Cultures Of Gingival Fibroblasts Research Articles

Cigarette smoke condensate stimulates urokinase production through the generation of reactive oxygen species and activation of the mitogen activated protein kinase pathways in human gingival fibroblasts

Tobacco smoking is a significant risk factor for periodontal disease. It has been suggested that smoking may alter connective tissue remodeling in the periodontium. In the present study, we investigated whether cigarette smoke condensate modulates the production of the serine protease urokinase in human gingival fibroblasts. Primary cultures of human gingival fibroblasts were stimulated with cigarette smoke condensate. Urokinase production was evaluated through casein zymography and western blotting. Plasmin activation was assessed by means of a radial diffusion assay. The roles of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and reactive oxygen species in cigarette smoke condensate-stimulated urokinase production were studied using distinct selective inhibitors (SP600125, PD98059, N-acetyl cysteine). Reactive oxygen species production was determined using a fluorometric assay. Activation of ERK and JNK pathways were evaluated using western blots. In gingival fibroblasts, cigarette smoke condensate potently stimulated urokinase production and plasmin activation. Cigarette smoke condensate-stimulated urokinase production was dependent on the activity of ERK/JNK pathways and was inhibited by the reactive oxygen species scavenger, N-acetyl cysteine. Cigarette smoke condensate strongly stimulated ERK and JNK phosphorylation and the generation of reactive oxygen species. Cigarette smoke condensate stimulates urokinase production and plasmin activation in gingival fibroblasts. Moreover, cigarette smoke condensate-stimulated urokinase production depends on both the activation of ERK/JNK pathways and on the generation of intracellular reactive oxygen species. These results show that cigarette smoke may alter connective tissue remodeling by inducing production of the urokinase-type plasminogen activator through specific signaling pathways.

In vitro efficacy of nisin Z against Candida albicans adhesion and transition following contact with normal human gingival cells

To investigate the nisin Z innocuity using normal human gingival fibroblast and epithelial cell cultures, and its synergistic effect with these gingival cells against Candida albicans adhesion and transition from blastospore to hyphal form. Cells were cultured to 80% confluence and infected with C. albicans in the absence or presence of various concentrations of nisin Z. Our results indicate that only high concentrations of nisin Z promoted gingival cell detachment and differentiation. Determination of the LD(50) showed that the fibroblasts were able to tolerate up to 80 microg ml(-1) for 24 h, dropping thereafter to 62 mug ml(-1) after 72 h of contact, compared to 160 microg ml(-1) after 24 h, and 80 microg ml(-1) after 72 h recorded by the gingival epithelial cells which displayed a greater resistance to nisin Z. The use of nisin Z even at low concentration (25 microg ml(-1)) at appropriate concentrations with gingival cells significantly reduced C. albicans adhesion to gingival monolayer cultures and inhibited the yeast's transition. These findings show that when used at non-toxic levels for human cells, nisin Z can be effective against C. albicans adhesion and transition and may synergistically interact with gingival cells for an efficient resistance against C. albicans. This study suggests the potential usefulness of nisin Z as an antifungal agent, when used in an appropriate range.

Tumor necrosis factor‐α‐stimulated membrane type 1‐matrix metalloproteinase production is modulated by epidermal growth factor receptor signaling in human gingival fibroblasts

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a collagenolytic enzyme involved in connective tissue remodeling. In periodontal tissues, either cytokines or growth factors regulate the production of proteolytic enzymes. Mice deficient in epidermal growth factor receptor (EGFR) show a reduced expression of MT1-MMP, suggesting that this receptor may play an important role in MT1-MMP production. The present study evaluated the role of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and EGFR in the production of MT1-MMP in gingival fibroblasts. Primary cultures of human gingival fibroblasts were cultured over plastic or a type I collagen matrix and stimulated with TNF-alpha and EGF. A selective EGFR inhibitor (AG1478) was used to interfere with this signaling pathway. Production of MT1-MMP and activation of proMMP-2 were studied using Western blot and gelatin zymography, respectively. Activation of EGFR signaling was assessed through immunoprecipitation and Western blot. Expression of EGFR ligands was determined through reverse transcriptase-polymerase chain reaction. Treatment of gingival fibroblasts cultured over a collagen matrix with TNF-alpha stimulated proMMP-2 activation and MT1-MMP production. However, after using AG1478, both responses were inhibited. Tumor necrosis factor-alpha induced EGFR transactivation and stimulated the expression of the mRNA for the EGFR ligands heparin binding-epidermal growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha). The present study shows that TNF-alpha may stimulate MT1-MMP production through transactivation of EGFR. Tumor necrosis factor-alpha may also modulate the expression of the EGFR ligands TGF-alpha and HB-EGF. Production of MT1-MMP by TNF-alpha requires interaction with EGFR, suggesting that tissue remodeling is controlled by cross-communication between diverse signaling pathways in gingival fibroblasts.

