Response Of Human Gingival Fibroblasts Research Articles

Characterization of Human Gingival Fibroblasts on Zirconia Surfaces Containing Niobium Oxide.

It was indicated that tetragonal zirconia polycrystal (TZP) containing yttria (Y2O3) and niobium oxide (Nb2O5) ((Y,Nb)-TZP) could be an adequate dental material to be used at esthetically important sites. The (Y,Nb)-TZP was also proved to possess its osteogenic potential comparable with those conventional dental implant material, titanium (Ti). The objective of the current study was to characterize cellular response of human gingival fibroblasts (HGFs) to smooth and rough surfaces of the (Y,Nb)-TZP disc, which were obtained by polishing and sandblasting, respectively. Various microscopic, biochemical, and molecular techniques were used to investigate the disc surfaces and cellular responses for the experimental (Y,Nb)-TZP and the comparing Ti groups. Sandblasted rough (Y,Nb)-TZP (Zir-R) discs had the highest surface roughness. HGFs cultured on polished (Y,Nb)-TZP (Zir) showed a rounded cell morphology and light spreading at 6 h after seeding and its proliferation rate significantly increased during seven days of culture compared to other surfaces. The mRNA expressions of type I collagen, integrin α2 and β1 were significantly stimulated for the Zir group at 24 h after seeding. The current findings, combined with the previous results, indicate that (Y,Nb)-TZP provides appropriate surface condition for osseointegration at the fixture level and for peri-implant mucosal sealing at the abutment level producing a suitable candidate for dental implantation with an expected favorable clinical outcome.

Veratric Acid Inhibits LPS-Induced IL-6 and IL-8 Production in Human Gingival Fibroblasts.

Veratric acid, one of the major benzoic acid isolated from vegetables and fruits, has been reported to have anti-inflammatory activity. The purpose of this study was to investigate the anti-inflammatory effects of veratric acid on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (HGFs). HGFs were pretreated with veratric acid 1h before LPS stimulation. The productions of IL-6 and IL-8 were detected by ELISA. The expression of iNOS, COX-2, PI3K, AKT, and NF-κB were detected by western blotting. The results showed that veratric acid inhibited LPS-induced IL-6 and IL-8 production, as well as iNOS and COX-2 expression. Veratric acid also inhibited LPS-induced NF-κB activation. In addition, veratric acid was found to suppress LPS-induced PI3K and AKT phosphorylation. In conclusion, the anti-inflammatory mechanism of veratric acid is due to its ability to inhibit PI3K/Akt/NF-κB signaling pathway.

Improved human gingival fibroblast response to titanium implants coated with ultraviolet-irradiated vitamin D precursor and vitamin E.

Ultraviolet (UV)-irradiated 7-dehydrocholesterol (7-DHC) and vitamin E (VitE)-coated titanium (Ti) implants have a beneficial effect on bone cells. Human gingival fibroblasts (HGFs) are the most abundant cells in periodontal tissues and are involved in the wound healing and repair. The objective of this study was to evaluate the response of HGFs to Ti implants coated with UV-irradiated 7-DHC and VitE, for improved soft-tissue integration of dental implants. Ti surfaces were coated with 7-DHC and VitE, irradiated with UV light and incubated for 48h at 23°C to allow cholecalciferol (D3 ) synthesis from 7-DHC onto the Ti surface. HGFs were cultured on the modified surfaces and the influence of the coating on these cells was evaluated through the analysis of: (i) biocompatibility; (ii) the mRNA levels of genes involved in the composition and turnover of the extracellular matrix, the inflammatory response, periodontal bone resorption and wound healing; and (iii) the levels of MMP-1 and TIMP-1 proteins. We found a beneficial effect of UV-irradiated 7-DHC:VitE-coated Ti implants on HGFs. Besides being biocompatible with HGFs, the UV-irradiated 7-DHC and VitE coating increased the levels of collagen III α1 and fibronectin mRNAs. and decreased the level of interleukin-8 mRNA. TIMP-1 was increased at both mRNA and protein levels in HGFs cultured on UV-irradiated 7-DHC:VitE-coated Ti implants. Finally, the UV-irradiated 7-DHC and VitE coating decreased the level of RANKL mRNA in HGFs. UV-irradiated 7-DHC:VitE-coated Ti implants have a positive effect on HGFs invitro by reducing the inflammatory response and extracellular matrix breakdown.

