Effect of electrochemically dealloying Ti6Al4V abutments on human gingival fibroblasts / 口腔疾病防治

Objective@#To investigate the effects of electrochemically dealloying of Ti6Al4V abutments on human gingival fibroblasts (HGFs) and to provide experimental evidence for surface modification of implant abutments.@*Methods@#The samples were divided into an NC group (negative control, no other treatment on a smooth surface), an NM-1 group (nanomesh-1, electrochemical dealloying treatment in 1 mol/L NaOH 1 h on 2 V voltage), and an NM-2 group (nanomesh-2, electrochemical dealloying treatment in 5 mol/L NaOH 1 h on 2 V voltage). The surface morphologies of the samples and the adhesion of HGFs on the sample surfaces were observed with scanning electron microscopy (SEM). The surface hydrophilicities of the samples were measured with a contact angle measuring instrument. The proliferation of HGFs on the different samples were evaluated with CCK-8, and the expression of adhesion-related genes, including collagen Ⅰ (COL1A1), collagen Ⅲ (COL3A1), fibronectin 1 (FN1), focal adhesion kinase (FAK), vinculin (VCL), integrin α2 (ITGA2), and integrin β1 (ITGB1), on the different samples was measured with qRT-PCR. The expression of vinculin on the surfaces of HGFs was observed via confocal laser scanning microscopy (CLSM) after immunofluorescent staining. Collagen fiber secretion and syntheses of HGFs from different samples were evaluated via Sirius red staining.@*Results@#SEM revealed the formation of ordered and uniform three-dimensional mesh structures on the surfaces of the NM-1 and NM-2 groups, with grid diameters of approximately 30 nm for the NM-1 group and approximately 150 nm for the NM-2 group. Compared with that of the NC group, the water contact angles of the NM-1 group and NM-2 groups were significantly lower (P<0.000 1). Cell proliferation in the NM-1 group was significantly greater than that in the NC group (P<0.01). Moreover, there was no significant difference in the water contact angles or cell proliferation between the NM-1 group and the NM-2 group. SEM revealed that HGFs were adhered well to the surfaces of all samples, while the HGFs in the NM-1 and NM-2 groups showed more extended areas, longer morphologies, and more developed pseudopodia than did those in the NC group after 24 h. qRT-PCR revealed that the expression levels of the adhesion-related genes COL1A1, COL3A1, FN1, FAK and VCL in the NM-1 group were significantly greater than those in the NC and NM-2 groups (P<0.01). The expression of vinculin protein in the NM-1 group was the highest, and the number of focal adhesions was greatest in the NM-1 group (P<0.01). The results of Sirius red staining showed that the NM-1 group had the highest secretion and syntheses of collagen fibers (P<0.000 1).@*Conclusion@#The three-dimensional nanomechanical structure of Ti6Al4V modified by electrochemical dealloying promoted the adhesion, proliferation, collagen fiber secretion and syntheses of HGFs, and electrochemical dealloying of Ti6Al4V with a grid diameter of approximately 30 nm obviously promoted HGF formation.

Effect and mechanism of reactive oxygen species-responsive nanoparticles on the regulation of human gingival fibroblast function and inflammation induced by lipopolysaccharide / 口腔疾病防治

