Human Gingival Fibroblast Cells Research Articles

Anabolic effects of low-intensity pulsed ultrasound on human gingival fibroblasts

Objective Low-intensity pulsed ultrasound (LIPUS) demonstrated anabolic effects on cementoblasts, odontoblasts, and periodontal ligament cells. However, LIPUS effect on human gingival fibroblasts (HGF) remains to be investigated. Therefore, we evaluated the in vitro effects of LIPUS on HGF proliferation and differentiation to test its feasibility for periodontal therapy. Design LIPUS treatment (1.5 MHz, 30 mW/cm 2) was applied to HGF in the experimental groups after 24-h of culture (5 or 10 min/day for 28 days) and omitted in the control. Changes in HGF activities were evaluated in response to LIPUS treatment in dose-dependent (5 and 10 min) and time-dependent (weeks 1–4) manner. The effects of LIPUS on HGF cell viability (MTT), proliferation (total DNA content and growth pattern), alkaline phosphatase (ALP) activity, and gene expression by reverse-transcriptase polymerase chain reaction (RT-PCR) were determined. Results Cell viability remained unchanged after LIPUS treatment during the 4 weeks of treatment as compared to the untreated control group which ensured a safe biological response. Both LIPUS treatments (5–10 min/day) did not yield any significant changes in the proliferation, and expression of proliferating cell nuclear antigen (PCNA) and collagen-I (COL-I). Conversely, LIPUS treatment enhanced osteogenic differentiation potential of HGF as determined by significant up-regulation of specific ALP activity and osteopontin (OPN) expression, with optimum effect following 3 weeks of 5 min/day LIPUS treatment. Conclusion LIPUS treatment at 30 mW/cm 2 selectively enhanced HGF differentiation but not proliferation. The ability of LIPUS to enhance HGF differentiation is promising for its application in cell-based periodontal therapy.

Characterization of recombinant human cementum protein 1 ( hrCEMP1): Primary role in biomineralization

Cementum protein 1 (CEMP1) has been recently cloned, and in vitro experiments have shown functions as regulator of cementoblast behavior and inducer of differentiation of non-osteogenic cells toward a cementoblastic/osteoblastic phenotype. In this study, we have produced a full-length human recombinant CEMP1 protein in a human gingival fibroblast cell line. The purified protein ( hrCEMP1) has a M r 50,000. Characterization of hrCEMP1 indicates that its secondary structure is mainly composed of β-sheet (55%), where random coil and alpha helix conformations correspond to 35% and 10%, respectively. It was found that hrCEMP1 is N-glycosylated, phosphorylated and possesses strong affinity for hydroxyapatite. Even more important, our results show that hrCEMP1 plays a role during the biomineralization process by promoting octacalcium phosphate (OCP) crystal nucleation. These features make CEMP1 a very good candidate for biotechnological applications in order to achieve cementum and/or bone regeneration.

Efficient gene transfer to periodontal ligament cells and human gingival fibroblasts by adeno-associated virus vectors

Objectives We explored for the first time the possibility to deliver a reporter gene (Green Fluorescence Protein) to human primary periodontal ligament (PDL) cells and human gingival fibroblasts (HGF) using shuttle vectors derived from adeno-associated virus (AAV). Since AAV transduction rates on other human primary fibroblasts have been previously shown to depend on the particular cell lineage and on the employed viral serotype, we determined the most effective AAV variant for periodontal cells comparing different vector types. Methods AAV serotypes 1–5 encoding GFP in single stranded (ss) and self-complementary (sc) vector genome conformations were used to infect primary HGF and PDL cells. Two days post-infection, the percentage of GFP expressing cells was determined by flow cytometry. Results Highest transduction rates for both cell types were achieved with self-complementary vectors derived from AAV-2, resulting in GFP expression in up to 86% of PDL cells and 50% of HGF. Transgene expression could be observed by optical microscopy for 2 months after infection. Lower but detectable rates were obtained with serotypes 1, 3 and 5. Conclusions The efficacy demonstrated here and the safety and versatility of AAV technology indicated in previous studies clearly suggest the potential of AAV vectors as tools for gene transfer to periodontal tissues.

