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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. [ABSTRACT FROM AUTHOR]

Gingival fibroblasts are a significant source of paracrine signals required to maintain periodontal homeostasis and to mediate pathological events linked to periodontitis and oral squamous cell carcinomas. Among the potential paracrine signals are stanniocalcin-1 (STC1), involved in oxidative stress and cellular survival; amphiregulin (AREG), a growth factor that mediates the cross-talk between immune cells and epithelial cells; chromosome 11 open reading frame 96 (C11orf96) with an unclear biologic function; and the inflammation-associated prostaglandin E synthase (PTGES). Gingival fibroblasts increasingly express these genes in response to bone allografts containing remnants of injured cells. Thus, the gene expression might be caused by the local release of damage-associated molecular patterns arising from injured cells. The aim of this study is consequently to use the established gene panel as a bioassay to measure the damage-associated activity of oral cell lysates. To this aim, we have exposed gingival fibroblasts to lysates prepared from the squamous carcinoma cell lines TR146 and HSC2, oral epithelial cells, and gingival fibroblasts. We report here that all lysates significantly increased the transcription of the entire gene panel, supported for STC1 at the protein level. Blocking TGF-β receptor 1 kinase with SB431542 only partially reduced the forced expression of STC1, AREG, and C11orf96. SB431542 even increased the PTGES expression. Together, these findings suggest that the damage signals originating from oral cells can change the paracrine activity of gingival fibroblasts. Moreover, the expression panel of genes can serve as a bioassay for testing the biocompatibility of materials for oral application. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-β 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-β and p38 signaling in gingival fibroblasts. [ABSTRACT FROM AUTHOR]

Zhaoming Deng,1,2 Lerong Yu,1,2 Yishen Kuang,1,2 Ziyao Zhou,1,2 Xiangwei Li1 1Department of Stomatology, The Fifth Affiliated Hospital of Sun Yat-Sen University, Zhuhai City, 519000, People’s Republic of China; 2Guangdong Provincial Key Laboratory of Biomedical Imaging, The Fifth Affiliated Hospital of Sun Yat-Sen University, Zhuhai City, 519000, People’s Republic of ChinaCorrespondence: Xiangwei Li, The Fifth Affiliated Hospital of Sun Yat-Sen University, East Meihua Road No. 52, Xiangzhou District, Zhuhai City, Guangdong Province, People’s Republic of China, Email lixiangwei@126.comBackground: 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 β 1/β 4/α 6, 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. Keywords: nanotubes, gingival fibroblast, cell behaviors, cell adhesion, gingival sealing

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. 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. 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. 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. • Biomaterial and surface treatment determine surface topography of (hybrid) ceramics. • Surface topography is the key modulator of GF function on (hybrid) ceramics. • Polishing and glazing promote STI-relevant GF functions compared to machining. • Polished and glazed HC, LS, 4- and 5Y-PSZ surfaces show comparable GF response. [ABSTRACT FROM AUTHOR]

To assess the antibacterial properties and cytotoxic effects of NeoPUTTY, TotalFill BC RRM (Bioceramic Root Repair Material) putty, and MTA Plus bioceramic root canal cements. Antimicrobial testing was performed using the tube dilution method against Enterococcus faecalis (ATCC 29212). The colony forming units (CFU) were digitally counted after 24, 48, and 72 hours of incubation using Promega Colony Counter. Cytotoxicity testing against human gingival fibroblasts was performed using MTT assay after 24, 48, and 120 hours. Readings were obtained on an automatic microplate spectrophotometer reader. Data showed parametric distribution and were analysed using two-way mixed model ANOVA followed by comparisons of estimated marginal means using multiple t-test with p-value adjustment using Tukey’s method. The significance level was set at p < 0.05. The three tested bioceramic cements decreased the CFU counts compared to the positive control. At 72 hours, NeoPUTTY showed a significant reduction in the CFU count compared to TotalFill putty and MTA Plus. NeoPUTTY showed significantly better antibacterial properties after 72 hours. At all tested time intervals, TotalFill putty was significantly more cytotoxic than the other cements. At 24 hours, NeoPUTTY and MTA Plus yielded a higher percentage of cell viability. At 120 hours, NeoPUTTY showed better cell viability than the other tested cements. All tested bioceramic cements showed good antibacterial properties and cytocompatibility. NeoPUTTY possesses prolonged antibacterial efficacy after 72 hours’ time, and similarly suggested to be biocompatible showing better cell viability even after 5 days when compared to the other cements. [ABSTRACT FROM AUTHOR]

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.

Background: In this study, we investigated the potential use of keratin for oral tissue regeneration. Keratin is well-known for its effectiveness in skin regeneration by promoting keratinization and enhancing the elasticity and activity of fibroblasts. Because of its structural stability, high storability, biocompatibility, and safety in humans, existing research has predominantly focused on its role in skin wound healing. Herein, we propose using keratin proteins as biocompatible materials for dental applications.Methods: To assess the suitability of alpha-keratin protein as a substrate for cell culture, keratin was extracted from human hair via PEGylation. Viabilities of primary human gingival fibroblasts (HGFs) and human oral keratinocytes (HOKs) were assessed. Fluorescence immunostaining and migration assays were conducted using a fluorescence microscope and confocal laser scanning microscope. Wound healing and migration assays were performed using automated software to analyze the experimental readout and gap closure rate. Results: We confirmed the extraction of alpha-keratin and formation of the PEG-g-keratin complex. Treatment of HGFs with keratin protein at a concentration of 5 mg/ml promoted proliferation and maintained cell viability in the test group compared to the control group. HOKs treated with 5 mg/ml keratin exhibited a slight decrease in cell proliferation and activity after 48 hours compared to the untreated group, followed by an increase after 72 hours. Wound healing and migration assays revealed rapid closure of the area covered by HOKs over time following keratin treatment. Additionally, HOKs exhibited changes in cell morphology and increased the expression of the mesenchymal marker vimentin. Conclusion: Our study demonstrated the potential of hair keratin for soft tissue regeneration, with potential future applications in clinical settings for wound healing.