Objective@#To investigate the effects of PssL-NAC reactive oxygen species (ROS)-responsive nanoparticles on intracellular ROS production, inflammatory factor levels, collagen production, cell function and Toll-like receptor 4 (TLR4), NF-κB nuclear factor-κB (p65) pathway protein expression in human gingival fibroblasts (HGFs) induced by Porphyromonas gingivalis-lipopolysaccharide (P.g-LPS).@*Methods@#This study was reviewed and approved by the ethics committee. PssL-NAC microspheres containing oil soluble antioxidant N-acetylcysteine (NAC) were obtained by connecting the hydrophobic end of polycaprolactone (PCL) and the hydrophilic end of polyethylene glycol (PEG) via thioketal (TK) bonds in response to ROS, and self loading in the aqueous and oil phases. After preparation of the PssL-NAC microspheres and aqueous NAC solution, successful synthesis of the nanoparticles was verified by transmission electron microscopy. Then, HGFs were exposed to P.g-LPS (0, 5, or 10 μg/mL), P.g-LPS (0, 5, or 10 μg/mL)+NAC, and P.g-LPS (0, 5, or 10 μg/mL)+PssL-NAC, and the ROS levels in the different groups were observed under confocal microscopy to determine the concentration of P.g-LPS for use in subsequent experiments. The groups were as follows: control group (no treatment), P.g-LPS group (HGFs treated with P.g-LPS), NAC group (HGFs treated with P.g-LPS and NAC), and PssL-NAC group (HGFs treated with P.g-LPS and PssL-NAC). Cell counting kit-8 (CCK-8) assays verified the biosafety of PssL-NAC. The ROS levels in the different groups were detected by DCFH-DA probes and observed via confocal microscopy. Real-time qPCR (RT-qPCR) was used to monitor the gene expression levels of the intracellular inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), collagen 1 (COL1) and collagen 3 (COL3). The effect of PssL-NAC on the migration of HGFs was observed via the scratch test. The protein expression of TLR4-NF-κB, and phosphorylated p65 (p-p65) in the TLR4-NF-κB pathway was evaluated by Western blot.@*Results@#PssL-NAC had no significant effect on HGF proliferation (P>0.05). At elevated P.g-LPS concentrations, PssL-NAC maintained intracellular ROS levels approximately twice those in the control group (P<0.001). PssL-NAC significantly decreased P.g-LPS-induced IL-6 (P<0.001) and TNF-α (P<0.001) gene expression and increased COL1 gene expression (P<0.001). After P.g-LPS stimulation, PssL-NAC restored cell migration to the control level (P>0.05) and decreased the protein expression of TLR4 (P<0.001), p65 (P = 0.006), and p-p65 (P = 0.017) in the TLR4-NF-κB pathway.@*Conclusion@#PssL-NAC maintains the appropriate intracellular ROS concentration, alleviates P.g-LPS-induced inflammation in HGFs through the TLR4-NF-κB pathway, and restores the cell functions of collagen production and migration in an inflammatory environment.

Ca mobilization and signaling pathways induced by rRgpB in human gingival fibroblast / 浙江大学学报·医学版

: To assess the () recombinant gingivalis gingipain R2 (rRgpB)-induced Ca mobilization in human gingival fibroblast (HGF) mediated by protease-activated receptor (PAR) and its downstream signal transduction pathways. : Flow cytometry was used to detect the expression of PAR in HGF. The proliferation of HGF was measured by CCK-8. The dynamic changes of intracellular Ca concentration in HGF induced by rRgpB and the blocking effect of PAR-1 antagonist were observed by laser confocal microscopy. Western blot was performed to determine the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK) and p65 in HGF. : PAR-1 and PAR-3 were expressed in HGF, and the rRgpB could promote the proliferation of HGF. rRgpB caused a transient increase in [Ca], which could be completely suppressed by vorapaxar, a PAR-1 antagonist. The phosphorylation levels of JNK, ERK1/2 and p65 were significantly up-regulated after the induction of rRgpB for and (all <0.05), which was completely inhibited by vorapaxar. However, the phosphorylation level of p38 MAPK had no significant change after rRgpB stimulation. : rRgpB causes an increase in [Ca] in HGF mediated by PAR-1. JNK, ERK1/2 and nuclear factor-κB may be involved in intracellular signal transduction after PAR-1 activation.

Sitagliptin inhibits lipopolysaccharide-induced inflammatory response in human gingival fibroblasts by blocking nuclear factor-κB signaling pathway / 华西口腔医学杂志

OBJECTIVES@#This study was performed to clarify the effects of sitagliptin on @*METHODS@#Healthy gingival samples were collected from the donors. HGFs were isolated with enzymic digestion method and identified. The effects of LPS and sitagliptin on cell viability were detected by cell-counting kit-8 (CCK8). The mRNA levels of inflammatory cytokines, namely, interleukin (IL)-6, IL-8, C-C motif ligand 2 (CCL2), and superoxide dismutase 2 (SOD2), were evaluated by quantity real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA) was used to measure the secretion protein levels of IL-6, IL-8, and CCL2. Western blot analysis was used to further investigate the activation of nuclear factor (NF)-κB signaling pathway. The effect of NF-κB pathway inhibitor BAY11-7082 on LPS-induced HGF inflammatory cytokines at the gene level was verified by qRT-PCR.@*RESULTS@#Low concentrations of sitagliptin (0.1, 0.25, and 0.5 µmol·L@*CONCLUSIONS@#Sitagliptin could significantly inhibit LPS-induced HGF inflammatory response by blocking the NF-κB signaling pathway activation.