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.

Attachment and growth behaviour of human gingival fibroblasts on titanium and zirconia ceramic surfaces

The attachment, growth behaviour and the genetic effect of human gingival fibroblasts (HGF) cultured on titanium and different zirconia surfaces were investigated. HGF cells were cultured on (1) titanium discs with a machined surface, (2) yttrium-stabilized tetragonal zirconia polycrystals (Y-TZP) with a smooth surface and (3) Y-TZP with 100 µm grooves. The cell proliferation activity was evaluated through a MTT assay at 24 h and 48 h, and the cell morphology was examined by SEM. The mRNA expression of integrin-β1, type I and III collagen, laminin and fibronectin in HGF were evaluated by RT-PCR after 24 h. From the MTT assay, the mean optical density values for the titanium and grooved zirconia surfaces after 48 h of HGF adhesion were greater than the values obtained for the smooth zirconia surfaces. SEM images showed that more cells were attached to the grooves, and the cells appeared to follow the direction of the grooves. The results of RT-PCR suggest that all groups showed comparable fibroblast-specific gene expression. A zirconia ceramic surface with grooves showed biological responses that were comparable to those obtained with HGF on a titanium surface.

Effect of zoledronic acid on oral fibroblasts and epithelial cells: a potential mechanism of bisphosphonate‐associated osteonecrosis

Osteonecrosis of the jaw secondary to bisphosphonate infusion (zoledronic acid-ZA) is assumed to be a bone disease. This study investigated the effects of ZA on soft tissues using oral mucosal cells as an in vitro model of soft tissue cell death in the pathogenesis of bone necrosis. Human gingival fibroblast and keratinocyte cell lines were exposed to different concentrations of ZA (0.25-3 micromol/l), using 1 micromol/l as the expected baseline concentration. A dose-response effect on apoptosis and cell proliferation [Terminal deoxynucleotidyl transferase-mediated dUTP-Biotin End Labelling and Annexin V or Coulter counter and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), respectively] was observed with increasing ZA concentrations; both reversed using siRNA against caspase 3 or 9. Gene expression analysis using RT(2) Profiler polymerase chain reaction Arrays demonstrated the differential expression of multiple genes involved in apoptosis including those that encode TNF, BCL-2, Caspase, IAP, TRAF and Death Domain families. Western blot analysis confirmed the presence of activated forms of caspase 3 and 9 and underexpression of survivin protein expression. This study demonstrated that low concentrations of ZA rapidly and directly affected the oral mucosal tissues though the induction of a gene-regulated apoptotic process. These findings support the potential for soft tissue injury as an initiating/potentiating event for osteonecrosis.

Indoleamine 2,3‐Dioxygenase Expression and Regulation in Chronic Periodontitis

Indoleamine 2,3-dioxygenase (IDO) is an intracellular tryptophan-oxidizing enzyme with immunosuppressive characteristics. Its expression and regulation in periodontal tissues are unknown. The aim of this study was to determine IDO expression in healthy gingiva and chronic periodontitis lesions. In addition, the effect of inflammatory cytokines and bacterial products on the expression and activity of DOI in human gingival fibroblasts (HGFs) was assessed. Human gingival tissue samples were obtained from patients who underwent periodontal surgery. IDO expression in healthy gingiva and periodontitis lesions was determined by immunohistochemistry. HGF cells were treated with interferon-gamma (IFN-gamma), interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharides from Porphyromonas gingivalis (PgLPS). IDO mRNA expression was determined by reverse transcription-polymerase chain reaction. The IDO enzymatic activity was determined by measuring the kynurenine level using a colorimetric method. In gingival tissues, IDO expression was detected in epithelial cells, fibroblasts, endothelial cells, and inflammatory mononuclear cells. IDO expression was higher in periodontitis lesions than in healthy gingiva. HGFs did not constitutively express IDO. IFN-gamma strongly induced IDO expression and activity in HGFs, in a dose-dependent manner. IL-1beta, TNF-alpha, and PgLPS were also able to induce IDO expression in HGF cells. IFN-gamma in combination with IL-1beta, TNF-alpha, or PgLPS showed enhanced IDO expression. IDO was expressed in human gingiva, and the expression was upregulated in chronic periodontitis. The increased IDO expression in periodontitis lesions may be due, in part, to the activation of HGFs by inflammatory cytokines and bacterial products.