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. [ABSTRACT FROM AUTHOR]

Background Nicotine, as the main component of cigarettes, is known to interfere with the proliferation of human gingival fibroblasts (HGFs) and can trigger oxidative stress. This study aimed to analyze the impact of nicotine on viability, expression of the antioxidant Nrf2, levels of the product of oxidative stress malondialdehyde (MDA), and the migration capacity of HGFs. Methods An experimental laboratory study used fibroblasts isolated from healthy human gingiva. The cells were grouped into the non-treatment control group (NTC), the solvent control (SC), and the treatment groups, exposed to nicotine at various concentrations for twenty-four hours. Cell viability was assesed using the cell counting kit-8 (CCK-8), Nrf2 expression was examined using ELISA, MDA level was measured using an MDA kit, and migration capacity was assessed using a scratch assay. Statistical analysis used one-way Anova or Kruskal-Wallis test. A p-value of

Gingival fibroblasts are a significant source of paracrine signals required to maintain periodontal homeostasis and to mediate pathological events linked to periodontitis and oral squamous cell carcinomas. Among the potential paracrine signals are stanniocalcin-1 (STC1), involved in oxidative stress and cellular survival; amphiregulin (AREG), a growth factor that mediates the cross-talk between immune cells and epithelial cells; chromosome 11 open reading frame 96 (C11orf96) with an unclear biologic function; and the inflammation-associated prostaglandin E synthase (PTGES). Gingival fibroblasts increasingly express these genes in response to bone allografts containing remnants of injured cells. Thus, the gene expression might be caused by the local release of damage-associated molecular patterns arising from injured cells. The aim of this study is consequently to use the established gene panel as a bioassay to measure the damage-associated activity of oral cell lysates. To this aim, we have exposed gingival fibroblasts to lysates prepared from the squamous carcinoma cell lines TR146 and HSC2, oral epithelial cells, and gingival fibroblasts. We report here that all lysates significantly increased the transcription of the entire gene panel, supported for STC1 at the protein level. Blocking TGF-β receptor 1 kinase with SB431542 only partially reduced the forced expression of STC1, AREG, and C11orf96. SB431542 even increased the PTGES expression. Together, these findings suggest that the damage signals originating from oral cells can change the paracrine activity of gingival fibroblasts. Moreover, the expression panel of genes can serve as a bioassay for testing the biocompatibility of materials for oral application.

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-β 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-β and p38 signaling in gingival fibroblasts.

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.

Background To investigate and compare the effect of four commercially used dental cement at 24 hours, 48 hours,72 hours (h) and 6 days on the cellular response of human gingival fibroblast (HGF). Methods 3 cement pellet samples were made for each 4-test cement (n=12). The cement used for this study were zinc phosphate (ZP), zinc oxide non-eugenol (ZOE), RelyX U200 (RU200), and glass ionomer cement (GIC). The cytotoxicity of peri-implant tissues was investigated using one commercial cell line. All processing was done following International Organization for Standardization (ISO) methods 10993-5 and 10993-12 (MTT assay Test). Cell cultures without dental cement were considered as control. Standard laboratory procedures were followed to permit cell growth and confluence over 48 hrs after sub-cultivation. Before being subjected to analysis, the cells were kept in direct contact with the cement samples for the suggested time period. To validate the results the specimens were tested three times each. Cell death and inhibition of cell growth were measured quantitatively. Results were analyzed using 1-way ANOVA (a=0.05) followed by Tukey B post hoc test. Results The study showed the dental cement test material was cytotoxic. ZOE, ZP, GIC, and RU200 were cytotoxic in decreasing order, respectively, significantly reducing cell viability after exposure to HGF (p Conclusions Within the limitations of this in-vitro cellular study, results indicated that HGF were vulnerable to the test the dental cement. The highest cytotoxicity was observed in ZOE, followed by ZP, GIC, and RU200.

Aims. The effect of polyphenolic fraction of Lonicera caerulea (PFLC) and alkaloid fraction of Macleaya cordata (AFMC) mix on the production of inflammatory mediators in human gingival fibroblasts pretreated with lipopolysaccharide (LPS) was investigated. In addition, protective effects of mucoadhesive paste containing combination of PFLC and AFMC (0.05% and 0.01%, respectively; n=15, Group A) and placebo (n=15, Group B) were evaluated in patients after surgical extraction of lower third molars. Methods. Gingival fibroblasts were pre-treated with LPS (10 µg/mL; 24 h) and PFLC/AFMC (25/0.25; 50/0.25; 100/0.25; 25/0.5; 50/0.5; 100/0.5 µg/mL) in serum-free medium was applied for 4 h. Then the interleukin-6 (IL-6), reactive oxygen species (ROS) generation, level of intracellular glutathione (GSH) and expression of cyclooxygenase-2 (COX-2) were evaluated. The study was a 6-day, single-center, randomized, double-blind and placebo-controlled trial consisting of two parallel treatment arms. A modified Oral health impact profile questionnaire including both general oral condition and extraction related questions, was used to evaluate the oral condition and other changes before (day 0) and on the days 1, 3 and 6 after surgical extraction. Results and Conclusion. The combination of PFLC with AFMC caused a reduction of ROS generation, reduced IL-6 production and suppressed the expression of COX-2. In group A the paste treatment contributed to improvement of oral health-related quality of life. Topical application of PFLC and AFMC into the extraction wound improved post-extraction site wound healing probably by antioxidant and anti-inflammatory mechanisms.

Background To investigate and compare the effect of four commercially used dental cement at 24 hours, 48 hours,72 hours (h) and 6 days on the cellular response of human gingival fibroblast (HGF). Methods 3 cement pellet samples were made for each 4-test cement (n=12). The cement used for this study were zinc phosphate (ZP), zinc oxide non-eugenol (ZOE), RelyX U200 (RU200), and glass ionomer cement (GIC). The cytotoxicity of peri-implant tissues was investigated using one commercial cell line. All processing was done following International Organization for Standardization (ISO) methods 10993-5 and 10993-12 (MTT assay Test). Cell cultures without dental cement were considered as control. Standard laboratory procedures were followed to permit cell growth and confluence over 48 hrs after sub-cultivation. Before being subjected to analysis, the cells were kept in direct contact with the cement samples for the suggested time period. To validate the results the specimens were tested three times each. Cell death and inhibition of cell growth were measured quantitatively. Results were analyzed using 1-way ANOVA (a=0.05) followed by Tukey B post hoc test. Results The study showed the dental cement test material was cytotoxic. ZOE, ZP, GIC, and RU200 were cytotoxic in decreasing order, respectively, significantly reducing cell viability after exposure to HGF (p

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.