Regulation of human gingival fibroblast gene expression on microgrooves: A DNA microarray study / 대한치과보철학회지

PURPOSE: We aimed to investigate the gene expression of human gingival fibroblasts on microgroove surface using DNA microarray. MATERIALS AND METHODS: Microgrooves were applied on grade II titanium discs to have 0/0 µm (NE0, control group), 60/10 µm (E60/10, experimental group) of respective width/depth by photolithography. The entire surface of the microgrooved Ti substrata was further acid etched and used as the two experimental groups in this study. Human gingival fibroblasts were cultured in the experimental group and the control group, and total RNA was extracted. The oligonucleotide microarray was performed to confirm the changes of various gene expression levels between experimental group and control group. Changes of gene expression level were determined at the pathway level by mapping the expression results of DNA chips, using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. RESULTS: Gene expression levels on E60/10 and NE0 were analyzed, there were 123 genes showing significant differences in expression more than 1.5 times on E60/10 microgrooved surface compared to NE0 surface, and 19 genes showing significant differences in expression more than 2 times. The KEGG pathway analysis confirmed the changes in gene expression levels under experimental conditions. Cell signaling, proliferation, and activity among the various gene expression results were identified. CONCLUSION: Microgrooved surfaces induce gene expression changes and related cell signaling. According to the results of this study, microgrooves can be used as the surface of various biomaterials which need to improve cell activity through gene expression changes and activation of cell signaling.

Fibroblast response to initial attachment and proliferation on titanium and zirconium surfaces / Respuesta de fibroblastos a la adhesión inicial y proliferación en superficies de titanio y zirconio

Introduction: In recent decades, dental implants have become one of the best options for comprehensive dental restoration; their placement is a multidisciplinary task that requires a solid understanding of biological, periodontal, surgical and prosthetic principles. Objective: The aim of this study was to quantify in vitro the adhesion and proliferation of human gingival fibroblasts (HGF) response on titanium (Ti) and zirconia (Zr) surfaces. Methodology: Samples of Ti and Zr were observed under atomic force microscopy (AFM). HGFs were inoculated in each sample to determine adhesion and cell proliferation. The reagent MTT was mixed with medium DMEM and inoculated in each plate; formazan was dissolved with dimethyl sulfoxide and analyzed at 540nm in a microplate spectrophotometer. The test was performed with three independent experiments. Data were analyzed with Kolmogorov-Smirnov tests (Lilliefors), Kruskal-Wallis tests and Mann-Whitney test comparisons. Results: Topography of the Zr plates showed greater roughness (Ra= 0.39 microns) than Ti (Ra= 0.049 microns). Quantification of HGF adhesion was significantly higher (p<0.05) in Ti, while proliferation showed no statistically significant differences between the groups. Conclusion: It is noteworthy that, even though Ti initially showed increased cell adhesion on the surface, after 24h Zr samples showed similar proliferation; this demonstrates that both surfaces have a comparable biological response.

Introducción: En las últimas décadas, los implantes dentales se han posicionado como una de las mejores opciones de restauración dental integral; su colocación es una tarea multidisciplinaria que requiere una sólida comprensión de los principios biológicos, periodontales, quirúrgicos y protésicos. Objetivo: El objetivo de este estudio fue cuantificar in vitro la adhesión y proliferación de la respuesta de fibroblastos gingivales humanos (HGF) en superficies de titanio (Ti) en contraste con superficies de zirconia (Zr). Metodología: Se observaron las muestras de Ti y Zr bajo microscopía de fuerza atómica (AFM). Los HGF fueron inoculados en cada muestra para determinar la adhesión y proliferación celular. El reactivo MTT se mezcló con medio DMEM y se inoculó en cada placa, el formazán se disolvió con dimetilsulfóxido y se analizó a 540nm en un espectrofotómetro de microplaca. El ensayo se realizó con tres experimentos independientes. Los datos fueron analizados con pruebas de Kolmogorov-Smirnov (Lilliefors), pruebas de Kruskal-Wallis y comparaciones de Mann-Withney. Resultados: La topografía de las placas de Zr mostraron una mayor rugosidad (Ra=0.39 micrones) en comparación con las de Ti (Ra=0.049 micrones). La cuantificación de la adhesión de HGF fue significativamente mayor (p<0.05) en el Ti, mientras que la proliferación no mostró diferencias estadísticamente significativas entre los grupos. Conclusión: Es importante mencionar que, a pesar de que el Ti mostró inicialmente una mayor adhesión celular sobre la superficie, después de 24 hrs las muestras de Zr mostraron una proliferación similar; lo que demuestra que ambas superficies poseen una respuesta biológica comparable.

Cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate on primary cultured human gingival fibroblasts / 华中科技大学学报（医学）（英德文版）

The aim of this study was to investigate the cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate (CHX) on human gingival fibroblasts (HGFs), and to evaluate the biosecurity of modified nonequilibrium plasma with 2% CHX as a new method of root canal treatment. Tissue samples taken from human gingiva were primarily cultured and passaged. Cells from passages 3-7 were used. HGFs were treated by modified nonequilibrium plasma with 2% CHX for 0 min (control group), 30 s, 1 min, 1.5 min, 3 min, 5 min, and 10 min, respectively, and then they were incubated for 0, 24, and 48 h. After that, cell counting kit-8 (CCK-8) assay was applied to analyze the cytotoxicity of modified nonequilibrium plasma with 2% CHX on HGFs. There was no significant difference between the 0 h group treated with the modified nonequilibrium plasma for 1 min and the control group (P>0.05). However, there were significant differences between all the other treated groups and the control group (P<0.05). When treated for 1.5 min or shorter, the cell viability was obviously increased; while treated for 3 min or longer, it was obviously reduced. Moreover, when successively cultured for 0, 24, and 48 h, cell viability was decreased at first and then increased in the 3-min-treated and 5-min-treated groups. The modified nonequilibrium plasma with 2% CHX was of no influence on cell viability in 1.5 min treatment, and it could be safely used on root canal treatment.

The effects of microgroove surface form of titanium on the adhesion and cell cycle progression of human gingival fibroblasts / 实用口腔医学杂志

Objective:To investigate the effect of microgroove surfaces of titanium on the adhesion and cell cycle progression of human gingival fibroblasts(HGFs).Methods:Microgroove titanium surfaces were fabricated on silicon plate by photolithography with parallel grooves:1 5,30 and 60 μm in width and 5 μm and 1 0 μm in depth,the groups were denoted as T1 5 /5,T1 5 /1 0,T30 /5,T30 /1 0, T60 /5 and T60 /1 0,respectively.Smooth titanium surfaces (T0)were used as the controls.Surface topography were observed by ES-EM.HGFs were cultured on the microgroove surfaces.Morphology of “contact guidance”was observed by immunofluorescence tech-nique.Cell cycle distribution was analyzed by Flow cytometry.Results:HGFs on the microgroove surfaces had “contact guidance”par-allel to the microgrooves,whereas the cells on T0 were oriented randomly.T60 /1 0 group had the highest percentage of S phase cells, followed by T30 group and T1 5 group,but still higher than that in the control group.In groups with higher groove width (T60 group and T30 group),the increase of groove depth benefited the increase of S phase percentage,while in T1 5 group,the increase of groove width decreased the S phase percentage.Conclusion:Surfaces of microgrooves with different dimensions achieved “contact guidance”for the cultured HGFs.The surfaces with increasing groove width and depth benefit the cell cycle progression.

The comparison on pluripotent differentiation between human gingival fibroblasts and human periodontal ligament cells in vitro / 天津医药

Objective To investigate the the multi-directional differentiation potential between pluripotent of human gingival fibroblasts (HGFs) and human periodontal ligament cells (HPDLCs). Methods HPDLCs and HGFs were obtained from the primary culture. HPDLCs and HGFs at 3rd-4th passage were cultured in osteogenic, adipogenic or chondrogenic me-dium. Cells without differentiation were taken as control. Alizarin red, Alcian blue and oil red O staining were performed to detect osteogenic differentiation, chondrogenic and adipogenic differentiation in vitro, respectively. Reverse transcription polymerase chain reaction (RT-PCR) was applied to examine the expression of osteocalcin (OCN), runt-related transcription factor 2 (RUNX2) and collagen 1 (Col 1), peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and collagen 10 (Col 10). Results HPDLCs and HGFs cultured in osteogenic medium showed massive calicium nodulus at day 28, but HP-DLCs formed more calicium nodulus than those of HGFs. The expressions of OCN, RUNX2 and Col 1 were significantly high-er in HPDLCs than those in HGFs (P<0.05). In chondrogenic medium both cells were found blue deposit at day 14, and the expression of Col 10 was significantly higher in HGFs than that of HPDLCs (P<0.01). Furthermore, in adipogenic medium HGFs showed more lipid-filled droplets stained with oil red O than HPDLCs at day 21. The expression of PPARγ2 was sig-nificantly higher in HGFs than that of HPDLCs (P<0.01). Conclusion HPDLCs has the better potency of osteogenic differ-etiation than HGFs, however, HGFs has the better potency of adipogenic and chondrogenic differentiation.

