present study aimed to establish a basis for the therapeutic application of 18α-GA to treat the gingival overgrowth induced by PHT. Human gingival fibroblasts from healthy donors were cultured to semi-confluence and then stimulated in serum-free DMEM containing PHT with or without 18α-GA for subsequent experiments. Apoptotic cells were detected by ELISA. Analysis of the distribution of cell cycle phases and the apoptotic cell population was performed by flow cytometry. The expression levels of mRNAs and proteins of apoptotic regulators were measured using reverse transcription-quantitative PCR and western blotting, respectively. Caspase (CASP) activities were assessed by an ELISA. Treatment with 18α-GA markedly increased the number of apoptotic cells, reduced BCL2 mRNA expression, increased CASP2 and receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1) domain containing adaptor with death domain, Fas (TNFRSF6)-associated via death domain, RIPK1, tumor necrosis factor receptor superfamily; member 1A, TNF receptor-associated factor 2, CASP2, CASP3 and CASP9 mRNA expression, and also upregulated the protein expression levels and activities of caspase-2, caspase-3 and caspase-9. These results demonstrated that 18α-GA induced apoptosis through the activation of the Fas and TNF pathways in the death receptor signaling pathway in gingival fibroblasts treated with PHT. 18α-GA exhibited therapeutic potential for the treatment of PHT-induced gingival overgrowth.

Biological Response of the Peri-Implant Mucosa to Different Definitive Implant Rehabilitation Materials.

BACKGROUND: Sealing the peri-implant tissue is a determining factor for long-term implant survival. In the transmucosal region, the cervical fraction of the prosthetic crown is in contact with these tissues, so mucointegration will also be influenced by the biomaterial used for the prosthetic restoration. This study aims to compare the tissue response generated by definitive restorative materials and polymeric materials from a histological point of view. METHODS: This study performed an observational prospective cohort study in which biopsies of the peri-implant mucosa were taken after placement of implant-supported prosthetic restorations made of different materials (zirconium oxide, lithium disilicate, and PMMA). RESULTS: A statistically significant difference was observed in the increase in the thickness of the non-keratinized epithelium when comparing the definitive materials (zirconium oxide/lithium disilicate) vs. the provisional material (PMMA) and in the number of collagen fibers when comparing zirconium oxide and lithium disilicate. CONCLUSIONS: This study found that zirconia is the material that presents the most adequate biological response of peri-implant tissues. It shows a lower intensity of inflammatory cellular content, a total normality in the number of collagen fibers (the arrangement of the fibers is normal in 90% of the cases), and vascular proliferation of connective tissue in 83% of the cases. These parameters make it a material with a predictable response. Similarly, only the following slight statistically significant differences between the definitive and provisional materials are observed, indicating that the biological response generated by the provisional material (PMMA) is not very different from that obtained with the placement of the definitive restoration.

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.

Albiflorin Inhibits Advanced Glycation End Products-Induced ROS and MMP-1 Expression in Gingival Fibroblasts.

BACKGROUND: Periodontitis is a common complication of diabetes, with advanced glycation end products (AGEs) playing a key role in its pathogenesis. Albiflorin, a monoterpene glycoside, has shown potential anti-inflammatory and antioxidant properties. This study aims to investigate the effects of albiflorin on AGEs-induced gingival fibroblasts and its underlying mechanisms. OBJECTIVE: This study aimed to evaluate the role of albiflorin in mitigating ROS production, inflammation, and MMP-1 expression in AGEs-induced gingival fibroblasts. METHODS: The viability of gingival fibroblasts treated with albiflorin and AGEs was assessed using CCK-8 assays. ROS levels were measured by DCF staining, and the expression of inflammatory markers and MMP-1 was evaluated by ELISA and qPCR. The involvement of the NF-κB and Nrf2 pathways was examined by immunoblotting. RESULTS: Albiflorin enhanced the viability of AGEs-induced gingival fibroblasts and reduced ROS production. It also decreased the expression of IL-6, IL-8, RAGE, and MMP-1, suggesting an anti- inflammatory effect. Mechanistically, albiflorin modulated the NF-κB and Nrf2 pathways in AGEs-induced gingival fibroblasts. CONCLUSION: Albiflorin exhibited protective effects against AGEs-induced oxidative stress and inflammation in gingival fibroblasts, highlighting its potential as a therapeutic agent for periodontitis in diabetic patients. The modulation of the NF-κB and Nrf2 pathways by albiflorin provides insight into its mechanism of action.