Role of Zoledronic Acid in the Apoptotic Cell Death of Oral Fibroblasts and Epithelial Cells: A Mechanism of Bisphosphonate-Associated Osteonecrosis

Purpose: Osteonecrosis of the jaw is reported in patients receiving IV zoledronic acid (ZA). The purpose of this study was to investigate the effects of ZA on oral mucosal cells using an in vitro model as an alternative mode for the induction of bone necrosis.Experimental Design: Human gingival fibroblast and keratinocyte cell lines were exposed to different concentrations (0.25–3μM) of ZA and siRNA versus caspase 3 or 9, using 1μM as the expected baseline concentration. Apoptotic effects were determined by TUNEL and Annexin V studies. Effects on cell proliferation were determined by Coulter Counter and MTS assay. Validation of the apoptotic effects of ZA were obtained via RT2 Profiler PCR Arrays of human apoptosis and Western blot.Results: A dose response effect on apoptosis and cell proliferation was observed with increasing ZA concentrations; both reversed using siRNA against caspase 3 or 9. Initial apoptotic effects were observed microscopically at 8 hours, with increasing apoptotic events measured out to 24 hours. Gene expression analysis demonstrated the differential expression of multiple genes involved in apoptosis including: TNF, Bcl-2, caspase, IAP, TRAF, and death domain families. Western blot analysis confirmed the presence of activated forms of caspase 3 and 9 and underexpression of survivin protein expression.Conclusions: This study demonstrates that low concentrations of ZA rapidly and directly affect the oral mucosal tissues though the induction of a gene-regulated apoptotic process. These findings provide the establishment of an oral cell based model supporting a soft tissue mechanism in the pathogenesis of osteonecrosis.

Influence of chlorine dioxide on cell death and cell cycle of human gingival fibroblasts

Objectives The effects of chlorine dioxide (ClO 2), sodium hypochlorite (NaOCl), and hydrogen peroxide (H 2O 2) on cell death and the cell cycle of human gingival fibroblast (HGF) cells were examined. Methods The inhibition of HGF cell growth was evaluated using a Cell Counting Kit-8. The cell cycle was assessed with propidium iodide-stained cells (distribution of cells in G 0/G 1, S, and G 2/M phases) using flow cytometry. The patterns of cell death (necrosis and apoptosis) were analyzed using flow cytometry with annexin V-FITC/PI staining. Results The lethal doses for 50% of the cells (LD 50) of ClO 2, NaOCl, and H 2O 2 were 0.16, 0.79, and 0.11 mM, respectively. All three dental disinfectants induced G 0/G 1 cell cycle arrest. H 2O 2 induced apoptosis at concentrations of 0.05 and 0.1 mM, while NaOCl and ClO 2 did not induce significant apoptosis at any concentration examined. Conclusions These results suggest that ClO 2 is sufficient for use as a dental disinfectant compared with H 2O 2 or NaOCl.