Human leukocyte histocompatibility antigen class II‐induced cytokines from human gingival fibroblasts promote proliferation of human umbilical vein endothelial cells: potential association with enhanced angiogenesis in chronic periodontal inflammation

The role of human leukocyte histocompatibility antigen (HLA) class II molecules on non-antigen-presenting cells has been a matter of controversy. We previously reported that HLA-II molecules on human gingival fibroblasts (GF) do not present antigens, but transduce signals into the cells, resulting in the expression of several cytokines, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation, normal T-cell expressed and secreted (RANTES) and IL-8. However, the exact role of these cytokines, as well as other cytokines which are potentially secreted from GF, in the pathogenesis of chronic periodontal inflammation is not fully understood. The aim of this study was to observe the effects of HLA-II-induced cytokines on the proliferation of human umbilical vein endothelial cells (HUVEC). Antibody-based cytokine-microarray analyses were performed to detect potential cytokines associated with angiogenesis. Next, cytokine productivity was confirmed by quantitative methods. Then, cell proliferation assay was performed to see whether these cytokines promoted the proliferation of HUVEC. Besides IL-6, MCP-1, RANTES and IL-8, growth-related gene product (GRO) was newly identified as an HLA-II-induced cytokine released from GF. This was confirmed by a quantitative method. Cell culture supernatant from HLA-II-stimulated GF cultures promoted the growth of HUVEC. Addition of anti-IL-8 neutralizing antibody, anti-CXC receptor (CXCR)1 antibody and anti-MCP-1 antibody inhibited the growth of HUVEC in a dose-dependent manner, while addition of anti-GROalpha antibody did not. The HLA-II-induced IL-8, via CXCR1, as well as MCP-1 from GF, promotes endothelial cell proliferation, which is possibly associated with enhanced angiogenesis in chronic periodontal lesions.

In vitro evaluation of various bioabsorbable and nonresorbable barrier membranes for guided tissue regeneration

BackgroundDifferent types of bioabsorbable and nonresorbable membranes have been widely used for guided tissue regeneration (GTR) with its ultimate goal of regenerating lost periodontal structures. The purpose of the present study was to evaluate the biological effects of various bioabsorbable and nonresorbable membranes in cultures of primary human gingival fibroblasts (HGF), periodontal ligament fibroblasts (PDLF) and human osteoblast-like (HOB) cells in vitro.MethodsThree commercially available collagen membranes [TutoDent® (TD), Resodont® (RD) and BioGide® (BG)] as well as three nonresorbable polytetrafluoroethylene (PTFE) membranes [ACE (AC), Cytoplast® (CT) and TefGen-FD® (TG)] were tested. Cells plated on culture dishes (CD) served as positive controls. The effect of the barrier membranes on HGF, PDLF as well as HOB cells was assessed by the Alamar Blue fluorometric proliferation assay after 1, 2.5, 4, 24 and 48 h time periods. The structural and morphological properties of the membranes were evaluated by scanning electron microscopy (SEM).ResultsThe results showed that of the six barriers tested, TD and RD demonstrated the highest rate of HGF proliferation at both earlier (1 h) and later (48 h) time periods (P < 0.001) compared to all other tested barriers and CD. Similarly, TD, RD and BG had significantly higher numbers of cells at all time periods when compared with the positive control in PDLF culture (P ≤ 0.001). In HOB cell culture, the highest rate of cell proliferation was also calculated for TD at all time periods (P < 0.001). SEM observations demonstrated a microporous structure of all collagen membranes, with a compact top surface and a porous bottom surface, whereas the nonresorbable PTFE membranes demonstrated a homogenous structure with a symmetric dense skin layer.ConclusionResults from the present study suggested that GTR membrane materials, per se, may influence cell proliferation in the process of periodontal tissue/bone regeneration. Among the six membranes examined, the bioabsorbable membranes demonstrated to be more suitable to stimulate cellular proliferation compared to nonresorbable PTFE membranes.