Evaluation in vitro of cytotoxicity of dentin desensitizers on human gingival fibroblasts / Evaluación in vitro de la citotoxicidad dedesensibilizantes dentinarios en fibroblastos gingivales humanos

The purpose of this study is to compare the cytotoxic effect of threematerials, which have been used for treating dental hypersensitivity. Materialand method: In vitro study. Clinpro (3M Co, St. Paul, MN. USA), Seal & Protect(Dentsply, DeTrey GmbH. Germany) and UltraEZ (Ultradent Products,Inc., S. South Jordan UT. USA) were used at concentrations of 0.1, 0.05, 0.01 and 0.001g/ml on human gingival fibroblasts. Furthermore, Clinpro and Seal & Protect were applied to this cell culture as polymerized disks. Toxicity was assessed at 24 and 48 hours by the use of the cell viability assay (MTT). Statistical analysis for cell viability was performed using two-way ANOVA and Tukey’s post hoc test. Statistical significance was set at 5 percent. Results: Seal & Protect and Clinpro were found to be highly toxic at 24 and 48 hours, reaching 70 percent toxicity at concentrations over 0.01g/ml. Seal & Protect and Clinpro polymerized disks were toxic at 24 and 48 hours. UltraEZ showed an increased between 46 percent and 67 percent in cell viability at 24 hours and between 8 percent and 45 percent at 48 hours. Statistical analysis showed differences between these three desensitizers when comparing concentration and control group (p<0.05). Discussion: UltraEZ did not have a cytotoxic effect and may be considered a compatible and safe material, whereas polymerized and non-polymerized Clinpro and Seal & Protect should be used with caution...

Introduccion: El proposito de este estudio es comparar el efecto citotoxico de tres materiales que se han utilizado para el tratamiento de la hipersensibilidad dental. Material y metodo: Estudio in-vitro. Los desensibilizantes dentinarios Clinpro (3M ESPE), Seal&Protect (Dentsply) yUltraEZ (Ultradent) fueron utilizados a concentraciones de 0,1; 0,05; 0,01 y 0,001 g/ml sobre cultivos celulares de fibroblastos gingivales humanos. Ademas, Clinpro y Seal&Protect se aplicaron a este cultivo celular como discos polimerizados. La toxicidad se evaluo a 24 y 48 horas mediante ensayo de viabilidad (MTT). El analisis estadistico para la viabilidad celular se realizo mediante ANOVA de dos vias seguido de analisis Tukey. La significancia estadistica se fijo al 5 por ciento. Resultados: Clinpro y Seal&Protect resultaron ser altamente toxicos a las 24 y 48 horas, alcanzando un 70 por ciento de toxicidad aconcentraciones superiores de 0,01 g/ml. Los discos polimerizadosde Clinpro y Seal&Protect fueron toxicos a 24 y 48 horas. UltraEZ produjo un aumento de la viabilidad celular entre un 46 por ciento y 67 por ciento a las 24 horas y entre un 8 por ciento y 45 por ciento a las 48 horas. El analisis estadistico mostro diferencias entreestos tres desensibilizantes al comparar la concentracion y su grupo control (p<0,05). Discusion: UltraEZ no tuvo efecto citotoxico y puede ser considerado como un material compatible y seguro para ser utilizado, mientras que Clinpro y Seal&Protect en su estado polimerizado y no polimerizado deberian ser utilizados con precaucion...