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-β target gene, IL11 expression was partially blocked by SB431542, a TGF-β 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. [ABSTRACT FROM AUTHOR]

Aim: Necroptosis participates in the pathogenesis of many inflammatory diseases, including periodontitis. Here, we aimed to investigate the role and mechanism of necroptosis inhibitors in attenuating periodontitis. Materials and Methods: The Gene Expression Omnibus (GEO) dataset GSE164241 was re‐analysed to identify the role of necroptosis in periodontitis. Gingival specimens from healthy subjects or periodontitis patients were collected to evaluate the expression level of necroptosis‐associated proteins. The therapeutic effect of necroptosis inhibitors on periodontitis was assessed in vivo and in vitro. Moreover, Transwell assays and Western blotting and siRNA transfection were used to identify the effects of necroptotic human gingival fibroblasts (hGFs) on THP‐1 macrophages. Results: Re‐analysis revealed that gingival fibroblasts (GFs) in periodontitis gingiva showed the highest area under the curve score of necroptosis. Elevated levels of necroptosis‐associated proteins were identified in GFs in periodontitis gingiva collected from patients and mice. In ligature‐induced periodontitis mice, local administration of receptor interacting protein kinase 3(RIPK3) inhibitor GSK′872 or sh‐mixed‐lineage kinase domain‐like pseudokinase (Mlkl) markedly abrogated necroptosis and rescued periodontitis. Analogously, necroptosis inhibitors alleviated the inflammatory response and release of damage‐associated molecular patterns in lipopolysaccharide‐ or LAZ (LPS + AZD'5582 + z‐VAD‐fmk, necroptosis inducer)‐induced GFs and then reduced THP‐1 cell migration and M1 polarization. Conclusions: Necroptosis in GFs aggravated gingival inflammation and alveolar bone loss. Necroptosis inhibitors attenuate this process by modulating THP‐1 macrophage migration and polarization. This study offers novel insights into the pathogenesis and potential therapeutic targets of periodontitis. [ABSTRACT FROM AUTHOR]

(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. [ABSTRACT FROM AUTHOR]

Background: To investigate and compare the effect of four commercially used dental cement at 24 hours, 48 hours,72 hours (h) and 6 days on the cellular response of human gingival fibroblast (HGF). Methods: 3 cement pellet samples were made for each 4-test cement (n=12). The cement used for this study were zinc phosphate (ZP), zinc oxide non-eugenol (ZOE), RelyX U200 (RU200), and glass ionomer cement (GIC). The cytotoxicity of peri-implant tissues was investigated using one commercial cell line. All processing was done following International Organization for Standardization (ISO) methods 10993-5 and 10993-12 (MTT assay Test). Cell cultures without dental cement were considered as control. Standard laboratory procedures were followed to permit cell growth and confluence over 48 hrs after sub-cultivation. Before being subjected to analysis, the cells were kept in direct contact with the cement samples for the suggested time period. To validate the results the specimens were tested three times each. Cell death and inhibition of cell growth were measured quantitatively. Results were analyzed using 1-way ANOVA (a=0.05) followed by Tukey B post hoc test. Results: The study showed the dental cement test material was cytotoxic. ZOE, ZP, GIC, and RU200 were cytotoxic in decreasing order, respectively, significantly reducing cell viability after exposure to HGF (p Conclusions: Within the limitations of this in-vitro cellular study, results indicated that HGF were vulnerable to the test the dental cement. The highest cytotoxicity was observed in ZOE, followed by ZP, GIC, and RU200.

Aims. The effect of polyphenolic fraction of Lonicera caerulea (PFLC) and alkaloid fraction of Macleaya cordata (AFMC) mix on the production of inflammatory mediators in human gingival fibroblasts pretreated with lipopolysaccharide (LPS) was investigated. In addition, protective effects of mucoadhesive paste containing combination of PFLC and AFMC (0.05% and 0.01%, respectively; n=15, Group A) and placebo (n=15, Group B) were evaluated in patients after surgical extraction of lower third molars. Methods. Gingival fibroblasts were pre-treated with LPS (10 µg/mL; 24 h) and PFLC/AFMC (25/0.25; 50/0.25; 100/0.25; 25/0.5; 50/0.5; 100/0.5 µg/mL) in serum-free medium was applied for 4 h. Then the interleukin-6 (IL-6), reactive oxygen species (ROS) generation, level of intracellular glutathione (GSH) and expression of cyclooxygenase-2 (COX-2) were evaluated. The study was a 6-day, single-center, randomized, double-blind and placebo-controlled trial consisting of two parallel treatment arms. A modified Oral health impact profile questionnaire including both general oral condition and extraction related questions, was used to evaluate the oral condition and other changes before (day 0) and on the days 1, 3 and 6 after surgical extraction. Results and Conclusion. The combination of PFLC with AFMC caused a reduction of ROS generation, reduced IL-6 production and suppressed the expression of COX-2. In group A the paste treatment contributed to improvement of oral health-related quality of life. Topical application of PFLC and AFMC into the extraction wound improved post-extraction site wound healing probably by antioxidant and anti-inflammatory mechanisms. [ABSTRACT FROM AUTHOR]