Surface modification of zirconia with polydopamine to enhance fibroblast response and decrease bacterial activity in vitro: A potential technique for soft tissue engineering applications

The quality of soft-tissue integration plays an important role in the short- and long-term success of dental implants. The aim of the present study was to provide a surface modification approach for zirconia implant abutment materials and to evaluate its influence on fibroblast behavior and oral bacteria adhesion, which are the two main factors influencing the quality of peri-implant soft-tissue seal. In this study, polydopamine (PDA)-coated zirconia was prepared and the surface characteristics were evaluated using scanning electron microscopy, atomic force microscopy, a contact-angle-measuring device, X-ray photoelectron spectroscopy, and Raman spectroscopy. The responses of human gingival fibroblasts (HGFs) to PDA-coated zirconia; i.e., adhesion, proliferation, morphology, protein synthesis, and gene expression, were analyzed. Additionally, the adhesion of Streptococcus gordonii and Streptococcus mutans to zirconia after PDA coating was assessed by scanning electron microscopy and live/dead staining. The material surface analyses suggested the successful coating of PDA onto the zirconia surface. The PDA coating significantly increased cell adhesion and proliferation compared with pristine zirconia. HGFs exhibited a high degree of spreading and secreted a high level of collagen type I on PDA-modified disks. Upregulation of integrin α5, β1, β3 and fibronectin was noted in HGFs cultured on PDA-coated zirconia. The number of adherent bacteria decreased significantly on zirconia after PDA coating. In summary, our result suggest that PDA is able to modify the surface of zirconia, influence HGFs' behavior and reduce bacterial adhesion. Therefore, this surface modification approach holds great potential for improving soft-tissue integration around zirconia abutments in clinical application.

Tormentic acid inhibits LPS-induced inflammatory response in human gingival fibroblasts via inhibition of TLR4-mediated NF-κB and MAPK signalling pathway

ObjectivePeriodontal disease is one of the most prevalent oral diseases, which is associated with inflammation of the tooth-supporting tissues. Tormentic acid (TA), a triterpene isolated from Rosa rugosa, has been reported to exert anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effects of TA on lipopolysaccharide (LPS)-stimulated human gingival fibroblasts (HGFs). MethodsThe levels of inflammatory cytokines such as interleukin (IL)-6 and chemokines such as IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). The expression of Toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), IκBα, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) was determined by Western blotting. ResultsThe results showed that Porphyromonas gingivalis LPS significantly upregulated the expression of IL-6 and IL-8. TA inhibited the LPS-induced production of IL-6 and IL-8 in a dose-dependent manner. Furthermore, TA inhibited LPS-induced TLR4 expression; NF-κB activation; IκBα degradation; and phosphorylation of ERK, JNK, and P38. ConclusionTA inhibits the LPS-induced inflammatory response in HGFs by suppressing the TLR4-mediated NF-κB and mitogen-activated protein kinase (MAPK) signalling pathway.

Differentiation, apoptosis, and GM-CSF receptor expression of human gingival fibroblasts on a titanium surface treated by a dual acid-etched procedure.

Analysis of the effects of titanium surface properties on the biological behavior of human gingival fibroblasts (HGFs). HGFs were in vitro cultured on a titanium surface modified by a dual acid-etched procedure and on a control machined surface. Cell adhesion, proliferation, apoptosis, production of certain extracellular matrix (ECM) proteins, and expression of granulocyte macrophage-colony stimulating factor receptor (GM-CSFR) were investigated using in each experiment a total of 18 samples for each titanium surface. Cell attachment at 3 h of culture was statistically significantly higher on the etched surface. HGF growth increased on both surfaces during the entire experimental period and at day 14 of culture cell proliferation was statistically significantly higher on the treated surface than on the control. No statistically significant differences in percentage of apoptosis events were observed between the surfaces. ECM protein production increased progressively over time on both surfaces. A statistically significant deposition was observed at day 7 and 14 for collagen I and only at day 14 for fibronectin and tenascin, when compared to the baseline. GM-CSFR registered a positive expression on both surfaces, statistically significant at day 14 on the etched surface in comparison with the machined one. Data showed that titanium surface microtopography modulates in vitro cell response and phenotypical expression of HGFs. The etched surface promoted a higher cell proliferation and differentiation improving the biological behavior of HGFs. Results suggest a possible beneficial effect of surface etching modification on peri-implant biological integration and soft tissue healing which is critical for the formation of a biological seal around the neck of dental implants.

