Core-Shell Electrospun Membranes Enable Antimicrobial and Immunomodulatory Local Therapy for Periodontitis.

Matrix metalloproteinases (MMPs) play a key role in inflammatory periodontal disease. Synergistically enhanced MMP-2 expression in a coculture of human gingival fibroblasts (HGFs) and human monocytic U937 cells was observed. Crosstalk between these two cells via the extracellular matrix metalloproteinase inducer (EMMPRIN) was demonstrated. Enzyme levels of MMP-2 in HGFs and direct coculture with U937 were examined by zymography. MMP-2 and EMMPRIN expressions of HGFs and U937 were determined in coculture and conditioned cultures (using supernatants from HGF- or U937-conditioned medium). The crosstalk was evaluated by EMMPRIN extrasupplement and EMMPRIN inhibition, through pretreatment of U937 with cyclosporine-A. Direct coculturing of HGFs and U937 enhanced MMP-2 enzyme level and mRNA expression. Coculturing also increased membranous EMMPRIN expression of U937, but not from HGFs. In conditioned cultures, mRNA expression of MMP-2 increased in HGFs which received U937-conditioned medium. Increased MMP-2 was not observed in U937 with HGF-conditioned medium, although mRNA expression of EMMPRIN increased. Enhanced MMP-2 was observed after administration of exogenous EMMPRIN in HGFs; however, reduced MMP-2 enzyme level was noted if EMMPRIN of cocultured U937 was inhibited. In the coculture of HGFs and U937, upregulated EMMPRIN expression in U937, which may be triggered by HGFs, can enhance MMP-2 expression in HGFs. Crosstalk between HGFs and U937 involving MMP-2 from HGFs was proposed; EMMPRIN from U937 may play a particular role.

In vitro evaluation of poly (vinyl alcohol)/collagen blended hydrogels for regulating human periodontal ligament fibroblasts and gingival fibroblasts

Cluster of differentiation 147 (CD147) is a multifunctional glycoprotein that functions as an inducer of matrix metalloproteinase (MMP) expression in fibroblasts. Synergistically enhanced MMP-2 expression was recently observed in the coculture of human gingival fibroblasts (HGFs) and U937 human monocytic cells; however, the responsible mechanisms have not yet been fully established. The aim of this study was to evaluate the release of soluble CD147 in HGFs after coculturing with U937 cells and its functional effect on the enhancement of MMP-2 expression in HGFs. Enzyme-linked immunosorbent assay was used to determine the amount of CD147 protein in media, whereas real-time polymerase chain reaction was performed to evaluate the mRNA levels of CD147 and MMP-2 in HGFs and U937 cells. The enzyme activities of MMP-2 released from cells were examined by zymography. Transwell coculturing and conditioned media treatments were selected to rule out the effect of direct contact of HGFs and U937 cells. The protein and mRNA expression of CD147 in HGFs were enhanced after transwell coculturing with U937 cells and exposure to U937-conditioned medium. MMP-2 enzyme activities in HGFs were also significantly increased by the coculturing methods. Administration of exogenous CD147 enhanced MMP-2 expression in HGFs, whereas treatment with cyclosporine-A, which inhibited CD147 expression, reduced U937-enhanced MMP-2 expression in HGFs. CD147 can interact with fibroblasts to stimulate the expression of MMPs associated with periodontal extracellular matrix degradation. This study has demonstrated that CD147 released from fibroblasts might play a role in monocyte-enhanced MMP-2 expression in HGFs.

Antimicrobial and anti-biofilm activity of a mucoadhesive hydrogel functionalized with aminochalcone on titanium surfaces and in Galleria mellonella model: In vitro and in vivo study.

The objective of this work was to determine the antimicrobial and antibiofilm properties of mineral trioxide aggregate (MTA) supplemented with bismuth lipophilic nanoparticles (BisBAL NPs). The antimicrobial activity of the composite MTA-BisBAL NPs was determined by the disk diffusion assay, while antibiofilm activity was analyzed by fluorescence microscopy. The cytotoxicity of MTA-BisBAL NPs was determined on human gingival fibroblasts by optical microscopy and crystal violet staining. MTA-BisBAL NPs inhibited the growth of Enterococcus faecalis, Escherichia coli, and Candida albicans and also detached the biofilm of fluorescent E. faecalis after 24 h of treatment. The addition of BisBAL nanoparticles did not significantly modify the physical properties of MTA, and cytotoxicity was not observed when MTA-BisBAL NPs was added on human gingival fibroblasts. Altogether these results suggest that BisBAL nanoparticles provide antimicrobial and antibiofilm activities to MTA while it retained their biophysical properties without cause side effects on human gingival fibroblasts.