Purpose: To detect the effect of Resolvin D1 (RvD1) on Developmental endothelial locus-1 (Del-1) and cytokine expressions of human gingival fibroblast cells exposed to Porphyromonas gingivalis lipopolysaccharide (P. gingivalis-LPS). Material and Methods: The effect of RvD1 on cell viability of human gingival fibroblasts exposed to P. gingivalis-LPS was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Meanwhile, the effect of RvD1 on Del-1 and cytokine (IL-1ß, IL-6, IL-8, IL-10, IL-17) expressions of human gingival fibroblasts exposed to P. gingivalis-LPS (1000 ng/mL) were studied by real-time PCR experiment, statistical analysis was performed using GraphPad Prism version 5 for Windows. Results: Cell viability assay results demonstrated that RvD1 concentrations upregulated cell number compared to control group at 24 and at 72 h. While RvD1 reduced the IL-6, IL-8, and IL-17 mRNA expressions, the IL-10 and Del-1 mRNA expressions increased in a time- and dose-dependent manner. Also, IL-1ß was not affected by RvD1 treatments. Conclusion: The increased expression of Del-1 and IL-10 by RvD1 down-regulated the pro-inflammatory cytokine expressions induced by P. gingivalis-LPS in gingival fibroblast. Resolvin D1displayed regulatory effects on gingival inflammation in P. gingivalis LPS-induced cell culture experiment. In particular, results of this study show that Del-1 induced by RvD1 may have therapeutic potential to modulate periodontal inflammation. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to determine the regulatory mechanism of bone marrow-derived mesenchymal stem cell (BM-MSC) differentiation mediated by humoral factors derived from human periodontal ligament (HPL) cells and human gingival fibroblasts (HGFs). We analyzed histone deacetylase (HDAC) expression and activity involved in BM-MSC differentiation and determined their regulatory effects in co-cultures of BM-MSCs with HPL cells or HGFs. Background: BM-MSCs can differentiate into various cell types and can, thus, be used in periodontal regenerative therapy. However, the mechanism underlying their differentiation remains unclear. Transplanted BM-MSCs are affected by periodontal cells via direct contact or secretion of humoral factors. Therefore, their activity is regulated by humoral factors derived from HPL cells or HGFs. Methods: BM-MSCs were indirectly co-cultured with HPL cells or HGFs under osteogenic or growth conditions and then analyzed for osteogenesis, HDAC1 and HDAC2 expression and activity, and histone H3 acetylation. BM-MSCs were treated with trichostatin A, or their HDAC1 or HDAC2 expression was silenced or overexpressed during osteogenesis. Subsequently, they were evaluated for osteogenesis or the effects of HDAC activity. Results: BM-MSCs co-cultured with HPL cells or HGFs showed suppressed osteogenesis, HDAC1 and HDAC2 expression, and HDAC phosphorylation; however, histone H3 acetylation was enhanced. Trichostatin A treatment remarkably suppressed osteogenesis, decreasing HDAC expression and enhancing histone H3 acetylation. HDAC1 and HDAC2 silencing negatively regulated osteogenesis in BM-MSCs to the same extent as that achieved by indirect co-culture with HPL cells or HGFs. Conversely, their overexpression positively regulated osteogenesis in BM-MSCs. Conclusion: The suppressive effects of HPL cells and HGFs on BM-MSC osteogenesis were regulated by HDAC expression and histone H3 acetylation to a greater extent than that mediated by HDAC activity. Therefore, regulation of HDAC expression has prospects in clinical applications for effective periodontal regeneration, mainly, bone regeneration. [ABSTRACT FROM AUTHOR]

Interactions between gingival fibroblasts (GFs) and oral pathogens contribute to the chronicity of inflammation in periodontitis. Epigenetic changes in DNA methylation are involved in periodontitis pathogenesis, and recent studies indicate that DNA methyltransferase (DNMT) inhibitors may protect against epithelial barrier disruption and bone resorption. To assess the impact of DNMT inhibition on GFs, cells were cultured with decitabine (5-aza-2’-deoxycytidine, DAC) for 12 days to induce DNA hypomethylation. We observed several potentially detrimental effects of DAC on GF biological functions. First, extended treatment with DAC reduced GF proliferation and induced necrotic cell death. Second, DAC amplified Porphyromonas gingivalis- and cytokine-induced expression and secretion of the chemokine CCL20 and several matrix metalloproteinases (MMPs), including MMP1, MMP9, and MMP13. Similar pro-inflammatory effects of DAC were observed in periodontal ligament fibroblasts. Third, DAC upregulated intercellular adhesion molecule-1 (ICAM-1), which was associated with increased P. gingivalis adherence to GFs and may contribute to bacterial dissemination. Finally, analysis of DAC-induced genes identified by RNA sequencing revealed increased expression of CCL20, CCL5, CCL8, CCL13, TNF, IL1A, IL18, IL33, and CSF3, and showed that the most affected processes were related to immune and inflammatory responses. In contrast, the genes downregulated by DAC were associated with extracellular matrix and collagen fibril organization. Our observations demonstrate that studies of DNMT inhibitors provide important insights into the role of DNA methylation in cells involved in periodontitis pathogenesis. However, the therapeutic potential of hypomethylating agents in periodontal disease may be limited due to their cytotoxic effects on fibroblast populations and stimulation of pro-inflammatory pathways. [ABSTRACT FROM AUTHOR]

Regenerative endodontic treatment based on tissue engineering has recently gained interest in contemporary restorative dentistry. However, low survival rates and poor potential differentiation of stem cells could undermine the success rate of pulp regenerative therapy. Human gingival fibroblast-conditioned medium (hGF-CM) has been considered a potential therapy for tissue regeneration due to its stability in maintaining multiple factors essential for tissue regeneration compared to live cell transplantation. This study aimed to investigate the potency of hGF-CM on stem cells from human dental pulp (DPSC) in pulp regeneration. A series of experiments confirmed that hGF-CM contributes to a significant increase in proliferation, migration capability, and cell viability of DPSC after H2O2 exposure. Moreover, it has been proved to facilitate the odontogenic differentiation of DPSC via qRT-PCR, ALP (alkaline phosphatase), and ARS (Alizarin Red S) staining. It has been discovered that such highly upregulated odontogenesis is related to certain types of ECM proteins (collagen and laminin) from hGF-CM via proteomics. In addition, it is found that the ERK pathway is a key mechanism via inhibition assay based on RNA-seq result. These findings demonstrate that hGF-CM could be beneficial biomolecules for pulp regeneration. [ABSTRACT FROM AUTHOR]