Highly Ordered Nanotube-Like Microstructure on Titanium Dental Implant Surface Fabricated via Anodization Enhanced Cell Adhesion and Migration of Human Gingival Fibroblasts.

Background: Titanium (Ti) surface with nanotubes array via anodization has been used in dental implants to enhance bone regeneration but little research was carried out to evaluate whether the presence of highly ordered or disorderly distributed nanotubes array on titanium surface would have an effect on cell behaviors of gingival fibroblasts. Methods: The present study fabricated nanotubes arrays with varied topography under different constant voltage of electrochemical anodization in fluorine-containing electrolyte. Human gingival fibroblasts (HGFs) from extracted third molar were harvested and co-cultured with titanium disks with different nanotubes topography. Then cell behaviors of gingival fibroblasts including cell proliferation, adhesive morphology and cell migration were estimated to investigate the influence of titanium nanotubes on cell biology. Besides, gene and protein expression of adhesion molecule (integrin ß1/ß4/α6, fibronectin, intracellular adhesion molecule-1 and collagen type I) were detected to evaluate the influence of different surfaces on cell adhesion. Results: Highly ordered arrays of nanotubes with pore diameter of 60 nm and 100 nm were fabricated under 30 and 40 V of anodization (TNT-30 and TNT-40) while disorderedly distributed nanotube arrays formed on the titanium surface under 50 V of anodization (TNT-50). Our results demonstrated that compared with raw titanium surface and disorderly nanotubes, surface with orderly nanotubes array increased cell area and aspect ratio, as well as cell migration ability in the early phase of cell adhesion (p<0.05). Besides, compared with raw titanium surface, gene and protein expression of adhesion molecules were upregulated in nanotubes groups to different extents, no matter whether in an orderly or disorderly array. Conclusion: Within the limitations of our study, we conclude that compared with raw titanium surface, the presence of nanotubes array on titanium surface could enhance cells adhesion and cell migration in the early phase. And compared with disorderly distributed nanotubes, highly ordered nanotubes array might provide a much more favorable surface for gingival fibroblasts to achieve a tight adhesion on the materials.

Gingival fibroblast response to (hybrid) ceramic implant reconstruction surfaces is modulated by biomaterial type and surface treatment.

OBJECTIVES: Surface characteristics of implant reconstructions determine the gingival fibroblast (GF) response and thus soft tissue integration (STI). However, for monolithic implant reconstructions it is unknown whether the (hybrid) ceramic biomaterial type and its surface treatment affect GF response. Therefore, this investigation examined the influence of the implant reconstruction biomaterials hybrid ceramic (HC), lithium disilicate ceramic (LS), 4 and 5 mol% yttria partially stabilized zirconiumdioxide ceramics (4/5Y-PSZ) and their surface treatment - machining, polishing or glazing - on surface characteristics and GF response. METHODS: After characterization of surface topography and wettability by scanning electron microscopy, interferometry and contact angle measurement, the adhesion, morphology, metabolic activity and proliferation of GFs from six donors was investigated by fluorescent staining and a resazurin-based assay at days 1, 3 and 7. Titanium (Ti) served as control. RESULTS: Biomaterial type and surface treatment affected the GF response in a topography-dependent manner. Smooth polished and glazed surfaces demonstrated enhanced GF adhesion and earlier proliferation onset compared to rough machined surfaces. Due to minor differences in surface topography of polished and glazed surfaces, however, the GF response was similar for polished and glazed HC, LS, 4- and 5Y-PSZ as well as Ti. SIGNIFICANCE: Within the limits of the present investigation, polishing and glazing of machined HC, LS and 4/5Y-PSZ can be recommended to support STI-relevant cell functions in GF. Since the GF response on polished and glazed HC, LS, 4- and 5Y-PSZ surfaces and the Ti control was comparable, this investigation proofed equal cytocompatibility of these surfaces in vitro.

Analysis of soft tissue integration-supportive cell functions in gingival fibroblasts cultured on 3D printed biomaterials for oral implant-supported prostheses.