Oral health diseases are common in all regions of the world. Mouth rinses are widely used generally by population as a port of daily oral care regimen. In addition to antimicrobial activity, mouth rinses possess certain cytotoxic effects. Electron-beam (E-beam) radiation is a form of ionizing energy known to induce structural, physical, and chemical changes in irradiated products. In this study, the modulatory effects of E-beam in irradiated mouth rinses were evaluated for its biological activities. The antimicrobial activities of nonirradiated and irradiated mouth rinses were evaluated for its antimicrobial and antibiofilm activities against oral pathogens, Enterococcus faecalis, Streptococcus mutans, Staphylococcus aureus, and Candida albicans. The antimicrobial activity was evaluated by disc diffusion method and antibiofilm activity was evaluated by O'Toole method. The cytotoxicity was evaluated against human gingival fibroblast (HGF) cells by 3-(4, 5 Dimethythiazol-yl)-2,5-Diphenyl-tetrazolium bromide assay. Colgate Plax (CP) exhibited the antimicrobial activity against the tested pathogens, and a significant (P< 0.05) increase was observed against S. aureus at 750 Gy irradiation. Further, CP significantly (P< 0.05) suppressed S. mutans, S. aureus, and C. albicans biofilm. Listerine (LS) inhibited S. mutans and C. albicans biofilm. Whereas irradiated CP and LS significantly (P< 0.05) suppressed the biofilm formed by oral pathogens. The suppression of biofilm by irradiated mouth rinses was dose- and species-dependent. There was no significant (P > 0.05) difference in the cytotoxicity of irradiated and nonirradiated mouth rinses on HGF cells. However, an increased percentage viability of HGF cells was observed by mouth rinses irradiated at 750 Gy.xs The E-beam irradiation enhanced the antibiofilm activity of mouth rinses without modifying the cytotoxicity.

Periodontal disease is a chronic inflammatory disease caused by periodontopathic bacteria accumulated in the gingival sulcus and periodontal pocket. Cigarette smoking is a well-established risk factor for periodontal disease, and periodontal tissues in smokers are chronically exposed to cigarette smoke on a long-term basis. In this study, we investigated the effects of long-term exposure to nicotine or cigarette smoke condensate (CSC) on cellular functions of human gingival fibroblasts (HGFs). In vitro-maintained HGFs were divided into two groups. The HGFs of the short-term and the long-term culture groups were cultured for 4 and 25days, respectively, in the presence or absence of nicotine, which is one of the main components of cigarette smoke, or CSC. The cellular proliferation and migration capacities of HGFs exposed to nicotine or CSC were evaluated by WST-1 and wound healing assays. The effects of exposure to nicotine or CSC on the expression of various extracellular matrix (ECM) components, inflammatory cytokines, and senescence-related genes were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The cellular senescence of HGFs exposed to nicotine or CSC was detected by the senescence-associated β-galactosidase (SA-β-gal) assay. To explore the senescence-associated microRNA (miRNA), we extracted miRNA from the HGFs and the expression profiles were examined by miRNA array. In short-term culture, no significant changes were observed. Long-term exposure of HGFs to nicotine or CSC significantly suppressed their cellular proliferation and migration and upregulated type Ⅰ collagen, type Ⅲ collagen, interleukin (IL)-6, IL-8, p16, p21, and p53mRNA expression, and IL-6 and IL-8 protein expression. Furthermore, long-term nicotine or CSC exposure significantly increased the percentage of SA-β-gal-positive HGFs. In addition, long-term nicotine or CSC exposure reduced miR-29b and miR-199a expression to less than 50% of that in the unstimulated HGFs. These data suggest that long-term smoking habits may reduce wound healing ability, modulate ECM protein homeostasis, stimulate the inflammatory response, and accelerate cellular senescence in HGFs, and consequently accelerate the progression of periodontal diseases.