Effect of Platelet‐Rich Plasma on Cell Adhesion, Cell Migration, and Myofibroblastic Differentiation in Human Gingival Fibroblasts

Platelet-rich plasma is a blood-derived fraction that contains a high concentration of platelets and growth factors. It was proposed that the use of this platelet concentrate stimulates tissue repair. However, little is known about the biologic response of gingival fibroblasts to platelet's derived growth factors. In the present study, we evaluated whether platelet-rich plasma modulated cell adhesion, cell migration, and myofibroblastic differentiation in primary cultures of human gingival fibroblasts. We studied the response of primary cultures of gingival fibroblasts to thrombin-activated platelet-rich plasma fractions. Cell adhesion was evaluated through a colorimetric assay. Cell spreading, actin cytoskeleton remodeling, and focal adhesion distribution were assessed through light and immunofluorescence microscopy. Cell migration was analyzed using a bicameral cell culture system. Smooth muscle actin production was studied through Western blotting. Exposure of gingival fibroblasts to platelet-rich plasma stimulated adhesion and spreading of cells on fibronectin matrices, the development of actin-enriched cellular extensions, and formation of focal adhesions. Platelet-rich plasma also promoted cell migration and invasion through a reconstituted basement membrane matrix. Differentiation into the myofibroblastic phenotype, assessed through the production of smooth muscle actin, was also stimulated by platelet-rich plasma preparations. Platelet-rich plasma may modulate several cell responses potentially involved in wound healing such as cell adhesion, cell migration, and myofibroblastic differentiation.

Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization

The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.

Effect of blue light on the proliferation of human gingival fibroblasts

Objectives Previous studies have reported that blue light, under conditions similar to those used for orthodontic bonding, influences several aspects of cellular physiology. The purpose of this study was to investigate the effect of the exposure to blue light curing sources, i.e. halogen, light emitting diode (LED) and plasma arc irradiation, on the proliferation of human gingival fibroblasts. Methods Primary cultures of human gingival fibroblasts were exposed to halogen, LED and plasma arc irradiation for 240, 180 and 120 s, respectively. The effect of blue light on DNA synthesis and cell proliferation was estimated by tritiated thymidine incorporation and direct cell counting, respectively. The possible involvement of an oxidative stress on the effect of blue light irradiation was studied by using N-acetyl-cysteine. Finally the formation of DNA double-strand breaks after irradiation was studied by immunofluorescence with an antibody against histone H 2A.x phosphorylated in Ser139. Results Blue light showed no immediate effect on the regulation of DNA synthesis. However, exposure of cells to these light sources inhibits cell proliferation measured one week after irradiation. This phenomenon is not attributed to the formation of DNA double strand breaks and cannot be annulled by N-acetyl-cysteine. Significance The results presented here indicate a mild inhibition of gingival fibroblasts’ proliferation after exposure to blue light and necessitate further study to clarify the exact mechanism underlying this effect.

A simple and established method of tissue culture of human gingival fibroblasts for gingival augmentation.

Recent advances in tissue engineering technology suggest its application in different medical fields, including periodontology. There are some reports of new non-enzymatic methods of isolating human gingival fibroblast for short-time cultivation in vitro to be used in autologous gingival augmentation. The aim of this study was to obtain a simple and established method of culturing human gingival fibroblasts. The authors developed a recurrent method that can be successfully used in autologous gingival augmentation.

A simple and established method of tissue culture of human gingival fibroblasts for gingival augmentation.

A simple and established method of tissue culture of human gingival fibroblasts for gingival augmentation.