In vitro induction of hydrolytic activity in human gingival and pulp fibroblasts by triethylene glycol dimethacrylate and monocyte chemotatic protein-1

Objective Some dental resin composite materials leach the monomer triethylene glycol dimethacrylate (TEGDMA), which could contribute to the inflammatory reaction. The purpose of this study was to examine the ability of TEGDMA to alter cytokine/growth factor secretion and enzymatic activity in monocyte derived macrophages (U937 cells), human gingival fibroblasts (HGFs), and human pulp fibroblasts (DPFs). Methods A human growth factor/cytokine antibody array was utilized to determine whether TEGDMA alters the expression of cytokines/growth factors from U937 cells, HGFs, and DPFs. To determine if TEGDMA alters the hydrolase activity of U937, DPF, and HGF cells, the hydrolysis of p-nitrophenyl butyrate was utilized in a spectrophotometric assay. Results TEGDMA exposure induced the expression of monocyte chemotatic protein-1 (MCP-1) from the U937 cells. Both the cell lysates and conditioned media from the HGFs showed increased hydrolase activity after exposure to a sublethal concentration of TEGDMA. MCP-1 alone increased the hydrolytic activity and the combination of MCP-1 and TEGDMA was additive in HGF conditioned media. The DPFs were also exposed to MCP-1 alone and followed by a sublethal concentration of TEGDMA. The effect of MCP-1 was greater than that of TEGDMA. Significance These results demonstrate that TEGDMA induces enzymatic activity and cytokine/growth factor expression in a cell-specific manner.

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.

A Comparative Study of Inhibitory Effect of Human Calprotectin on the Growth of Human Gingival Fibroblast and Foreskin Fibroblast

In the present study, cytotoxicity effects of calprotectin on Human Gingival Fibroblast (HGF) and Human Foreskin Fibroblast (HFFF) were compared. For these evaluations, both cells were exposed to the different concentrations of calprotectin, for 24, 48 and 72 h. Cell proliferation was assessed using MTT assay. Our results revealed that growth inhibition of calprotectin on HGF and HFFF occur in a dose- and time-dependent manner. Results of this investigation showed that sensitivity of HGF cells to cytotoxic effect of human calprotectin was more than HFFF. The results indicate that drug resistance process is different for the two kinds of fibroblast cells.

<i>In vitro</i> cytotoxicity of “mswaki” fibre on human gingival fibroblasts

Aim: This study determined the in vitro cytotoxicity of mswaki fibres on human gingival fibroblasts (HGF). Methods: Two types of “mswaki” twigs (Salvadora persica and Euclea natalensis) were used. Each twig was swabbed with 70% ethanol, the bark was then removed and approximately 1cm pieces of fibre were cut and stored separately. Two HGF cell lines (GW3 and GW6) were cultured, harvested and seeded into 12mm tissue culture wells at a concentration of 3-5 X 105cells.ml-1 and allowed to form a monolayer by incubating them for 24h at 37oC in a humidified atmosphere of 5% CO2 and of 95% air. To determine cytotoxicity, cut pieces of both twigs were placed into the tissue culture wells with established HGF and excess media was removed for closer proximity. Using an inverted microscope, cell reactions were observed at 10 and 30min and 1, 2, 4, 24, and 48h and cell lysis determined when a change in cell morphology was observed. Results: None of the employed HGF cell lines showed any change in cell morphology for either of the inoculated mswaki fibres during the study period. Cells appeared healthy and no changes in morphology were observed indicative of cell lysis. Cell proliferation for both cell lines at 24 and 48h showed no significant difference from controls regardless of the presence of mswaki fibre. Conclusion: Both miswaki fibres were observed not to have exhibited any cytotoxic effect hence proof of their suitability as a traditional toothbrush in the maintenance of oral hygiene. Keywords : In vitro , cytotoxicity, mswaki, human gingival fibroblasts Tanzania Dental Journal Vol. 14 (2) 2007: pp. 59-64

New triterpene glycosides of Clematis montana and their cytotoxic activities

Two new triterpene glycosides designated as montanosides 1 and 2 were isolated from methanolic extract of Clematis montana. Their structures have been elucidated as 3-O-β-D-glucopyranosyl-oleanolιc acid-28-O-Β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl- (1→2)]-β-D-xylopyranoside and 3-0-β-D-glucopyranosyl-oleanolic acid-28-O-β-D-glucopyranosyl.(1→4)-[β-D-glucopyranosyl.(1→3)-α-L-rhamnopyranosyl-(1→2)]-β-D-sylopyranoside respectively by NMR and MS data. Cytotoxic activity of the compounds were evaluated against various squamous carcinoma cells (HSC-2) and human gingival fibroblast cells (HGF). Both of the compounds showed potent cytotoxic activity against the test cells.