Complementing Confocal Detection of Antibody‐labeled Lysophosphatidic Acid Receptors in Human Gingivae with Label‐free Second Harmonic Generation Confocal Microscopy Detection of Collagen

We have found that lysophosphatidic acid (LPA) controls the healing and [Ca2+]i responses of human gingival fibroblasts via the LPA1 and LPA3 receptor subtypes, and that LPA3 may modulate their inflammatory responses. We have used immunofluorescence to detect LPA receptor (LPAR) distribution in human gingivae, but that technique does not afford visualization of the state of collagen fiber organization. We hypothesized that using the second harmonic generation (SHG) technique would enable detection of changes in the appearance, collagen fibril density, and fibril organizational structure of human gingiva, and that this technique would complement visualizing LPAR expression by confocal microscopy. Pieces of intact, paraformaldehyde‐fixed, methanol:acetone (1:1) de‐lipidated attached gingivae [~1.25 mm thick (from IRB‐approved, consented donors) normal or with moderate‐severe periodontal disease] were used. Confocal analysis using anti‐LPA1 and anti–LPA3 antibodies confirmed that LPA3 is primarily found in the epithelium, and that nuclear LPA1 expression in the sub‐epithelial connective tissue is greatly diminished in periodontal disease. SHG analysis clearly showed the normal collagen structure and subsequent reduction in the abundance and organization in the collagen fibrils of periodontal gingivae. This multidisciplinary study shows that SHG effectively complements confocal microscopy as a tool to help understand periodontal changes at the level of receptors and collagen organization.

Influence of histamine on the expression of CCL20 in human gingival fibroblasts.

Histamine plays an important role during allergic and inflammatory reactions, and it has been suggested to influence periodontal inflammation. The aim of this study was to investigate the effects of histamine on the expression of the antimicrobial peptide C-C chemokine ligand 20 (CCL20) in human gingival fibroblasts (HGFs) when exposed to toll-like receptor (TLR) agonists. Monolayers of HGFs from three different donors were exposed to histamine, alone, and in combination with Pam3CSK4 (a TLR2 agonist) or lipopolysaccharide (LPS) from Escherichia coli (a TLR4 agonist), for 2, 4, 6 or 12 h. In another experimental group, cells were pretreated with a specific histamine-1 receptor antagonist (H1R) antagonist, cetirizine. Real-time PCR analysis was performed to detect expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), CCL20 and interleukin-8 (IL8) genes. The levels of CCL20 and IL-8 protein were determined by ELISA. In HGFs, histamine induced expression of CCL20 and IL8 genes in a time-dependent manner (p < 0.05). Combined stimulation with histamine and Pam3CSK4 or LPS led to a significant amplification in expression of CCL20 and IL-8 when compared with treatment with each stimulant alone (p < 0.05), and this effect was mediated via pathways involving the H1R (p < 0.05). The results of this study suggest a sensitizing effect of histamine on early innate immune responses of HGFs when simultaneously exposed to bacterial virulence factors.

Preventive Effects of Protocatechuic Acid on LPS-Induced Inflammatory Response in Human Gingival Fibroblasts via Activating PPAR-γ.

Protocatechuic acid (PCA), a major metabolite of anthocyanins, has been shown to have antioxidant, antitumoral, and anti-inflammatory activities. In this study, we investigated the anti-inflammatory effects of PCA on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (HGFs). The effects of PCA on LPS-induced inflammatory cytokines IL-6 and chemokines IL-8 in HGFs were detected by ELISA. The expression of NF-κB and PPAR-γ was detected by Western blot analysis. Our results demonstrated that PCA suppressed IL-6 and IL-8 production in LPS-stimulated HGFs. We also found that PCA inhibited LPS-induced NF-κB activation. Furthermore, the inhibition of PCA on LPS-induced IL-6 and IL-8 production can be reversed by PPAR-γ antagonist GW9662. In conclusion, the anti-inflammatory mechanism of PCA is associated with activating PPAR-γ, thereby inhibiting LPS-induced inflammatory response.