Do MMP-1 Levels of Gingival Fibroblasts Have a Role in the Gingival Overgrowth of Cyclosporine-Treated Patients?

The aim of this study was to compare the matrix metalloproteinase-1 (MMP-1) levels in gingival fibroblast cultures derived from two groups of renal transplant patients receiving cyclosporine (CsA) who exhibit gingival overgrowth and who have healthy periodontium. Gingival fibroblasts obtained from four patients with CsA-induced gingival overgrowth (CsA-GO) and four patients who receive CsA but have healthy periodontium were incubated with increasing concentrations of CsA and cultured for 72 hours. Expression levels of MMP-1 in all the groups were measured four times at 0, 24, 48, and 72 hours by the Rapid Collagenase Assay Kit. No significant difference was seen at baseline. As the CsA concentration and the duration in the cell media increased, the CsA-GO showed that fibroblasts displayed significantly suppressed MMP-1 levels with respect to the baseline, at which fibroblasts from CsA patients with healthy periodontium exhibited the same result as at the highest CsA concentration. Results of this study indicated that CsA therapy did not have a significant effect on MMP-1 levels. Since the overall pathogenesis of drug-induced gingival hyperplasia has been accepted as multifactorial, down-regulation of MMP-1 expression may play a minor role.

Culture of Human Gingival Fibroblasts on a Biodegradable Scaffold and Evaluation of Its Effect on Attached Gingiva: A Randomized, Controlled Pilot Study

An adequate width of attached gingiva is necessary to maintain healthy periodontium, especially in orthodontics or restorative treatments in periodontics. The purpose of this study was to evaluate the width of attached gingiva after clinical application of a cultured gingival graft compared to a periosteal fenestration technique. This study was conducted on nine patients (18 sites) with insufficient attached gingiva adjacent to at least two teeth in contralateral quadrants of the same jaw. A small portion (approximately 3 x 2 x 1 mm) of attached gingiva (epithelial + connective tissue) was removed with a surgical blade. After culture of gingival fibroblasts, 2 x 10(5) cells in 250 microl nutritional medium were added to 250 microl collagen gel. One tooth in each patient was randomized to receive a periosteal fenestration technique for gingival augmentation (control) or a tissue-engineered mucosal graft (test). Clinical parameters measured at baseline and 3 months included width of keratinized tissue, probing depth, and width of attached gingiva. An increased amount of keratinized tissue was seen at all treated sites after 3 months. The mean increased amount of attached gingiva was 2.8 mm at test sites and 2 mm at control sites; this difference was significant (P < 0.05). Based on the results of this investigation, the tissue-engineered mucosal graft is safe and capable of generating keratinized tissue.

TGF-β1 Enhances the Expression of α–Smooth Muscle Actin in Cultured Human Pulpal Fibroblasts: Immunochemical and Ultrastructural Analyses

Transforming growth factor-beta 1 (TGF-beta1) has been related to induce the expression of alpha-smooth muscle actin (alpha-SMA) in fibroblasts during repair. Because pulpal fibroblasts seem to be somewhat different from other fibroblasts, the present study investigated in vitro whether TGF-beta1 enhances the expression of alpha-SMA in human pulpal fibroblasts. TGF-beta1 was added in doses between 5-10 ng/mL to cultures of both dental pulp and gingival human fibroblasts. The expression of alpha-SMA was analyzed by immunofluorescence and Western blotting, whereas the ultrastructure was evaluated by electron microscopy. In addition, the expression of tenascin, osteonectin, and vimentin was also investigated. Both cell types were immunoreactive for alpha-SMA even without TGF-beta1. When TGF-beta1 was added to cell cultures, the expression of alpha-SMA increased dramatically in pulpal fibroblasts, independent of the concentration used. It was confirmed by the Western blotting analysis. Ultrastructure revealed myofilaments and indented nuclei in both fibroblasts treated with TGF-beta1. Tenascin and osteonectin were only immunolabeled in pulpal fibroblasts treated or not with TGF-beta1. Both fibroblast types were positive for vimentin. The present findings showed that TGF-beta1 up-regulated the expression of alpha-SMA, thus inducing pulpal fibroblasts to acquire the myofibroblast phenotype.