Nanocrystalline Diamond as a Coating for Joint Implants: Cytotoxicity and Biocompatibility Assessment

Nanocrystalline diamond (NCD) coatings combine a very low surface roughness with the outstanding diamond properties, such as superlative hardness, low self‐friction coefficient, high wear and corrosion resistance, and biotolerance, which are ideal features for applications in medicine (knee and hip replacement) and surgical tools. The present work presents a comprehensive study of the cytotoxicity and biocompatibility of NCD films grown by hot‐filament chemical vapour deposition (HFCVD) technique, aiming such future applications. Cytotoxicity was evaluated in vitro by seeding human gingival fibroblasts on the NCD surface for 14 days, while specific biocompatibility was assessed on samples seeded with human bone marrow‐derived osteoblasts during 21 days. The NCD coatings proved to be noncytotoxic in the preliminary human gingival fibroblast cell cultures, as denoted by a notable sequence of cell attachment, spreading, and proliferation events. In the specific biocompatibility assay envisaging bone tissue applications, NCD coatings induced human osteoblast proliferation and the stimulation of differentiation markers, compared to standard polystyrene tissue culture plates.

Jatropha curcas latex inhibits the release of collagenase by gingival fibroblast

Folkloric use of J. curcas latex among others are to cure tooth pain, bleeding gum and as anti-inflammatory drug. Collagenase is a neutral protease released by activated macrophage and also by fibroblasts in small amounts. The aim of this study was to evaluate the effect of J. curcas latex on collagenase released by fibroblasts. Four doses of J. curcas latex from 37.5-300 g/ml were added to 3 human gingival primary fibroblast cell culture. After 1 to 4 days of incubation, collagenase in the supernatant was assayed with collagen. The degradation products were then separated by SDS-PAGE and the density of ¾ αA bands were measured semi quantitatively by Adobe Photo computer program. Result showed that J. curcas latex decreased collagenase released by human gingival fibroblast, and increasing dose inhibits more. It may be concluded that the latex of J. curcas inhibits the release of collagenase by human gingival fibroblast. (Med J Indones 2007; 16:219-23)

Apoptosis of gingival fibroblasts in periodontitis.

Introduction: Human gingival fibroblasts (HGF) have an important role in the periodontal immune response. The fibroblasts alter their normal behavior in response to pro-inflammatory cytokines. It is believed that HGF can be diminished and/or eliminated by means of apoptosis. The purpose of this study was to determine and to quantify apoptosis of HGF in periodontium biopsies from healthy and chronic periodontitis patients. Methods: A clinical cross-sectional study in people with healthy periodontium (S), gingivitis (G) and chronic periodontitis (PC) patients was carried out. The periodontal biopsies were obtained and immunostained by means of: hematoxylin-eosin, caspase-3, vimentin and caspase-vimentin double-staining for specific visualization of apoptotic fibroblasts. Histopathological and digital analyses were performed. Descriptive statistics were applied to categorical and nominal variables. Results: Total cell population of HGF had an average of 430±67.6 cells/field in healthy people, and a significantly progressive decrease in gingivitis (270±37.1) and chronic periodontitis groups (206.5±69.8) (p< 0.05). As for total population of inflammatory cells, an increase was noticed in gingivitis (191.8±50.1) and a decrease in periodontitis (109.3±21.7) without statistical significance. The expression of apoptotic HGF per field increased accordingly to the severity of the disease [28±16 in health (6.5%); 31±17 in gingivitis (11.5%) and 51±24 in periodontitis (24.8%), p< 0.001]. Similar findings were observed for inflammatory cells with different percentage expression [17±13 in health (23%); 28±19 in gingivitis (14.6%) and 47±35 in periodontitis (43.1%), p< 0.05]. The relationship between the percentage of expression of apoptotic cells and probing pocket depth was proportional but not significant (p>0.5, r²=0.02); while for the inflammatory cells a significant relationship was observed (p< 0.05, r²=0.2018). Conclusions: The results in this report conclude that HGF and inflammatory cells show apoptosis (caspase-3 expression), and apoptotic cells are significantly increased in gingivitis and chronic periodontitis groups.