Acanthoic acid inhibits LPS-induced inflammatory response in human gingival fibroblasts.

Periodontitis is a chronic disease that affects the gums and destroys connective tissue. Acanthoic acid (AA), a diterpene in Acanthopanax koreanum, has been reported to have anti-inflammatory activities. The aim of this study was to investigate the anti-inflammatory effects of AA on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (HGFs). HGFs were treated with Porphyromonas gingivalis LPS in the presence or absence of AA. The production of inflammatory cytokines IL-8 and IL-6 were measured by ELISA. The expression of NF-κB and TLR4 were detected by Western blotting. The results showed that AA inhibited LPS-induced IL-8 and IL-6 production in a dose-dependent manner. In addition, AA inhibited LPS-induced TLR4 expression and NF-κB activation. In conclusion, AA inhibits LPS-induced inflammatory response in HGFs through inhibition TLR4-mediated NF-κB signaling pathway.

Nitric oxide-mediated cytotoxic effect induced by zoledronic acid treatment on human gingival fibroblasts

The aim of our work was to evaluate the role of nitric oxide (NO) in the in vitro response of human gingival fibroblasts (HGFs) to 1, 5, 10, and 100μM doses of zoledronic acid (ZA), a bisphosphonate largely used in the clinical practice and for which several adverse effects are reported. Phase contrast microscopy and live/dead staining were used to evaluate HGFs morphology; cell viability, collagen type I and interleukin 6 (IL-6) secretion were evaluated by 3-[4,5-dimethyl-thiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT) and enzyme-linked immunosorbent assay (ELISA) assays. Reactive oxygen species (ROS) production and mitochondrial membrane potential were evaluated by flow cytometry; NO production and NOS activity by spectrophotometric analysis; endothelial NOS (eNOS) and neuronal NOS (nNOS) expression by immunofluorescence. Viable fibroblasts are evidenced in control sample while floating dead cells and cells close to detachment phase in ZA-treated sample, in agreement with decreased level of collagen type I. Control sample shows higher number of viable cells respect to ZA-treated one and ROS production increases when ZA is added. Released NO in ZA-treated sample appears higher and NO overproduction is related to increased nNOS activity. IL-6 secretion level is higher in ZA-treated sample than in control one. Our results suggest ROS involvement in NO overproduction, due to nNOS recruitment, both at low and high doses. In turn, NO release seems to be able to trigger the inflammatory response only when high doses are administered, thus confirming the ZA cytotoxic effect on HGFs. The knowledge of ZA-mediated cytotoxicity mechanisms on HGFs allows to better understand drug pharmacological activity.

Suppression of inflammatory responses of human gingival fibroblasts by gingipains from Porphyromonas gingivalis.

The interaction between human gingival fibroblasts (HGFs) and Porphyromonas gingivalis plays an important role in the development and progression of periodontitis. Porphyromonas gingivalis possesses several virulence factors, including cysteine proteases, the arginine-specific (Rgp) and lysine-specific (Kgp) gingipains. Studying the mechanisms that P. gingivalis, and its derived virulence, use to propagate and interact with host cells will increase the understanding of the development and progression of periodontitis. In this study, we aimed to elucidate how P. gingivalis influences the inflammatory events in HGFs regarding transforming growth factor-β1 (TGF-β1 ), CXCL8, secretory leucocyte protease inhibitor (SLPI), c-Jun and indoleamine 2,3-dioxygenase (IDO). HGFs were inoculated for 6 and 24 h with the wild-type strains ATCC 33277 and W50, two gingipain-mutants of W50 and heat-killed ATCC 33277. The P. gingivalis regulated CXCL8 and TGF-β1 in HGFs, and the kgp mutant gave significantly higher immune response with increased CXCL8 (P < 0.001) and low levels of TGF-β1 . We show that HGFs express and secrete SLPI, which was significantly suppressed by P. gingivalis (P < 0.05). This suggests that by antagonizing SLPI, P. gingivalis contributes to the tissue destruction associated with periodontitis. Furthermore, we found that P. gingivalis inhibits the expression of the antimicrobial IDO, as well as upregulating c-Jun (P < 0.05). In conclusion, P. gingivalis both triggers and suppresses the immune response in HGFs. Consequently, we suggest that the pathogenic effects of P. gingivalis, and especially the activity of the gingipains on the inflammatory and immune response of HGFs, are crucial in periodontitis.