Karol P Steckiewicz,1 Piotr Cieciórski,2 Ewelina Barci&nacute;ska,1 Maciej Ja&sacute;kiewicz,3 Magdalena Narajczyk,4 Marta Bauer,3 Wojciech Kamysz,3 El&zdot;bieta Megiel,2 Iwona Inkielewicz-Stepniak1 1Department of Pharmaceutical Pathophysiology, Faculty of Pharmacy, Medical University of Gdansk, Gdansk, Poland; 2Faculty of Chemistry, University of Warsaw, Warsaw, Poland; 3Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Gdansk, Gdansk, Poland; 4Laboratory of Electron Microscopy, Faculty of Biology, University of Gdansk, Gdansk, PolandCorrespondence: Iwona Inkielewicz-Stepniak Tel +48 58 349 1516Fax +48 58 349 1517Email iinkiel@gumed.edu.plPurpose: Periodontal disease (PD), defined as oral inflammation caused by dental plaque, is an emerging problem. PD may lead to tooth loss, and treatment options are limited. In this study, we designed, synthesized, and characterized silver nanoparticles (AgNPs) conjugated with chlorhexidine (AgNPs-CHL) or metronidazole (AgNPs-PEG-MET) to determine whether they can be used to treat PDs.Materials and Methods: AgNPs were synthesized and characterized by transmission electron microscopy, UV-vis spectrometry, thermogravimetric analyses, and dynamic light scattering. We determined the safety and the antimicrobial and anti-inflammatory properties of synthesized AgNPs in an in vitro model of periodontitis. Antimicrobial properties were determined by measuring the minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) on reference strains of bacteria and fungi. Human gingival fibroblast (HGF-1), murine macrophage (RAW264.7) and human foetal osteoblast (hFOB1.19) cells were used in the study. Lipopolysaccharide (LPS) was used to induce inflammation. Cytokine levels were measured using an enzyme-linked immunosorbent assay; metalloproteinase expression was measured using Western blotting.Results: The synthesized AgNPs were spherical and narrow-dispersed with an average diameter of 13.4 nm ± 3.0 nm in the case of AgNPs-CHL and 3.72 nm ± 0.72 nm in the case of AgNPs-PEG-MET. Both types of AgNPs were active against bacteria and fungi. AgNPs-CHL proved to be a more potent antimicrobial agent, although they were more cytotoxic than AgNPs-PEG-MET; however, both demonstrated beneficial properties in nontoxic concentrations. AgNPs-CHL and AgNPs-PEG-MET decreased the production of proinflammatory cytokines IL-1β, IL-6, IL-8 and TNFα. Both agents also decreased the levels of metalloproteinases MMP3 and MMP8, which may indicate that they will inhibit tissue degradation.Conclusion: AgNPs-CHL and AgNPs-PEG-MET may be possible therapeutic options for PD, as they have antibacterial and anti-inflammatory properties. However, to fully understand the potential of AgNPs, our in vitro findings must be evaluated in an in vivo model.Keywords: drug delivery platforms, gingival fibroblast, macrophages, osteoblasts, periodontal disease, periodontitis

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-β target gene, IL11 expression was partially blocked by SB431542, a TGF-β 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.

(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.

Interactions between gingival fibroblasts (GFs) and oral pathogens contribute to the chronicity of inflammation in periodontitis. Epigenetic changes in DNA methylation are involved in periodontitis pathogenesis, and recent studies indicate that DNA methyltransferase (DNMT) inhibitors may protect against epithelial barrier disruption and bone resorption. To assess the impact of DNMT inhibition on GFs, cells were cultured with decitabine (5-aza-2’-deoxycytidine, DAC) for 12 days to induce DNA hypomethylation. We observed several potentially detrimental effects of DAC on GF biological functions. First, extended treatment with DAC reduced GF proliferation and induced necrotic cell death. Second, DAC amplified Porphyromonas gingivalis- and cytokine-induced expression and secretion of the chemokine CCL20 and several matrix metalloproteinases (MMPs), including MMP1, MMP9, and MMP13. Similar pro-inflammatory effects of DAC were observed in periodontal ligament fibroblasts. Third, DAC upregulated intercellular adhesion molecule-1 (ICAM-1), which was associated with increased P. gingivalis adherence to GFs and may contribute to bacterial dissemination. Finally, analysis of DAC-induced genes identified by RNA sequencing revealed increased expression of CCL20, CCL5, CCL8, CCL13, TNF, IL1A, IL18, IL33, and CSF3, and showed that the most affected processes were related to immune and inflammatory responses. In contrast, the genes downregulated by DAC were associated with extracellular matrix and collagen fibril organization. Our observations demonstrate that studies of DNMT inhibitors provide important insights into the role of DNA methylation in cells involved in periodontitis pathogenesis. However, the therapeutic potential of hypomethylating agents in periodontal disease may be limited due to their cytotoxic effects on fibroblast populations and stimulation of pro-inflammatory pathways.

Abstract Background Milk is a rich source of natural growth factors that may support oral tissue homeostasis and wound healing. We had shown earlier that blocking TGF-β receptor type I kinase with the inhibitor SB431542 abolished the expression of IL11 and other genes in human gingival fibroblasts exposed to the aqueous fraction of milk. Our aim was to identify the entire signature of TGF-β receptor type I kinase-dependent genes regulated by the aqueous fraction of human milk. Result RNAseq revealed 99 genes being strongly regulated by milk requiring activation of the SB431542-dependent TGF-β receptor type I kinase. Among the SB431542-dependent genes is IL11 but also cadherins, claudins, collagens, potassium channels, keratins, solute carrier family proteins, transcription factors, transmembrane proteins, tumor necrosis factor ligand superfamily members, and tetraspanin family members. When focusing on our candidate gene, we could identify D609 to suppress IL11 expression, independent of phospholipase C, sphinosine-1 phosphate synthesis, and Smad-3 phosphorylation and its nuclear translocation. In contrast, genistein and blocking phosphoinositide 3-kinases by wortmannin and LY294002 increased the milk-induced IL11 expression in gingival fibroblasts. Conclusion Taken together, our data revealed TGF-β receptor type I kinase signaling to cause major changes of the genetic signature of gingival fibroblasts exposed to aqueous fraction of human milk.