To date, it is unknown whether 3D printed fixed oral implant-supported prostheses can achieve comparable soft tissue integration (STI) to clinically established subtractively manufactured counterparts. STI is mediated among others by gingival fibroblasts (GFs) and is modulated by biomaterial surface characteristics. Therefore, the aim of the present work was to investigate the GF response of a 3D printed methacrylate photopolymer and a hybrid ceramic-filled methacrylate photopolymer for fixed implant-supported prostheses in the sense of supporting an STI. Subtractively manufactured samples made from methacrylate polymer and hybrid ceramic were evaluated for comparison and samples from yttria-stabilized tetragonal zirconia polycrystal (3Y-TZP), comprising well documented biocompatibility, served as control. Surface topography was analyzed by scanning electron microscopy and interferometry, elemental composition by energy-dispersive x-ray spectroscopy, and wettability by contact angle measurement. The response of GFs obtained from five donors was examined in terms of membrane integrity, adhesion, morphogenesis, metabolic activity, and proliferation behavior by a lactate-dehydrogenase assay, fluorescent staining, a resazurin-based assay, and DNA quantification. The results revealed all surfaces were smooth and hydrophilic. GF adhesion, metabolic activity and proliferation were impaired by 3D printed biomaterials compared to subtractively manufactured comparison surfaces and the 3Y-TZP control, whereas membrane integrity was comparable. Within the limits of the present investigation, it was concluded that subtractively manufactured surfaces are superior compared to 3D printed surfaces to support STI. For the development of biologically optimized 3D printable biomaterials, consecutive studies will focus on the improvement of cytocompatibility and the synthesis of STI-relevant extracellular matrix constituents.

Synergistic effect of zoledronate and compressive force suppresses proliferation and differentiation of human gingival fibroblasts.

We investigated the effects of zoledronate (ZA) and compressive force, separately and in combination, on the proliferation and differentiation of human gingival fibroblasts (HGFs) to verify the mechanism underlying medication-related osteonecrosis of the jaw (MRONJ). The addition of 100 µM ZA markedly inhibited cell proliferation. Expression of type I collagen, fibroblast growth factor 2, and connective tissue growth factor genes, was decreased by ZA and compressive force. Similar results were observed for collagen expression by using Sirius red staining. These results, together with clinical findings that MRONJ is more common in cases with excessive mechanical stress on the oral mucosa, suggest that bisphosphonates such as ZA and mechanical stress may act in conjunction as risk factors for the development of MRONJ by affecting homeostasis of the oral mucosal tissues, including HGFs.

Novel approach to soft tissue regeneration: in vitro study of compound hyaluronic acid and horizontal platelet-rich fibrin combination

Abstract Objective This study aims to develop a compound biomaterial to achieve effective soft tissue regeneration. Methodology Compound hyaluronic acid (CHA) and liquid horizontal-platelet-rich fibrin (H-PRF) were mixed at a ratio of 1:1 to form a CHA-PRF gel. Human gingival fibroblasts (HGFs) were used in this study. The effect of CHA, H-PRF, and the CHA-PRF gel on cell viability was evaluated by CCK-8 assays. Then, the effect of CHA, H-PRF, and the CHA-PRF gel on collagen formation and deposition was evaluated by qRT‒PCR and immunofluorescence analysis. Finally, qRT‒PCR, immunofluorescence analysis, Transwell assays, and scratch wound-healing assays were performed to determine how CHA, H-PRF, and the CHA-PRF gel affect the migration of HGFs. Results The combination of CHA and H-PRF shortened the coagulation time of liquid H-PRF. Compared to the pure CHA and H-PRF group, the CHA-PRF group exhibited the highest cell proliferation at all time points, as shown by the CCK-8 assay. Col1a and FAK were expressed at the highest levels in the CHA-PRF group, as shown by qRT‒PCR. CHA and PRF could stimulate collagen formation and HGF migration, as observed by fluorescence microscopy analysis of COL1 and F-actin and Transwell and scratch healing assays. Conclusion The CHA-PRF group exhibited greater potential to promote soft tissue regeneration by inducing cell proliferation, collagen synthesis, and migration in HGFs than the pure CHA or H-PRF group. CHA-PRF can serve as a great candidate for use alone or in combination with autografts in periodontal or peri-implant soft tissue regeneration.

Mouse gingival single-cell transcriptomic atlas identified a novel fibroblast subpopulation activated to guide oral barrier immunity in periodontitis.