Transcriptional profiling of human gingival fibroblasts in response to multi-species in vitro subgingival biofilms.

Periodontitis is an infectious inflammatory disease that destroys the tooth-supporting tissues. It is initiated by complex subgingival biofilms, triggering an inflammatory response by the juxtaposed gingival tissue. The range of transcriptional events initiated in the gingiva following biofilm challenge is not fully elucidated. By employing gene microarray technology, this study aimed to characterize the overall transcriptional changes (more than two-fold regulation) of cultured human gingival fibroblasts in response to a 10-species in vitro subgingival biofilm model (BF), over a challenge period of 6 h. The relative involvement of the three 'red complex' species in these transcriptional events was evaluated by omitting these species from the biofilm composition (BF-RC). When compared with the unchallenged control, challenge with BF and BF-RC differentially regulated 386 and 428 genes, respectively, with an overlap of 52-75%. Interestingly, the expression of only three genes was significantly different between the BF and BF-RC challenged groups. There was also a strong overlap of the affected signalling pathways and gene ontology processes. These signalling pathways involved primarily the immune response, and included toll-like receptors, interleukin-1, interleukin-17 and heat-shock proteins 60 and 70. In conclusion, subgingival biofilms elicited a large number of transcriptional changes in gingival fibroblasts, while the presence of the 'red complex' in the biofilm did not yield any substantial differences. These findings show a uniform 'non-specific' transcriptional response of host cells to subgingival biofilms, and denote that redundancies may exist in the virulence properties of individual bacterial species within a polymicrobial biofilm community.

Biological responses of human gingival fibroblasts (HGFs) in an innovative co-culture model with Streptococcus mitis to thermosets coated with a silver polysaccharide antimicrobial system.

This study sought to evaluate the in vitro biological response of human gingival fibroblasts (HGFs) co-coltured with Streptococcus mitis to bisphenol A glycidylmethacrylate/triethylene glycol dimethacrylate (BisGMA/TEGDMA) thermosets coated with Chitlac-nAg, a nanocomposite system with antimicrobial properties. To avoid bacterial adhesion to dental devices and to reduce cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilizing silver nanoparticles, which have well-known antimicrobial properties, with a polyelectrolyte solution containing Chitlac. Cytotoxicity, cell morphology, cell migration and inflammatory interleukine-6 (IL-6) and prostaglandin E2 (PGE2) secretion were evaluated. Our results showed that the cytotoxicity exerted on HGFs by our nanocomposite material was absent in our co-culture model, where fibroblasts are able to adhere and migrate. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rises up 20%. Moreover the presence of S. mitis reduced this release in a greater amount with Chitlac-nAg coated thermosets. The secretion of IL-6 was significant in both Chitlac and Chitlac-nAg coated thermosets, but PGE2 production was minimal, suggesting that the IL-6 production was not related to an inflammatory response. Co-culture and the addiction of saliva did not influence IL-6 and PGE2 secretion. Data obtained in the present work suggest that Chitlac n-Ag coated thermosets could significantly improve the success rates of restorative dentistry, since they limit bacterial adhesion and are not toxic to HGFs.

Nitric Oxide-mediated cytotoxic effect induced by zoledronic acid treatment on Human Gingival Fibroblasts

Zoledronic acid (ZA) belongs to bisphosphonates (BPs), drugs administered to treat resorptive bone diseases. Although ZA is largely used in the clinical practice, significant adverse effects of ZA, such as osteonecrosis of the jaw (ONJ), were recorded. The aim of this work was to evaluate the role of Nitric Oxide (NO) in the in vitro response of Human Gingival Fibroblasts (HGFs) to 1, 5, 10 and 100μM ZA. HGFs morphology was evaluated through phase contrast microscopy and live/ dead staining; MTT and ELISA assays were applied to measure cell viability, Collagen Type I and IL6 secretion. ROS production and mitochondrial membrane potential were evaluated by flow cytometry; NO production and NOS activity by spectrophotometric analysis; eNOS and nNOS expression by fluorescence microscopy. Viable fibroblasts are evidenced in control sample while floating dead cells and cells close to detachment phase in ZA treated sample along with decreased level of Collagen Type I. Control sample shows higher number of viable cells respect to ZA treated one and ROS production increases when ZA is added. Released NO in ZA treated sample appears higher and NO overproduction is related to increased nNOS activity. IL 6 secretion level is higher in ZA treated sample than in control one. Our results suggest ROS involvement in NO overproduction, due to nNOS recruitment, both at low and high doses. In turn, NO release seems to be able to trigger the inflammatory response only when high doses are administered.