Aging is a gradual and progressive deterioration of integrity acrossmultiple organ systems that negatively affects gingival wound healing. The cellular responses associated with wound healing, such as collagen synthesis, cell migration, proliferation, and collagen contraction, have been shown to be lower in gingival fibroblasts (the most abundant cells from the connective gingival tissue) in aged donors than young donors. Cellular senescence is one of the hallmarks of aging, which is characterized by the acquisition of a senescenceassociated secretory phenotype that is characterized by the release of proinflammatory cytokines, chemokines, growth factors, and proteases which have been implicated in the recruitment of immune cells such as neutrophils, T cells and monocytes. Moreover, during aging, macrophages show altered acquisition of functional phenotypes in response to the tissue microenvironment. Thus, inflammatory and resolution macrophage-mediated processes are impaired, impacting the progression of periodontal disease. Interestingly, salivary antimicrobial peptides, such as histatins, which are involved in various functions, such as antifungal, bactericidal, enamel-protecting, angiogenesis, and re-epithelization, have been shown to fluctuate with aging. Several studies have associated the presence of Porphyromonas gingivalis, a key pathogen related to periodontitis and apical periodontitis, with the progression of Alzheimer's disease, as well as gut, esophageal, and gastric cancers. Moreover, herpes simplex virus types 1 and 2 have been associated with the severity of periodontal disease, cardiovascular complications, and nervous system-related pathologies. This reviewencompasses the effects of aging on periodontal tissues, how P. gingivalis and HSV infections could favor periodontitis and their relationship with other pathologies. [ABSTRACT FROM AUTHOR]

To evaluate and compare the cytotoxicity of multiple clear aligner systems (Invisalign, Eon, SureSmile, and Clarity). A cytotoxicity assessment was carried out by immersing three sets of aligners from the included four systems in normal saline for 1 month at 37°C. The solutions were then diluted to three different concentrations (5%, 10%, and 20% volume/volume). Gingival fibroblasts were exposed to the solution after being seeded to 96-well microplates for 48 hours, and the medium was substituted with an MTT solution (MTT: 3-[4,5-dime- thylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). Optical density was then measured to determine cell viability and evaluate cytotoxicity subsequently. Cytotoxicity comparison showed no statistically significant difference among the four included systems. However, when cell viability of each system was compared with the control, a significant difference was reported at the 10% and 20% solution concentrations. The Clarity system had the lowest toxicity across all solution concentrations. The thermoplastic materials used by all tested systems (Invisalign, Eon, SureSmile, and Clarity) presented some degree of toxicity (slight to moderate), with statistically significant mean differences compared with the control. [ABSTRACT FROM AUTHOR]

Steckiewicz KP, Cieciórski P, Barcińska E, et al. Int J Nanomedicine. 2022;17:495–517. Our authors have advised that the funding source number listed in the Acknowledgment section on page 513 was incorrect. The sentence should read from “This work was supported by PRELUDIUM grant 2017/27/N/NZ7/0267 from the Polish National Science Centre…” to “This work was supported by PRELUDIUM grant 2017/27/N/NZ7/02675 from the Polish National Science Centre…” The authors apologize for this oversight.

Aging is a gradual and progressive deterioration of integrity across multiple organ systems that negatively affects gingival wound healing. The cellular responses associated with wound healing, such as collagen synthesis, cell migration, proliferation, and collagen contraction, have been shown to be lower in gingival fibroblasts (the most abundant cells from the connective gingival tissue) in aged donors than young donors. Cellular senescence is one of the hallmarks of aging, which is characterized by the acquisition of a senescence-associated secretory phenotype that is characterized by the release of pro-inflammatory cytokines, chemokines, growth factors, and proteases which have been implicated in the recruitment of immune cells such as neutrophils, T cells and monocytes. Moreover, during aging, macrophages show altered acquisition of functional phenotypes in response to the tissue microenvironment. Thus, inflammatory and resolution macrophage-mediated processes are impaired, impacting the progression of periodontal disease. Interestingly, salivary antimicrobial peptides, such as histatins, which are involved in various functions, such as antifungal, bactericidal, enamel-protecting, angiogenesis, and re-epithelization, have been shown to fluctuate with aging. Several studies have associated the presence of Porphyromonas gingivalis, a key pathogen related to periodontitis and apical periodontitis, with the progression of Alzheimer’s disease, as well as gut, esophageal, and gastric cancers. Moreover, herpes simplex virus types 1 and 2 have been associated with the severity of periodontal disease, cardiovascular complications, and nervous system-related pathologies. This review encompasses the effects of aging on periodontal tissues, how P. gingivalis and HSV infections could favor periodontitis and their relationship with other pathologies.

Medication-related osteonecrosis of the jaw (ONJ) is a rare but significant adverse side effect of antiresorptive drugs. Bisphosphonate-related ONJ (BRONJ) is the most prevalent condition due to the extensive use of the drug in cancer and osteoporosis treatment. Nitrogen-containing bisphosphonates suppress osteoclastic resorption by inhibiting farnesyl pyrophosphate synthase in the mevalonate pathway, leading to deficiency of the substrate for GTPase prenylation. The bone remodelling process is uncoupled, subsequently impairing bone healing and causing ONJ. Targeted administration of geranylgeraniol (GGOH) represents a promising approach to mitigate BRONJ because GGOH is a substrate for GTPase prenylation. In the current review, the in vitro effects of GGOH on osteoclasts, osteoblasts and other related cells of the jaw are summarised. We also present and appraise the current in vivo evidence of GGOH in managing BRONJ in animal models. Lastly, several considerations of using GGOH in the clinical management of BRONJ are highlighted. As a conclusion, GGOH is a promising topical agent to manage BRONJ, pending more research on an effective delivery system and validation from a clinical trial. [ABSTRACT FROM AUTHOR]

Medication-related osteonecrosis of the jaw (ONJ) is a rare but significant adverse side effect of antiresorptive drugs. Bisphosphonate-related ONJ (BRONJ) is the most prevalent condition due to the extensive use of the drug in cancer and osteoporosis treatment. Nitrogen-containing bisphosphonates suppress osteoclastic resorption by inhibiting farnesyl pyrophosphate synthase in the mevalonate pathway, leading to deficiency of the substrate for GTPase prenylation. The bone remodelling process is uncoupled, subsequently impairing bone healing and causing ONJ. Targeted administration of geranylgeraniol (GGOH) represents a promising approach to mitigate BRONJ because GGOH is a substrate for GTPase prenylation. In the current review, the in vitro effects of GGOH on osteoclasts, osteoblasts and other related cells of the jaw are summarised. We also present and appraise the current in vivo evidence of GGOH in managing BRONJ in animal models. Lastly, several considerations of using GGOH in the clinical management of BRONJ are highlighted. As a conclusion, GGOH is a promising topical agent to manage BRONJ, pending more research on an effective delivery system and validation from a clinical trial.