BackgroundIL-32 was recently found to be elevated in the tissue of rheumatoid arthritis and inflammatory bowel disease. Periodontitis is a chronic inflammatory disease caused by polymicrobial infections that result in soft tissue destruction and alveolar bone loss. Although IL-32 is also thought to be associated with periodontal disease, its expression and possible role in periodontal tissue remain unclear. Therefore, this study investigated the expression patterns of IL-32 in healthy and periodontally diseased gingival tissue. The expression of IL-32 in cultured human gingival fibroblasts (HGF) as well as effects of autocrine IL-32 on IL-8 production from HGF were also examined.MethodsPeriodontal tissue was collected from both healthy volunteers and periodontitis patients, and immunofluorescent staining was performed in order to determine the production of IL-32. Using real-time PCR and ELISA, mRNA expression and protein production of IL-32 in HGF, stimulated by Porphyromonas gingivalis (Pg), were also investigated.ResultsContrary to our expectation, the production of IL-32 in the periodontitis patients was significantly lower than in the healthy volunteers. According to immunofluorescent microscopy, positive staining for IL-32 was detected in prickle and basal cell layers in the epithelium as well as fibroblastic cells in connective tissue. Addition of fixed Pgin vitro was found to suppress the otherwise constitutive expression of IL-32 mRNA and protein in HGF. However, recombinant IL-32 in vitro inhibited the expression of IL-8 mRNA by HGF stimulated with Pg. Interestingly, anti-IL-32 neutralizing antibody upregulated the IL-8 mRNA expression in non-stimulated HGF, indicating that constitutive expression of IL-32 in HGF suppressed IL-8 mRNA expression in the absence of bacterial stimulation.ConclusionThese results indicate that IL-32 is constitutively produced by HGF which can be suppressed by Pg and may play a role in the downregulation of inflammatory responses, such as IL-8 production, in periodontal tissue.

Histatins, a family of salivary proteins, have antimicrobial activity. Candida albicans, which is killed by histatins, induces oral candidiasis in individuals with compromised immune systems. Although the functional significance of histatins has been documented, their biological and physiological functions against host cells have not been clarified. In this study, we found that histatin 3, a member of the histatin family, binds to heat shock cognate protein 70 (HSC70). These proteins were co-localized in the cytoplasm and nucleus in human gingival fibroblasts following non-heat and heat shock. Histatin 3 induced stimulation of DNA synthesis and cell survival in human gingival fibroblasts in a dose-dependent manner. This DNA synthesis was found to be dependent on HSC70 by knockdown experiments. The effect of heat shock on DNA synthesis induced by histatin 3 was approximately 2-fold higher than that of non-heat shock. When the histatin 3 uptake into cells was inhibited by monodansylcadaverine or when histatin 3 binding to HSC70 was precluded by 15-deoxyspergualin, DNA synthesis by histatin 3 was approximately 2-fold less than that without monodansylcadaverine or 15-deoxyspergualin. Although HSC70 directly bound to p27(Kip1) (a cyclin-dependent kinase inhibitor), histatin 3 increased the binding between those proteins but not with a peptide capable of binding to HSC70. Moreover histatin 3 prevented ATP-dependent dissociation of HSC70-p27(Kip1). ATP was unable to form a histatin 3-HSC70(D10N)-p27(Kip1) complex (HSC70(D10N) is a mutant attenuating ATPase activity). These findings suggest that histatin 3 may be involved in cell proliferation through the regulation of HSC70 and p27(Kip1) in oral cells.

Antimicrobial activities of natural plant compounds against endodontic pathogens and biocompatibility with human gingival fibroblasts

Emulsion-loading-driven transitions in microstructure and release kinetics of sodium alginate films incorporating a succinylated pea protein-stabilized cinnamon essential oil Pickering emulsion.

Effects of Nitrogen-containing Bisphosphonates on the Response of Human Peripheral Blood Mononuclear Cells and Gingival Fibroblasts to Bacterial Components