Opposite effects of TGF‐β1 and IFN‐γ on transdifferentiation of myofibroblast in human gingival cell cultures

Previously, we have shown that myofibroblasts, the main cell type associated with interstitial fibrosis, may be implicated with the gingival overgrowth observed in hereditary gingival fibromatosis (HGF) patients. The goal of this study was to determine whether transforming growth factor-beta1 (TGF-beta1) stimulates myofibroblast generation in gingival fibroblast cultures. Moreover, we analysed how interferon-gamma (IFN-gamma) interferes in this process. Fibroblast cultures from normal gingiva and myofibroblast cells from HGF were included in this study. To determine the effects of TGF-beta1 and IFN-gamma stimulation in these cells, the expression of the specific myofibroblast marker smooth muscle isoform of alpha-actin (alpha-SMA) was examined by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunofluorescence. Enzyme-linked immunosorbent assay (ELISA) for type I collagen was performed to measure the myofibroblast activity. Our results demonstrated that TGF-beta1 promotes a dose- and time-dependent increase in the expression of alpha-SMA, whereas IFN-gamma blocks it and markedly prevents the fibroblast-myofibroblast switch induced by TGF-beta1 on normal gingiva cultures. IFN-gamma altered HGF myofibroblasts metabolism with a decrease of both alpha-SMA and type I collagen expression. Additionally, IFN-gamma treatment stimulated SMAD7 expression and inhibited connective tissue growth factor, which has been considered a key molecule to promote the transdifferentiation of myofibroblasts via TGF-beta1 activation. These findings demonstrate that TGF-beta1 induces gingival fibroblast-myofibroblast transdifferentiation, whereas IFN-gamma blocks this process. More importantly, this study suggests that IFN-gamma may be clinically effective in attenuating excessive accumulation of extracellular matrix produced by myofibroblasts in HGF.

Influence of different light curing units on the cytotoxicity of various dental composites

Objectives To evaluate the dependence of the toxicity of various dental composites on the use of high- and low-power light curing units (LCUs). Methods The composites Filtek Z 250, Durafill VS, Solitaire 2 and Grandio were polymerized using different light densities from three LCUs, namely Heliolux II, Swiss Master Light (SML) and a prototype LED. The toxicity of polymerized samples was tested by exposing them to the cell culture medium up to 28 days. The extracts of the composites were collected daily and used for incubation in human gingival fibroblasts cultures. Results Slow, low-intensity curing using the LED or the Heliolux II showed similar characteristics for all four composites, regarding the cell viability rate of human gingival fibroblasts. After 1 day of storage suboptimal results could be observed for the SML/Durafill and optimal results for SML/Grandio combination (approximately 100% cell viability). In addition, the composite Solitaire the SML yielded significantly better results than the other LCUs (cell viability, p ≤ 0.001: SML 60.5%, Heliolux 44.5%, LED 44.2%). Furthermore, the combination of the SML with Z 250 composite showed, after the first day and up to day 28, statistically significantly higher cell viability rates than the combination with the LED or Heliolux II. Significance This study shows that the combination of a high power LCU with some composites positively influences the HGF cell viability effected by the investigated composite extracts. Moreover, there is further indication that a reduction of composite toxicity is possible if the curing mode is adapted to the used composite.