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. [ABSTRACT FROM AUTHOR]

Aim(s): The aim of the study was to fabricate a nanospike surface on a titanium alloy surface using a newly established method of low-energy helium ion bombardment. Various methods to achieve nanospike formation on titanium have been introduced recently, and their antibacterial properties have been mainly investigated with respect to Escherichia coli and Staphylococcus aureus. Oral pathogens such as Porphyromonas gingivalis play an important role in the development of peri-implantitis. For that reason, the antibacterial properties of the novel, nanostructured titanium surface against P. gingivalis were assessed, and a possible effect on the viability of gingival fibroblasts was evaluated. Materials and Methods: Helium sputtering was employed for developing titanium surfaces with nanospikes of 500 nm (ND) in height; commercially available smooth-machined (MD) and sandblasted and acid-etched titanium disks (SLA) were used as controls. Surface structure characterization was performed through scanning electron microscopy (SEM) and atomic force microscopy (AFM). Following incubation with P. gingivalis, antibacterial properties were determined via conventional culturing and SEM. Additionally, the viability of human gingival fibroblasts (HGFs) was tested through MTT assay, and cell morphology was assessed through SEM. Results: SEM images confirmed the successful establishment of a nanospike surface with required heights, albeit with heterogeneity. AFM images of the 500 nm nanospike surface revealed that the roughness is dominated by large-scale hills and valleys. For frame sizes of 5 × 5 μm and smaller, the average roughness is dominated by the height of the titanium spikes. ND successfully induces dysmorphisms within P. gingivalis cultures following the incubation period, while conventional culturing reveals a 17% and 20% reduction for ND compared to MD and SLA, respectively. Moreover, the nanospike surfaces did not affect the viability of human growth fibroblasts despite their sharp surface. Conclusion(s): This study successfully developed a novel titanium-nanospike-based structuration technique for titanium surfaces. In addition, the nanospikes did not hinder gingival fibroblast viability. Enhanced antimicrobial effects for such a novel nanospike-based resurfacing technique can be achieved through further optimizations for nanospike spacing and height parameters. [ABSTRACT FROM AUTHOR]

Aim of the study We studied the effects of fibroblasts autophagy on the apoptosis of pro-B murine lymphocytes when were co-cultured. Material and methods Study of literature suggested that released autophagy-derived metabolites from fibroblasts might affect the apoptotic behavior of lymphocytes when there is a close interaction of these cells. The experimental plan of the actual studies was represented by induced effects of the presence of gingival fibroblasts in co-culture on NAK-1 receptors-activated pro-lymphocytes B apoptosis. Results We clearly showed that murine pro-B lymphocyte apoptosis induced by indol-3-carbinol could be significantly enhanced by the presence of gingival fibroblasts in co-culture. Furthermore, the addition of 3-methyl-adenine in the medium for 24 hours blocked the enhancing effects induced by fibroblasts on the apoptotic index of pro-B lymphocytes. Conclusions So, the enhancing apoptotic effects of fibroblasts on pro-B lymphocytes are related to released autophagy-derived metabolites. Their real nature remains to be established through future researches involving this complex system.

The aim of this study is to synthesize and investigate antimicrobial and cytotoxic activity of 3 H-quinazolinone (1), and its derivatives, which are 3-(4-hydroxyphenyl)-3,4-dihydroquinazolin-4-one(2),3-(3-hydroxyphenyl)-3,4-dihydroquinazolin-4-one(3), 3-(pyridin-3-ylmethy)-3,4-dihydroquinazolin-4-one(4) and derivatives of 2-methyl-3H-quinazolinone, which are 2-methyl-3-phenyl-3,4-dihydroquinazolin-4-one(6), 2-methyl-3(4-hydroxyphenyl)-3,4- dihydroquinazolin-4-one(7),2-methyl-3(4-nitrophenyl)-3,4-dihydroquinazolin-4-one(8), compounds. Compounds 2 and 3 were synthesized newly. All compounds were characterized via infrared, 1H-NMR, 13C-NMR, elemental and mass spectral analysis. The compounds were screened for their antimicrobial activity in vitro against E. faecalis ATCC 29212, P. aeruginosa ATCC 27853, S. aureus ATCC 29213, E. Coli ATCC 25922 bacteria and compared with commercial antibiotics (Ampicillin and Gentamicin). The determination of the antimicrobial activity was done by using the broth microdilution methods. The antimicrobial test results indicated that the compounds (1, 4, 6, and 8) have MIC values against P. aeruginosa ATCC 27853 lower than ampicillin. But clear cytotoxic effects on proliferation of the gingival fibroblasts applications were observed in 1, 4, 6, and 8 when compared to the control group. [ABSTRACT FROM AUTHOR]

Background: Milk is a rich source of natural growth factors that may support oral tissue homeostasis and wound healing. We had shown earlier that blocking TGF-β receptor type I kinase with the inhibitor SB431542 abolished the expression of IL11 and other genes in human gingival fibroblasts exposed to the aqueous fraction of milk. Our aim was to identify the entire signature of TGF-β receptor type I kinase-dependent genes regulated by the aqueous fraction of human milk. Result: RNAseq revealed 99 genes being strongly regulated by milk requiring activation of the SB431542-dependent TGF-β receptor type I kinase. Among the SB431542-dependent genes is IL11 but also cadherins, claudins, collagens, potassium channels, keratins, solute carrier family proteins, transcription factors, transmembrane proteins, tumor necrosis factor ligand superfamily members, and tetraspanin family members. When focusing on our candidate gene, we could identify D609 to suppress IL11 expression, independent of phospholipase C, sphinosine-1 phosphate synthesis, and Smad-3 phosphorylation and its nuclear translocation. In contrast, genistein and blocking phosphoinositide 3-kinases by wortmannin and LY294002 increased the milk-induced IL11 expression in gingival fibroblasts. Conclusion: Taken together, our data revealed TGF-β receptor type I kinase signaling to cause major changes of the genetic signature of gingival fibroblasts exposed to aqueous fraction of human milk. [ABSTRACT FROM AUTHOR]