Effect of microgroove surface morphology on the adhesion and proliferation of human gingival fibroblasts / 实用口腔医学杂志

Objective:To investigate the effects of microgroove surface morphology on the adhesion and proliferation of the human gin-gival fibroblasts(HGFs).Methods:Microgroove surfaces of titanium were fabricated by photolithography with parallel grooves of 15,30 or 60 μm in width and 5 μm or 10 μm in depth.The groups of the samples were denoted as T15 /5,T15 /10,T30 /5,T30 /10,T60 /5 and T60 /10.Smooth titanium surface(T0)was used as the control.The surface topography was observed by enviroment SEM(ES-EM).HGFs were cultured on the topographically modified surfaces.Morphology was observed by SEM.Cell proliferation was examined by CCK-8 kit.Results:HGFs on the microgroove surfaces had “contact guidance”parallel to the microgrooves,whereas the cells on T0 were oriented randomly.Cell proliferation was promoted and kept for longer period on T60 /10 surface.Conclusion:Surfaces of mi-crogrooves with increasing groove width and depth may achieve “contact guidance”for HGFs and promote cell proliferation.

Cytotoxic effects of the extractions of three gingival retraction cords with different extraction time / 实用口腔医学杂志

Objective:To evaluate the toxic effect of 3 different gingival retraction cords.Methods:DMEMextraction of DL-adren-aline HCl,aluminium sulphate and non-drug retraction cords with the extraction time of 5,10,15 and 30 min were respectively pre-pared and were used to culture human gingival fibroblasts(HGFs)in vitro respectively.Cell proliferation was tested by MTT assay. Cell apoptosis was examined by Annexin/PI method.Results:The 3 gingival retraction cord extractions inhibited the roliferation,pro-moted the apoptosis of HGFs(P <0.05),the effects were related to the extraction time.Conclusion:The 3 retraction cords have time-dependant cytotoxity.

Staurosporine Induces ROS-Mediated Process Formation in Human Gingival Fibroblasts and Rat Cortical Astrocytes

In the present study, we investigated the effect of staurosporine on the formation of cellular processes in human gingival fibroblasts and rat astrocytes. Staurosporine caused a rapid induction of process formation in human gingival fibroblasts and rat astrocytes in a concentration dependent manner. The process formation of human gingival fibroblasts and rat astrocytes was prevented by the pretreatment with N-acetylcysteine, suggesting that staurosporine-induced ROS production was responsible for the process formation. Colchicine, a microtubule depolymerizing agent, inhibited the staurosporine-induced process formation, whereas cytochalasin D, an actin filament breakdown agent, failed to suppress the formation of cellular processes. This result indicated that polymerization of microtubule, and not actin filament, was responsible for the formation of cellular processes induced by staurosporine. In support of this hypothesis, Western blot analysis was conducted using anti-tubulin antibody, and the results showed that the amount of polymerized microtubule was increased by the treatment with staurosporine while that of depolymerized beta-tubulin in soluble fraction was decreased. These results indicate that staurosporine induces ROS-mediated, microtubule-dependent formation of cellular processes in human gingival fibroblasts and rat astrocytes.

Study on differentiation pluripotency of human gingival fibroblasts induced in vitro / 天津医药

Objective To investigate the pluripotency of human gingival fibroblasts (hGFs), and provide a novel cell source for tissue engineering. Methods With informed consent from volunteers, fresh and healthy gingiva were collected. The hGFs were obtained from the gingiva by tissue culture. The third passage of hGFs was cultured in osteogenic medium, chondrogenic medium and adipogenic medium. Cells without differentiation were taken as control. Cells were examined by al?kaline phosphatase (ALP) staining, Alizarin red staining, Alcian blue staining and oil red O staining for detecting of the abili?ty of differentiation pluripotency. Real-time polymerase chain reaction was applied to examine the expression of osteogenic marker genes ALP, runt-related transcript factor 2 (Runx2), chondrogenic marker aggrecan (AGR) and adipogenic marker peroxisome proliferator-activated receptor gamma 2 (PPARγ2). Results The hGFs cultured in osteogenic medium showed massive violet deposit at day 7 and calcium nodulus at day 28, meanwhile, the expressions of ALP and Runx2 were higher than those of control (P<0.01). In chondrogenic group cells were found blue deposit at day 14. In adipogenic group lipid-filled droplets stained with oil red O were found in cells at day 14. However, hGFs in control group had no any positive stain?ing. Furthermore, expressions of AGR and PPARγ2 were significantly higher than those of control (P<0.01). Conclusion Human gingival fibroblasts have the pluripotency of osteogenic, adipogenic and chondrogenic differentiation.