Differential chemokine response of fibroblast subtypes to complement C1q

The pathogenesis of periodontitis includes an inappropriate activation of the classical complement cascade (C') with accumulation of inflammatory C' products in fluids and tissues. Our hypothesis is that in vivo the C' product, C1q, may act as a regulatory component of the innate immune response of distinct matrix fibroblasts to the inflammatory environment. This study analyzed the C1q induction of pro-inflammatory cytokine secretion in fibroblast subtypes derived from distinct periodontal tissues, and identified a mechanism of the cell response. Primary human gingival fibroblast, periodontal ligament fibroblast, and granulation tissue fibroblast cultures were treated for 24 h with C1q. Protein arrays assessed the secretory profile of constitutive and C1q-inducible pro-inflammatory cytokines, and enzyme-linked immunosorbent assays were used to quantify the kinetics of each inducible cytokine. Granulation tissue fibroblast cultures were unresponsive to C1q challenge. In contrast, periodontal ligament fibroblasts responded with a release of monocyte chemoattractant protein (MCP)-1, interleukin-6, interleukin-8, and macrophage inflammatory protein (MIP)-1beta higher than the basal level by 8.2-, 7.0-, 3.8-, and 7.2-fold, respectively. Human gingival fibroblast cultures increased secretion of these chemokines by 5.2-, 4.5-, 3.0-, and 9.8-fold, respectively. Inhibitor studies revealed that C1q-inducible release of chemokines by the human gingival fibroblast and periodontal ligament cultures was contingent upon p38 mitogen-activated protein kinase activity. The ability of C1q to stimulate secretion of pro-inflammatory chemokines depends upon which specific fibroblast subtype is involved. Targeting C1q-activated intracellular signaling pathways may be an effective means to inhibit the production of chemokines that promote inflammatory cell infiltration into gingival and periodontal ligament tissues.

The effect of chelerythrine on cell growth, apoptosis, and cell cycle in human normal and cancer cells in comparison with sanguinarine

We compared the effects of chelerythrine (CHE) and sanguinarine (SA) on human prostate cancer cell lines (LNCaP and DU-145) and primary culture of human gingival fibroblasts. CHE and SA treatment of cell lines for 24 h resulted in (1) inhibition of cell viability in a dose-dependent manner in all tested cells (as evaluated by MTT test and bromodeoxyuridine incorporation assay); (2) dose-dependent increase in DNA damage in all tested cells (as evaluated by DNA comet assay); (3) changes in apoptosis (assessed by western blot analysis and TUNEL assay); and (4) significant induction of cyclin kinase inhibitors p21(Waf1/Cip1) and p27(Kip1) in prostate cancer cells (identified by western blot analysis). Our study demonstrates that CHE had significant cytotoxic effect, independent of p53 and androgen status, on human prostate cancer cell lines. Normal gingival fibroblasts and DU-145 cells were more sensitive to the treatment with both alkaloids than were LNCaP cells. CHE and SA may be prospective natural molecules for use in the treatment of prostate cancer owing to their involvement in apoptosis and cell cycle regulation.

Antioxidant Effects of Glutathione and IGF in a Hyperglycaemic Cell Culture Model of Fibroblasts: Some Actions of Advanced Glycaemic end Products (AGE) and Nicotine

The aim of this investigation was to establish potential oxidative effects of glucose, advanced glycation end products (AGE) and nicotine (N) in a fibroblast cell culture model using the anti-oxidants glutathione (G) and insulin like growth factor (IGF). Assays of androgen metabolites were used as biomarkers of healing in this context. Confluent monolayer cultures of human gingival fibroblasts were established in 24 well multiwell plates and incubated in Eagle's MEM for 24h using two radiolabelled androgen substrates 14C-testosterone/14C-4-androstenedione. The established effective concentrations of G1000, glutathione and AGE were used alone and in combination with nicotine and insulin-like growth factor. The medium was then solvent extracted for steroid metabolites, evaporated to dryness and subjected to thin layer chromatography in a benzene acetone solvent system 4:1 v/v for separation of formed metabolites. The metabolites were quantified, using a radioisotope scanner. Significant reduction in the yields of DHT in response to G1000, AGE and nicotine (n=6; p <0.003) were overcome by glutathione (n=6; p <0.002). The stimulatory effect of IGF when combined with AGE was further enhanced by the antioxidant effect of glutathione (n=6; p <0.003). Glucose, AGE and nicotine had a significant inhibitory effect on the yields of the androgen biomarker DHT, overcome by the antioxidant glutathione and IGF, suggestive of an oxidant role for the former agents and an anti-oxidant one for the latter. These agents affected yields of androgen metabolites, biomarkers of oxidative stress and repair, with potential implications on healing in uncontrolled diabetic smokers.