Differential response of human gingival fibroblasts to titanium‐ and titanium‐zirconium‐modified surfaces

Gingival fibroblasts are responsible for the constant adaptation, wound healing and regeneration of gingival connective tissue. New titanium-zirconium (TiZr) abutment surfaces have been designed to improve soft tissue integration and reduce implant failure compared with titanium (Ti). The aim of the present study was first to characterize a primary human gingival fibroblast (HGF) model and secondly to evaluate their differential response to Ti and TiZr polished (P), machined (M) and machined + acid-etched (modMA) surfaces, respectively. HGF were cultured on tissue culture plastic or on the different Ti and TiZr surfaces. Cell morphology was evaluated through confocal and scanning electron microscopy. A wound healing assay was performed to evaluate the capacity of HGF to close a scratch. The expression of genes was evaluated by real-time RT-PCR, addressing: (i) extracellular matrix organization and turnover; (ii) inflammation; (iii) cell adhesion and structure; and (iv) wound healing. Finally, cells on Ti/TiZr surfaces were immunostained with anti-ITGB3 antibodies to analyze integrin β3 production. Matrix metalloproteinase-1 (MMP1) and inhibitor of metallopeptidases-1 (TIMP1) production were analyzed by enzyme-linked immunosorbent assays. On tissue culture plastic, HGF showed no differences between donors on cell proliferation and on the ability for wound closure; α-smooth muscle actin was overexpressed on scratched monolayers. The differentiation profile showed increased production of extracellular matrix components. Ti and TiZr showed similar biocompatibility with HGF. TiZr increased integrin-β3 mRNA and protein levels, compared with Ti. Cells on TiZr surfaces showed higher MMP1 protein than Ti surfaces, although similar TIMP1 protein production. In this in vitro experiment, P and M surfaces from both Ti and TiZr showed better HGF growth than modMA. Taking into account the better mechanical properties and bioactivity of TiZr compared with Ti, the results of the present study show that TiZr is a potential clinical candidate for soft tissue integration and implant success.

Human gingival fibroblast functions are stimulated by oxidized nano-structured titanium surfaces

ObjectivesThe aim of this study was to analyze the features of an oxidized titanium implant surface and to evaluate its effects on the response of human gingival fibroblasts. Methods10mm×10mm×1mm turned (control) and oxidized (test) titanium samples (P.H.I. s.r.l., Italy) were examined by scanning electron microscopy and atomic force microscopy and characterized by height, spatial and hybrid roughness parameters. Primary cultures of human gingival fibroblasts were seeded on titanium samples, and cell morphology, adhesion, proliferation and extracellular matrix deposition, in terms of type I collagen synthesis, were evaluated. ResultsControl and test surfaces appeared considerably different at the microscopic analyses: turned samples were grooved, whereas oxidized surfaces showed a more complex micro- and nano-scaled texture, as evidenced by roughness parameters. Cell adhesion and proliferation rate, as well as collagen synthesis, were greater on oxidized vs turned surfaces. ConclusionsAlthough both control and test samples were in the range of average roughness proper of smooth surfaces, they exhibited significantly different topographic properties in terms of height and, mostly, hybrid parameters. Furthermore, oxidized surfaces enhanced human gingival fibroblast adhesion, proliferation and extracellular matrix deposition, and this could be due to the different structure at micro- and nano-scale levels. Clinical significanceOxidized nanostructured titanium surfaces could have a significant clinical utilization in virtue of their affinity for soft tissue attachment at the implant neck and/or at the transmucosal portion of the prosthetic abutment.