Aim of the study We explored the relationships between the gingival fibroblasts and pro-B lymphocytes apoptosis in vitro because the current medical literature is lacking such data. Material and methods The aim of our study was represented by the effects of gingival fibroblasts on the apoptosis of Nur77 receptors-activated pro-lymphocytes in vitro, also in the presence of some retinoid derivatives. Gingival fibroblasts were obtained from male rats. Pro-B cells of murine type in cultures were used for actual experiments. The apoptosis of pro-B cells was induced using a combination of cytosporone B (50 µM) and cyclosporine A (1 µM). The apoptotic effects were revealed using the classical flow cytometry techniques. Results We clearly showed that pro-B lymphocyte apoptosis induced by the combination of cytosporone B (Nur77 agonist) and cyclosporine A in the presence of gingival fibroblasts could be slightly reduced by 13-cis retinoic acid. The inhibitory effects of 13-cis retinoic acid were powerful than those of tazarotenic acid. The effects of 13-cis retinoic acid and tazarotenic acid were evaluated toward the effects of the combination of cytosporone B and cyclosporine A. Conclusions The intrinsic pathways of retinoid derivatives remain to be established by further experiments

The enamel renal syndrome (ERS) is a rare disorder featured by amelogenesis imperfecta , gingival fibromatosis and nephrocalcinosis. ERS is caused by bi-allelic mutations in the secretory pathway pseudokinase FAM20A. How mutations in FAM20A may modify the gingival connective tissue homeostasis and cause fibromatosis is currently unknown. We here analyzed conditioned media of gingival fibroblasts (GFs) obtained from four unrelated ERS patients carrying distinct mutations and control subjects. Secretomic analysis identified 109 dysregulated proteins whose abundance had increased (69 proteins) or decreased (40 proteins) at least 1.5-fold compared to control GFs. Proteins over-represented were mainly involved in extracellular matrix organization, collagen fibril assembly, and biomineralization whereas those under-represented were extracellular matrix-associated proteins. More specifically, transforming growth factor-beta 2, a member of the TGFβ family involved in both mineralization and fibrosis was strongly increased in samples from GFs of ERS patients and so were various known targets of the TGFβ signaling pathway including Collagens, Matrix metallopeptidase 2 and Fibronectin. For the over-expressed proteins quantitative RT-PCR analysis showed increased transcript levels, suggesting increased synthesis and this was further confirmed at the tissue level. Additional immunohistochemical and western blot analyses showed activation and nuclear localization of the classical TGFβ effector phospho-Smad3 in both ERS gingival tissue and ERS GFs. Exposure of the mutant cells to TGFB1 further upregulated the expression of TGFβ targets suggesting that this pathway could be a central player in the pathogenesis of the ERS gingival fibromatosis. In conclusion our data strongly suggest that TGFβ -induced modifications of the extracellular matrix contribute to the pathogenesis of ERS. To our knowledge this is the first proteomic-based analysis of FAM20A-associated modifications. [ABSTRACT FROM AUTHOR]

• Secondary caries is the main reason for failure of composite fillings. • New antibacterial composites have proven to modify biofilm formation in qualitative and quantitative ways. • New antiadhesive composites have shown to diminish bacterial adhesion. • Biocompatibility was tested using LDH, BrdU, MTT, TGF-ß1, IL-8. • Antibacterial composites damage human gingival fibroblasts; antiadhesives do not. Aim of this study was to evaluate the biocompatibility of four experimental antiadhesive and antibacterial dental filling composites on human gingival fibroblasts (HGFs). For these experimental resin composites a delivery system based on novel polymeric hollow beads, loaded with Tego Protect (Aa1), Dimethicone (Aa2), Irgasan (Ab1) and methacrylated polymerizable Irgasan (Ab2) as active agents was used. The cultured HGFs' cell integrity, proliferation, viability, collagen synthesis and cytokine release were measured. For this purpose, human gingival fibroblasts were treated with eluates from all four composites and compared with an experimental standard composite (ST). Eluate extraction times 24 h and 168 h were chosen. Statistical analysis was conducted via a mixed model. Both antibacterial composites reduced proliferation, collagen and cytokine synthesis significantly (p < 0.05), increasing with time of elution. Ab1 did also have a damaging effect on the membrane and on cell viability. Overall, it can be concluded that the antiadhesive composites showed clear advantages over the antibacterial composites in terms of biocompatibility. This study also continues to show the potential of the new poly-pore system, as it can be used for a variety of other applications in future composite mixtures. [ABSTRACT FROM AUTHOR]

The enamel renal syndrome (ERS) is a rare disorder featured by amelogenesis imperfecta, gingival fibromatosis and nephrocalcinosis. ERS is caused by bi-allelic mutations in the secretory pathway pseudokinase FAM20A. How mutations in FAM20A may modify the gingival connective tissue homeostasis and cause fibromatosis is currently unknown. We here analyzed conditioned media of gingival fibroblasts (GFs) obtained from four unrelated ERS patients carrying distinct mutations and control subjects. Secretomic analysis identified 109 dysregulated proteins whose abundance had increased (69 proteins) or decreased (40 proteins) at least 1.5-fold compared to control GFs. Proteins over-represented were mainly involved in extracellular matrix organization, collagen fibril assembly, and biomineralization whereas those under-represented were extracellular matrix-associated proteins. More specifically, transforming growth factor-beta 2, a member of the TGFβ family involved in both mineralization and fibrosis was strongly increased in samples from GFs of ERS patients and so were various known targets of the TGFβ signaling pathway including Collagens, Matrix metallopeptidase 2 and Fibronectin. For the over-expressed proteins quantitative RT-PCR analysis showed increased transcript levels, suggesting increased synthesis and this was further confirmed at the tissue level. Additional immunohistochemical and western blot analyses showed activation and nuclear localization of the classical TGFβ effector phospho-Smad3 in both ERS gingival tissue and ERS GFs. Exposure of the mutant cells to TGFB1 further upregulated the expression of TGFβ targets suggesting that this pathway could be a central player in the pathogenesis of the ERS gingival fibromatosis.In conclusion our data strongly suggest that TGFβ -induced modifications of the extracellular matrix contribute to the pathogenesis of ERS. To our knowledge this is the first proteomic-based analysis of FAM20A-associated modifications.