Retinoic Acid Increases the Cell Cycle Progression of Human Gingival Fibroblasts by Increasing Cyclin E and CDK 2 Expression and Decreasing p21WAF1/CIP1 and p16INK4A Expression

Retinoic acid plays an important role in the regulation of cell growth and differentiation. In our present study, we evaluated the effects of all-trans retinoic acid (RA) on cell proliferation and on the cell cycle regulation of human gingival fibroblasts (HGFs). Cell proliferation was assessed using the MTT assay. Cell cycle analysis was performed by flow cytometry, and cell cycle regulatory proteins were determined by western blot. Cell proliferation was increased in the presence of a 0.1 nM to 1 microM RA dose range, and maximal growth stimulation was observed in cells exposed to 1 nM of RA. Exposure of HGFs to 1 nM of RA resulted in an augmented cell cycle progression. To elucidate the molecular mechanisms underlying cell cycle regulation by RA, we measured the intracellular levels of major cell cycle regulatory proteins. The levels of cyclin E and cyclin-dependent kinase (CDK) 2 were found to be increased in HGFs following 1 nM of RA treatment. However, the levels of cyclin D, CDK 4, and CDK 6 were unchanged under these conditions. Also after exposure to 1 nM of RA, the protein levels of p21WAF1/CIP1 and p16INK4A were decreased in HGFs compared with the control group, but the levels of p53 and pRb were similar between treated and untreated cells. These results suggest that RA increases cell proliferation and cell cycle progression in HGFs via increased cellular levels of cyclin E and CDK 2, and decreased cellular levels of p21WAF1/CIP1 and p16INK4A.

4-phenylbutyric Acid Regulates Collagen Synthesis and Secretion Induced by High Concentrations of Glucose in Human Gingival Fibroblasts

High glucose leads to physio/pathological alterations in diabetes patients. We investigated collagen production in human gingival cells that were cultured in high concentrations of glucose. Collagen synthesis and secretion were increased when the cells were exposed to high concentrations of glucose. We examined endoplasmic reticulum (ER) stress response because glucose metabolism is related to ER functional status. An ER stress response including the expression of glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), inositol requiring enzyme alpha (IRE-1alpha) and phosphoreukaryotic initiation factor alpha (p-eIF-2alpha) was activated in the presence of high glucose. Activating transcription factor 4 (ATF-4), a downstream protein of p-eIF-2alpha as well as a transcription factor for collagen, was also phosphorylated and translocalized into the nucleus. The chemical chaperone 4-PBA inhibited the ER stress response and ATF-4 phosphorylation as well as nuclear translocation. Our results suggest that high concentrations of glucose-induced collagen are linked to ER stress and the associated phosphorylation and nuclear translocation of ATF-4.

Evaluation of in vitro human gingival fibroblast seeding on acellular dermal matrix

The acellular dermal matrix (ADM) was introduced in periodontology as a substitute for the autogenous grafts, which became restricted because of the limited source of donor's tissue. The aim of this study was to investigate, in vitro, the distribution, proliferation and viability of human gingival fibroblasts seeded onto ADM. ADM was seeded with human gingival fibroblasts for up to 21 days. The following parameters were evaluated: cell distribution, proliferation and viability. Results revealed that, at day 7, fibroblasts were adherent and spread on ADM surface, and were unevenly distributed, forming a discontinuous single cell layer; at day 14, a confluent fibroblastic monolayer lining ADM surface was noticed. At day 21, the cell monolayer exhibited a reduction in cell density. At 7 days, about to 90 percent of adherent cells on ADM surface were cycling while at 14 and 21 days this proportion was significantly reduced. A high proportion of viable cell was detected on AMD surface both on 14 and 21 days. The results suggest that fibroblast seeding onto ADM for 14 days can allow good conditions for cell adhesion and spreading on the matrix; however, migration inside the matrix was limited.