Periodontitis, one of the most common non-communicable diseases, is characterized by chronic oral inflammation and uncontrolled tooth supporting alveolar bone resorption. Its underlying mechanism to initiate aberrant oral barrier immunity has yet to be delineated. Here, we report a unique fibroblast subpopulation activated to guide oral inflammation (AG fibroblasts) identified in a single-cell RNA sequencing gingival cell atlas constructed from the mouse periodontitis models. AG fibroblasts localized beneath the gingival epithelium and in the cervical periodontal ligament responded to the ligature placement and to the discrete topical application of Toll-like receptor stimulants to mouse maxillary tissue. The upregulated chemokines and ligands of AG fibroblasts linked to the putative receptors of neutrophils in the early stages of periodontitis. In the established chronic inflammation, neutrophils, together with AG fibroblasts, appeared to induce type 3 innate lymphoid cells (ILC3s) that were the primary source of interleukin-17 cytokines. The comparative analysis of Rag2-/- and Rag2-/-Il2rg-/- mice suggested that ILC3 contributed to the cervical alveolar bone resorption interfacing the gingival inflammation. We propose the AG fibroblast-neutrophil-ILC3 axis as a previously unrecognized mechanism which could be involved in the complex interplay between oral barrier immune cells contributing to pathological inflammation in periodontitis.

Effect of ascorbic acid on cellular respiration with mitochondrial reductase in gingival fibroblast.

Vitamin C or L-ascorbic acid has diverse functions in the body, especially healing promotion in tissue injury via participating in the hydroxylation reactions required for collagen formation. Systemic administration of vitamin C plays an important role on gingival fibroblast proliferation and functions. Whether local or rinsing administration of vitamin C alters gingival fibroblast wound healing behavior remains unclear. Therefore, it is of interest to investigate the effect of vitamin C on gingival fibroblast behavior utilizing cell culture. Primary human gingival fibroblasts isolated from gingival tissue were rinsed with medium containing various concentrations of vitamin C. Cell migration, cell viability was assessed using MTT assay. The viability assay showed >95% live cells, and no significant (P > 0.05) difference in these values was observed at different concentrations at 24 hrs. The levels of cell proliferation were not significantly different among the control and experimental groups in 24 hrs experimental time-points (p > 0.05). Vitamin C is nontoxic and can be used for further experiments to validate for clinical application. This was further confirmed with morphological examination after 24hrs incubation on control and experimental group drugs observed under the phase contrast microscope.

Gingival Fibroblasts Are Sensitive to Oral Cell Lysates Indicated by Their IL11 Expression.

Damaged cells that appear as a consequence of invasive dental procedures or in response to dental materials are supposed to release damage-associated signals. These damage-associated signals not only support tissue regeneration but might also contribute to unwanted fibrosis. The aim of this study was to identify a molecular target that reflects how fibroblasts respond to necrotic oral tissue cells. To simulate the cell damage, we prepared necrotic cell lysates by sonication of the osteocytic cell line IDG-SW3 and exposed them to gingival fibroblasts. RNAseq revealed a moderate increase in IL11 expression in the gingival fibroblasts, a pleiotropic cytokine involved in fibrosis and inflammation, and also in regeneration following trauma. Necrotic lysates of the human squamous carcinoma cell lines HSC2 and TR146, as well as of gingival fibroblasts, however, caused a robust increase in IL11 expression in the gingival fibroblasts. Consistently, immunoassay revealed significantly increased IL11 levels in the gingival fibroblasts when exposed to the respective lysates. Considering that IL11 is a TGF-ß target gene, IL11 expression was partially blocked by SB431542, a TGF-ß receptor type I kinase inhibitor. Moreover, lysates from the HSC2, TR146, and gingival fibroblasts caused a moderate smad2/3 nuclear translocation in the gingival fibroblasts. Taken together and based on IL11 expression, our findings show that fibroblasts are sensitive to damaged oral tissue cells.

Cytocompatibility and Wound Healing Activity of Chitosan Thiocolchicoside Lauric Acid Nanogel in Human Gingival Fibroblast Cells.

AIM: To investigate the cytocompatibility effect and wound healing activity of chitosan thiocolchicoside lauric acid (CTL) nanogel using human gingival fibroblast (hGF) cells. MATERIALS AND METHODS: hGF cells were established from gingival tissue as per the standard cell isolation protocol. The cytocompatibility effect was assessed using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay. A scratch wound healing assay was performed to assess the wound-healing potential of CTL nanogel. For the nuclear morphological changes analysis, acridine orange staining was used in gingival fibroblast cells. The stained nuclei were viewed under a fluorescent microscope. ANOVA with posthoc analysis was performed using GraphPad Prism 5 software (Dotmatics, Boston, Massachusetts). The significance level (p-value) was expressed as <0.05.  Results: CTL nanogel did not show any significant cytotoxicity at concentrations 10-80 µl/ml (p<0.05). CTL nanogel at a concentration of 40µl/ml has a cytocompatibility effect on hGF cells and increases cell viability. In vitro scratch wound healing assay resulted in faster wound healing and cell migration with CTL nanogel when compared to the control group. CONCLUSION: CTL nanogel has a significant effect on cell proliferation at various concentrations, which suggests its use as a safe and effective drug delivery system.