Periodontitis, a chronic inflammatory disease, is characterized by increased expression of interleukin (IL)-1 and other inflammatory mediators resulting in extensive osteoclast formation and bone loss. Expression of receptor activator of nuclear factor kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), by osteoblasts is important to regulate osteoclast differentiation. The aim of the present study was to investigate the regulatory effects of IL-1 on RANKL and OPG production by mesenchymal fibroblasts in periodontal tissue. Human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) were stimulated with IL-1alpha with or without protein synthesis inhibitor cycloheximide (CHX), protein kinase A (PKA) inhibitors, protein kinase C (PKC) inhibitors and prostaglandin E(2) (PGE(2)) inhibitor. In some experiments, the cultured cells were directly stimulated with either PKA or PKC activators. In HGF, IL-1alpha-stimulated OPG mRNA expression was high and could be reduced by CHX. PKA inhibitor completely abrogated IL-1alpha-induced OPG mRNA expression and OPG production. Endogenous PGE(2) further enhanced IL-1alpha-induced OPG production in HGF. In PDL, RANKL mRNA expression was greatly augmented by IL-1alpha. IL-1alpha induced OPG mRNA expression and protein production. PKC inhibitor partially reduced IL-1alpha-induced OPG production and PKC activator enhanced OPG production in PDL. The IL-1alpha-stimulated OPG mRNA expression in HGF was greater than PDL. These results provide new evidence for the possible osteoclastogenesis-inhibitory function of HGF through PKA activity pathway. PDL utilized PKC for OPG production. Thus, we emphasize that HGF and PDL have different characteristics of host defence mechanism against inflammatory process.

Applying multifunctional coatings employing strontium (Sr) ions on titanium (Ti) surfaces is a useful and biocompatible method to improve osseointegration and prevent tissue infections through antimicrobial activity. Nonetheless, the effectiveness of Sr coating on the adhesion and viability of human gingival fibroblasts (HGFs) to Ti surfaces remains unclear. The study aimed to evaluate the effect of Sr coating on the adhesion and viability of HGFs to Ti surfaces. The Ti wafers were divided into two groups based on Sr coating: uncoated Ti (control) and Sr-coated Ti. The Magnetron sputtering technique was used for Sr coating on Ti surfaces. The HGFs were seeded onto the surfaces and cultured for 48 and 96 hours before the cell adhesion and viability of the attached HGFs were assessed. The adhesion of HGFs was analyzed using the attached cell numbers at 48 h and 96 h, and the morphology at 24 h and 72 h. The cytotoxic effect on HGFs was assessed after 24 and 72 hours of incubation using cell viability assay. Student's t-test was used for statistical analysis. The number of cells attached to Sr-coated surfaces was significantly greater than those attached to uncoated Ti surfaces after 48 hours (P<0.0001) and 96 hours (P=0.0002). Sr-coated and uncoated Ti surfaces were not cytotoxic to HGFs, with the cell viability ranging from 92% to 105% of the untreated control HGFs. There were no significant differences in cell viability between Sr-coated and uncoated Ti surfaces at 24 hours (P=0.3675) and 72 hours (P=0.0982). Sr-coated Ti surfaces induce adhesion of HGFs compared to uncoated Ti surfaces. Further, Sr-coated and uncoated Ti surfaces show no cytotoxic effect on the attached HGFs.

Periodontitis is a bacterial infection characterized by chronic gingival inflammation, which leads to the loss of tooth-supporting tissues. Dense infiltration of activated memory T cells and high levels of their cytokines were consistently detected in periodontal lesions. Although T cells have been considered to be central to both progression and control of chronic inflammatory periodontal diseases, the definite contribution in local immunoregulation has not been fully clarified. Recent observations showed the presence of IL-17, a novel T cell cytokine, and IFN-gamma in the inflamed periodontal tissues. Therefore, in this present study we investigated the immunostimulatory role of IL-17 and IFN-gamma on human gingival fibroblasts (HGF) which were obtained from clinically healthy periodontal tissues. Various concentrations of IL-17, IFN-gamma, or the combination of these two cytokines were added to HGF cultures. The expression of CD40 and HLA-DR was assessed by flow cytometry and IL-8 production was determined by ELISA. Our results demonstrated that IFN-gamma markedly up-regulated HLA-DR and minimally up-regulated CD40 expression on HGF. IL-17 did not induce the expression of both molecules and did not enhance IFN-gamma-induced CD40 and HLA-DR expression on HGF. Unlike IFN-gamma, IL-17 induced IL-8 production from HGF. When combined, IFN-gamma synergistically enhanced IL-17-induced IL-8 production. This enhancement was detected in all HGF cell lines at a higher dose of IL-17 (500 ng/ml) whereas at lower doses (5 and 50 ng/ml), heterogeneous response was observed. The findings of heterogeneity in IL-8 production by HGF in response to the combination of IL-17 and IFN-gamma are interesting and may explain the variation in host response in disease susceptibility or disease progression in periodontitis. Clearly, further investigation into this issue is needed.