A matriz dérmica acelular (MDA) foi introduzida na Periodontia como um substituto para enxertos autógenos, os quais se tornaram restritos devido à quantidade limitada de tecido doador. O objetivo deste estudo foi verificar, in vitro, a distribuição, proliferação e viabilidade de fibroblastos gengivais humanos cultivados em MDA. Fibroblastos gengivais foram cultivados sobre MDA por até 21 dias. Os seguintes parâmetros foram avaliados: distribuição, proliferação e viabilidade celular. Os resultados revelaram que, aos 7 dias, os fibroblastos estavam aderidos e espraiados na superfície da MDA, e estavam distribuídos de forma desigual, formando uma camada celular descontínua; aos 14 dias, uma monocamada confluente de fibroblastos revestindo a superfície da MDA foi observada. Aos 21 dias, a monocamada celular exibiu uma redução na densidade celular. Aos 7 dias, cerca de 90 por cento das células aderidas na superfície da MDA estavam no ciclo celular, enquanto que aos 14 e 21 dias esse número reduziu significativamente. Uma maior proporção de células viáveis foi detectada na superfície da MDA tanto aos 14 quanto aos 21 dias. Os resultados sugerem que fibroblastos cultivados sobre a MDA por 14 dias permitem boas condições de adesão e espraiamento das células sobre a matriz, porém, a migração de células para o interior da matriz foi limitada.

PPARgamma Inhibits Inflammation through the Suppression of ERK1/2 Kinase Activity in Human Gingival Fibroblasts

Periodontal disease is a major oral disorder and comprises a group of infections that lead to inflammation of the gingiva and the destruction of periodontal tissues. PPARgamma plays an important role in the regulation of several metabolic pathways and has recently been implicated in inflammatory response pathways. However, its effects on periodontal inflammation have yet to be clarified. In our current study, we evaluated the anti-inflammatory effects of PPARgamma on periodontal disease. Human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) showed high levels of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase-2 (MMP-2), and -9 (MMP-9). Moreover, these cells also showed upregulated activities for extracellular signal regulated kinase (ERK1/2), inducible nitric oxide synthase (iNOS) and cyclooxygnase-2. However, cells treated with Ad/PPARgamma and rosiglitazone in same culture system showed reduced ICAM-1, VCAM-1, MMP-2, -9 and COX-2. Finally, the anti-inflammatory effects of PPARgamma appear to be mediated via the suppression of the ERK1/2 pathway and consequent inhibition of NF-kB translocation. Our present findings thus suggest that PPARgamma indeed has a pivotal role in gingival inflammation and may be a putative molecular target for future therapeutic strategies to control chronic periodontal disease.

Pro-inflammatory cytokine expression in human gingival fibroblasts by Tannerella forsythia whole bacteria, membrane proteins, and lipopolysaccharide / 대한치주과학회지

PURPOSE: The purpose of this study was to investigate induction of cytokine expression in human gingival fibroblasts (HGFs) by whole cell and the components of T. forsythia. MATERIAL AND METHODS: After HGFs were treated with lipopolysaccharide (LPS), membrane protein isolated from T. forsythia or culture media of T. forsythia, the induction of interleukin (IL)-1, IL-6 and IL-8 was examined with real-time PCR and ELISA. Their induction ability of cytokines was compared with whole bacteria. RESULT: The expression of IL-6 and IL-8 was significantly induced in HGFs by whole bacteria and membrane protein. The expression of IL-1beta was induced by membrane protein of T. forsythia, not by whole bacteria. LPS and condition media of T. forsythia slightly activated HGFs. CONCLUSION: The membrane protein of T. forsythia could be one of virulence factors.

Expression of mRNA for matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gingival and periodontal ligament fibroblasts treated with lipopolysaccharide from Prevotella intermedia / 대한치주과학회지

Matrix metalloproteinases (MMPs) are a family of host-derived proteolytic enzymes and implicated in the remodeling and degradation of extracellular matrix under both physiological and pathological conditions. Connective tissue degradation in periodontal diseases is thought to be due to excessive MMP activities over their specific inhibitors. The effects of lipopolysaccharide (LPS) from Prevotella intermedia, one of the major putative pathogens of periodontitis, on the expression of mRNA for MMPs and tissue inhibitors of metalloproteinases (TIMPs) in human gingival and periodontal ligament fibroblasts were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of mRNAs encoding MMP-1, -2, -3, -10, and -14 was increased in human gingival fibroblasts treated with P. intermedia LPS, whereas MMP-11 and TIMP-2 mRNA expression was decreased in these cells stimulated with LPS. P. intermedia LPS increased the MMP-1, -2, -10, -11, and -14 mRNA expression and decreased TIMP-1 and -2 mRNA expression in human periodontal ligament fibroblasts. These findings imply that P. intermedia LPS may play an important role in the connective tissue degradation in periodontitis.