Proliferative effect of cannabidiol in human gingival fibroblasts via the mitogen-activated extracellular signal-regulated kinase (MEK) 1/2.

BACKGROUND AND OBJECTIVES: Cannabidiol exerts its anti-inflammatory and anti-oxidant activities in various human cells. However, its proliferative effect has not been extrapolated to human gingival fibroblasts (HGFs). This study aimed to determine the proliferative and promigratory effects of cannabidiol in HGFs and to elucidate the signaling mechanism(s). MATERIALS AND METHODS: HGFs, characterized by their CD73, CD90, and CD105 expressions by flow cytometry, were treated with cannabidiol at 0.01-30 µM. The cytotoxicity was determined by the MTT assay, while the proliferative effect was examined by the BrdU assay, immunoblot and immunofluorescence for cyclin D1 and Ki-67 expressions, respectively, and cell cycle analysis. The promigratory effect of cannabidiol was investigated by a wound healing assay. Phosphorylation of the p38 MAPK, JNK, and ERK upon treatment with cannabidiol was explored, and their involvement in cell proliferation and cyclin D1 and Ki-67 expressions was studied using pharmacological inhibitors. RESULTS: No toxicity was found in HGFs treated with any doses of cannabidiol up to 30 µM. The mean percentage of cell proliferation was significantly enhanced by treatment with cannabidiol at 3 or 10 µM (p < .001), consistent with upregulated expressions of cyclin D1 and Ki-67 and increased percentages of HGFs in the S and G2/M phases. Moreover, treatment with cannabidiol significantly induced cell migration (p < .05). The p38 MAPK and ERK1/2 were significantly activated by cannabidiol (p < .05), but only pretreatment with UO126, a MEK1/2 inhibitor, significantly inhibited cell proliferation and cyclin D1 and Ki-67 expressions (p < .05). CONCLUSION: Treatment with cannabidiol at non-toxic doses promotes HGFs' proliferation and migration.

Behaviour of the Peri-Implant Soft Tissue with Different Rehabilitation Materials on Implants.

(1) Background: Mucointegration seems to gain interest when talking about success in the maintenance of dental implants. As we well know, collagen fibres cannot be inserted due to the lack of root structure on the implant surface, so the structural integration of peri-implant tissues that provide a firm seal around implants seems to be of interest when it comes to ensuring the survival of dental implants. To achieve a good epithelial barrier, the physicochemical characteristics of the surfaces of the restorative materials are of vital importance; therefore, the objective of this study is to analyse the histological behaviour of the peri-implant soft tissues in three different restorative materials. (2) Methods: Histological analysis of biopsied peri-implant keratinised mucosa, inflammatory epithelium and connective tissue in contact with a reinforced composite (BRILLIANT Crios), a cross-linked polymethylmethacrylate (TELIO CAD), and a hybrid ceramic (Vita Enamic), restored on a customised Atlantis-type abutment (Dentsply Sirona) between 60 and 180 days after restoration. (3) Results: A greater number of cells per mm2 of keratinised epithelium is observed in the reinforced composite, which could indicate greater surface roughness with greater inflammatory response. In this way, the greater number of lymphocytes and the lateral cellular composition of the inflammatory cells confirm the greater inflammatory activity towards that material. The best material to rehabilitate was hybrid ceramic, as it shows a better cellular response. (4) Conclusions: Knowing the limitations of the proposed study, despite the fact that greater inflammation is observed in the reinforced composite relative to the other materials studied, no statistically significant differences were found.

Egg white-derived peptide KPHAEVVLR promotes wound healing in rat palatal mucosa via PI3K/AKT/mTOR pathway.

KPHAEVVLR (KR-9) is a peptide derived from egg white hydrolyzed, which has been found to accelerate skin wound healing in mice. However, the effect of KR-9 on wound healing on palatal mucosa in rats remains unknown, and the mechanism through which KR-9 promotes wound healing should be further explored. Herein, we aimed to investigate the effect and mechanism of KR-9 peptide on palatal mucosa wound healing. Our results showed that KR-9 reduced the wound area of palatal mucosa in rats and promoted human gingival fibroblasts(HGFs) migration and proliferation.The peptide can enter into cytoplasm. It also increased the phosphorylation of PI3K, AKT, and mTOR protein. The effect of KR-9 on HGFs migration and proliferation could be reversed by PI3K inhibitor. These results demonstrated that KR-9 peptide facilitated wound healing of palatal mucosa in rats by promoting HGFs migration and proliferation, which was mediated by PI3K/AKT/mTOR signaling pathway. This data proves that KR-9 might be used as a potential agent for wound healing treatment.