Induction of the myofibroblastic phenotype in human gingival fibroblasts by transforming growth factor‐β1: role of RhoA‐ROCK and c‐Jun N‐terminal kinase signaling pathways

Myofibroblastic differentiation is an important event in gingival wound healing and chronic inflammation. Transforming growth factor-beta 1 (TGF-beta1) is a potent growth factor that has been implicated in this process. Gingival myofibroblasts have an increased ability to remodel the extracellular matrix and this feature has been associated with changes in the distribution of F-actin and the expression of the myofibroblast marker alpha-smooth muscle actin. In the present study we have analyzed the role of TGF-beta1 and the signaling routes activated by this factor in the cytoskeletal changes that characterize the myofibroblastic differentiation process in human gingival fibroblasts. The signalling pathways involved in myofibroblastic differentiation were studied in primary cultures of human gingival fibroblasts using several signal transduction inhibitors. RhoA activation was analyzed through a pull-down assay. Distribution of focal adhesions and actin cytoskeleton was assessed by means of immunofluorescence and western blot. A cell adhesion assay was performed in TGF-beta1-stimulated cells. Smooth muscle actin expression was studied through western blot and immunofluorescence. c-Jun N-terminal kinase phosphorylation was assessed through western blot. Our observations show that TGF-beta1 activated the GTPase RhoA, a key regulator of the actin cytoskeleton. As a consequence of this event, this growth factor stimulated the generation of actin stress fibers and the reinforcement of vinculin-enriched focal adhesions. These responses were blocked after inhibiting ROCK, the main target of RhoA activation. TGF-beta1 also stimulated the adhesion of fibroblasts over fibronectin, an extracellular matrix molecule involved in myofibroblastic differentiation. Finally, induction of the myofibroblast marker alpha-smooth muscle actin by TGF-beta1 was abolished by the c-Jun N-terminal protein kinase inhibitor SP600125, suggesting a role for this signaling pathway during the induction of this phenotype. We propose that TGF-beta1 may promote the differentiation of myofibroblasts through the stimulation of cell spreading and adhesion, the reinforcement of focal adhesions, the maturation of the actin cytoskeleton, and the induction of alpha-smooth muscle actin. Activity of RhoA-ROCK and c-Jun N-terminal protein kinase signaling pathways are probably involved in these cellular events.

Synergism between nifedipine and cyclosporine A on the incorporation of [35S]sulfate into human gingival fibroblast cultures in vitro

We assessed the effects of cyclosporine A and nifedipine on the in vitro incorporation of [(35)S]sulfate into gingival fibroblast cell cultures derived from responder and nonresponder subjects who had received an organ transplant followed by a therapeutic regimen using a combination of those drugs. Gingival fibroblasts were isolated from responder and nonresponder subjects and maintained in vitro. Prior to cell harvest, gingival interleukin-1beta concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Cells were untreated or exposed to either 10(-7)-10(-10) m nifedipine or 100-500 ng/ml cyclosporine A. Incorporation of [(3)H]proline or [(35)S]sulfate into the cell cultures was determined by liquid scintillation analysis. In addition, the effects of 400 ng/ml cyclosporine A + 10(-7) m nifedipine and 400 ng/ml cyclosporine A + 10(-10) m nifedipine on incorporation of [(35)S]sulfate into the cell cultures was determined. Data were compared by factorial analysis of variance (anova) and a posthoc Tukey's test. Gingiva from responders contained significantly more interleukin-1beta than gingiva from nonresponders (p < 0.01). The cell cultures derived from responders incorporated significantly more [(35)S]sulfate than those derived from nonresponders following exposure to either cyclosporine A or 10(-7) m nifedipine. In addition, the exposure of fibroblasts derived from gingival overgrowth to either 400 ng/ml cyclosporine A + 10(-7) m nifedipine or 400 ng/ml cyclosporine A + 10(-10) m nifedipine significantly increased or decreased, respectively, the incorporation of [(35)S]sulfate into the cultures. The therapeutic combination of cyclosporine A and nifedipine could be a significant risk factor for gingival overgrowth in subjects susceptible to either agent. The mechanism for overgrowth could include edema secondary to increased sulfated-glycosaminoglycan (sGAG) synthesis by fibroblasts, but further investigation is required.