Actinobacillus actinomycetemcomitans adheres to human gingival fibroblasts and modifies cytoskeletal organization

Adherence of Actinobacillus actinomycetemcomitans to human gingival fibroblast cells induces cytoskeletal reorganization. A. actinomycetemcomitans is considered a pathogenic bacteria involved in localized aggressive periodontitis. Studies with epithelial cells have shown an adherent capacity of bacteria that is increased under anaerobic conditions. For adherence to take place, there is a need for interaction between extracellular vesicles and bacterial fimbriae. However, molecular events associated with the adherence process are still unknown. The aim of this study was to investigate whether A. actinomycetemcomitans adherence to human gingival fibroblasts promotes cytoskeletal reorganization. Adherence was determined with light microscopy and scanning electron microscopy. For F-actin visualization, cells were treated with fluorescein-isothiocyanate-phalloidin and samples were examined with epifluorescence optics. Fluorescent was recorded on Kodak T-Max 400 film. We showed that A. actinomycetemcomitans adheres to human gingival fibroblast primary cultures, this property stimulating an increase in the intracellular calcium levels. In human gingival fibroblast primary cultures, we observed that maximal A. actinomycetemcomitans adherence took place 1.5h after culture infection occurred and remained for 6h. The adherence was associated with morphologic alterations and an increased in the intracellular calcium levels. These experiments suggest that A. actinomycetemcomitans adherence cause morphological alterations, induce actin stress fibers and recruitment of intracellular calcium levels.

ヒト歯肉上皮細胞と繊維芽細胞の共培養系のGene Chip, IPA解析

ヒト歯肉上皮細胞と繊維芽細胞の共培養系のGene Chip, IPA解析

Effects of laser irradiation on the release of basic fibroblast growth factor (bFGF), insulin like growth factor-1 (IGF-1), and receptor of IGF-1 (IGFBP3) from gingival fibroblasts

Various studies have shown biostimulation effects of laser irradiation by producing metabolic changes within the cells. Little is known about the biological effect of laser irradiation on the oral tissues. Among the many physiological effects, it is important to recognize that low-level laser therapy (LLLT) may affect release of growth factors from fibroblasts. Therefore, the aim of the present study was to determine whether the laser irradiation can enhance the release of basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), and receptor of IGF-1 (IGFBP3) from human gingival fibroblasts (HGF). The number of all samples in the study were 30, and the samples were randomly divided into three equal groups; In the first group (single dose group), HGF were irradiated with laser energy of 685 nm, for 140 s, 2 J/cm(2) for one time, and in the second group, energy at the same dose was applied for two consecutive days (double dose group). The third group served as nonirradiated control group. Proliferation, viability, and bFGF, IGF-1, IGFBP3 analysis of control and irradiated cultures were compared with each other. Both of the irradiated groups revealed higher proliferation and viability in comparison to the control group. Comparison of the single-dose group with the control group revealed statistically significant increases in bFGF (p < 0.01) and IGF-1 (p < 0.01), but IGFBP3 increased insignificantly (p > 0.05). When the double dose group was compared with the control group, significant increases were determined in all of the parameters (p < 0.01). In the comparison of the differences between the two irradiated groups (one dose and two doses), none of the parameters displayed any statistically significant difference (p > 0.05). In both of the laser groups, LLLT increased the cell proliferation and cell viability. The results of this study showed that LLLT increased the proliferation of HGF cells and release of bFGF, IGF-1, and IGFBP3 from these cells. LLLT may play an important role in periodontal wound healing and regeneration by enhancing the production of the growth factors.