A Sequential Micro-Immunotherapy Medicine Increases Collagen Deposition in Human Gingival Fibroblasts and in an Engineered 3D Gingival Model under Inflammatory Conditions.

Periodontal therapies use immune mediators, but their side effects can increase with dosage. Micro-immunotherapy (MI) is a promising alternative that employs immune regulators at low and ultralow doses to minimize adverse effects. In this study, the effects of 5 capsules and the entire 10-capsule sequence of the sequential MI medicine (MIM-seq) were tested in two in vitro models of periodontitis. Firstly, human gingival fibroblasts (hGFs) exposed to interleukin (IL)-1ß to induce inflammation were treated with five different capsules of MIM-seq for 3 days or with MIM-seq for 24 days. Subsequently, MIM-seq was analyzed in a 3D model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus. Simultaneously, a non-IL-1ß-treated control and a vehicle were included. The effects of the treatments on cytotoxicity, collagen deposition, and the secreted levels of IL-1α, IL-6, prostaglandin E2 (PGE2), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinases-1 (TIMP-1) were evaluated. None of the tested items were cytotoxic. The complete sequence of MIM-seq decreased PGE2 release and restored collagen deposition levels induced by IL-1ß treatment in hGFs exposed to IL-1ß. MIM-seq treatment restored collagen production levels in both models. These promising preclinical findings suggest that MIM-seq should be further investigated for periodontitis treatment.

"Efficacy of cytotoxic effect of green tea catechins on the human periodontal fibroblasts and human dental pulp fibroblasts -An in vitro study".

Background: Inflammation of tooth-supporting tissue and the pulp tissue is followed by wound healing and regeneration process that involves the specific type of connective tissue cells, the fibroblasts. During periodontitis and pulpitis, the inflammation of the tissue causes damage to the fibroblasts. These fibroblasts secrete collagen proteins and maintain the structural framework; along with this the inflammatory process moves toward healing where in the specific cells such as the fibroblast cells play important roles. Green tea catechins epigallocatechin-3-gallate (EGCG) being one of the major catechins is known to have multiple beneficial effects on human fibroblasts. Objective: To assess the in vitro cytotoxicity of green tea catechins on the human periodontal ligament (PDL) fibroblasts and human dental pulp fibroblasts. Materials and Methods: Human PDL fibroblasts (hPDLFs) and human dental pulp fibroblasts were isolated from the two extracted premolar teeth that were indicated for orthodontic treatment. The fibroblasts were then seeded in 96 well tissue culture plate for cell viability study. EGCG was used at different concentration to treat the cells. After 48 h; (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) assay was performed to determine the cell viability. Results: The vitality of hPDLFs and human dental pulp fibroblasts was found to be inversely proportional to EGCG concentrations. Conclusions: hPDLFs have shown 37% proliferation at lowest concentration of EGCG used and human dental pulp fibroblasts have shown 99% viability at lowest concentration of EGCG used.

Magnolia kobus Extract Suppresses Porphyromonas gingivalis LPS-Induced Proinflammatory Cytokine and MMP Expression in HGF-1 Cells and Regulates Osteoclastogenesis in RANKL-Stimulated RAW264.7 Cells.

Clinical prevention is of utmost importance for the management of periodontal diseases. Periodontal disease starts with an inflammatory response in the gingival tissue, and results in alveolar bone destruction and subsequent tooth loss. This study aimed to confirm the anti-periodontitis effects of MKE. To confirm this, we studied its mechanism of action using qPCR and WB in LPS-treated HGF-1 cells and RANKL-induced osteoclasts. We found that MKE suppressed proinflammatory cytokine protein expression by inhibiting the TLR4/NF-κB pathway in LPS-PG-induced HGF-1 cells and blocking ECM degradation by regulating the expression of TIMPs and MMPs. We also confirmed that TRAP activity and multinucleated cell formation were reduced in RANKL-stimulated osteoclasts after exposure to MKE. These results were confirmed by inhibiting TRAF6/MAPK expression, which led to the suppression of NFATc1, CTSK, TRAP, and MMP expression at the gene and protein levels. Our results confirmed that MKE is a promising candidate for the management of periodontal disease based on its anti-inflammatory effects and inhibition of ECM degradation and osteoclastogenesis.