Effects of Chitosan Particles in Periodontal Pathogens and Gingival Fibroblasts

Chitosan is a naturally derived polymer with antimicrobial and anti-inflammatory properties. However, studies evaluating the role of chitosan in the control of periodontal pathogens and the responses of fibroblasts to inflammatory stimuli are lacking. In the present study, we analyzed whether chitosan particles may inhibit the growth of periodontal pathogens and modulate the inflammatory response in human gingival fibroblasts. Chitosan particles were generated through ionic gelation. They inhibited the growth of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans at 5 mg/mL. Conversely, IL-1β strongly stimulated PGE2 protein levels in gingival fibroblasts, and chitosan inhibited this response at 50 µg/mL. IL-1β–stimulated PGE2 production was dependent on the JNK pathway, and chitosan strongly inhibited this response. IL-1β stimulated NF-κB activation, another signaling pathway involved in PGE2 production. However, chitosan particles were unable to modify NF-κB signaling. The present study shows that chitosan exerts a predominantly anti-inflammatory activity by modulating PGE2 levels through the JNK pathway, which may be useful in the prevention or treatment of periodontal inflammation.

MicroRNA-146 inhibits pro-inflammatory cytokine secretion through IL-1 receptor-associated kinase 1 in human gingival fibroblasts

BackgroundAlthough various microRNAs (miRNAs) regulate immune and inflammatory responses, the function of miRNAs in periodontitis has not been clearly illuminated. In this study, we measured miRNA-146 (miRNA-146a and miRNA-146b-5p) expression and explored its regulatory function in the inflammatory response in human gingival fibroblasts (HGFs).MethodsmiRNA-146a and miRNA-146b-5p expression was measured by performing real-time polymerase chain reaction in HGFs after Porphyromonas gingivalis (p.g) lipopolysaccharide (LPS) stimulation. After the HGFs were transfected with miRNA-146a and miRNA-146b-5p inhibitor, the expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) were detected by western blot and quantitative PCR. A luciferase assay was used to detect whether miRNA-146 could directly bind to the 3’-UTR of IRAK1.ResultsThe expression levels of miRNA-146a and miRNA-146b-5p significantly increased in the P.g LPS-stimulated HGFs compared to the non-stimulated HGFs. The inhibition of miRNA-146a and miRNA-146b-5p resulted in increased IL-1β, IL-6 and TNF-α secretion. The mRNA and protein levels of IRAK1, but not TRAF6, also increased. We further found that miRNA-146a and miRNA-146b-5p directly bound to the IRAK1 3’-UTR.ConclusionOur data suggest that miRNA-146 inhibits pro-inflammatory cytokine secretion through IRAK1 in HGFs, which indicates that miRNA-146 functions as a negative regulator of periodontal inflammation.

The expression and regulation of matrix metalloproteinase-3 is critically modulated by Porphyromonas gingivalis lipopolysaccharide with heterogeneous lipid A structures in human gingival fibroblasts

BackgroundPorphyromonas gingivalis lipopolysaccharide (LPS) is a crucial virulence factor strongly associated with chronic periodontitis which is the primary cause of tooth loss in adults. It exhibits remarkable heterogeneity containing tetra-(LPS1435/1449) and penta-(LPS1690) acylated lipid A structures. Human gingival fibroblasts (HGFs) as the main resident cells of human gingiva play a key role in regulating matrix metalloproteinases (MMPs) and contribute to periodontal homeostasis. This study investigated the expression and regulation of MMPs1-3 and tissue inhibitors of MMP-1 (TIMP-1) in HGFs in response to P. gingivalis LPS1435/1449 and LPS1690 and hexa-acylated E. coli LPS as a reference. The expression of MMPs 1–3 and TIMP-1 was evaluated by real-time PCR and ELISA.ResultsThe MMP-3 mRNA and protein were highly upregulated in P. gingivalis LPS1690- and E. coli LPS-treated cells, whereas no induction was observed in P. gingivalis LPS1435/1449-treated cells. On the contrary, the expression of MMP-1 and −2 was not significantly affected by P. gingivalis LPS lipid A heterogeneity. The TIMP-1 mRNA was upregulated in P. gingivalis LPS1435/1449- and E. coli LPS-treated cells. Next, signal transduction pathways involved in P. gingivalis LPS-induced expression of MMP-3 were examined by blocking assays. Blockage of p38 MAPK and ERK significantly inhibited P. gingivalis LPS1690-induced MMP-3 expression in HGFs.ConclusionThe present findings suggest that the heterogeneous lipid A structures of P. gingivalis LPS differentially modulate the expression of MMP-3 in HGFs, which may play a role in periodontal pathogenesis.