Effect of 1,25(OH)2 D3 and 20(OH)D3 on interleukin-1ß-stimulated interleukin-6 and -8 production by human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Vitamin D-1,25(OH)2 D3 or 1,25D3-maintains healthy osseous tissue, stimulates the production of the antimicrobial peptide cathelicidin and has anti-inflammatory effects, but it can cause hypercalcemia. Evidence links diminished serum levels of 1,25D3 with increased gingival inflammation. Periodontitis progression is associated with increased local production of inflammatory mediators by immune cells and gingival fibroblasts. These include interleukin (IL)-6, a regulator of osteoclastic bone resorption, and the neutrophil chemoattractant IL-8, both regulated by signaling pathways, including NF-κB and MAPK/AP-1. The objectives were to determine the effects of 1,25D3 or a non-calcemic analog, 20-hydroxyvitamin D3 -20(OH)D3 or 20D3-on IL-1ß-stimulated IL-6 and IL-8 production, and NF-κB and MAPK/AP-1 activation, by human gingival fibroblasts. MATERIAL AND METHODS: Human gingival fibroblasts were incubated ± IL-1ß, with or without exposure to 1,25D3 or 20D3. IL-6 and IL-8 in culture supernatants were measured by enzyme-linked immunosorbent assay. NF-κB (p65) and AP-1 (phospho-cJun) and were measured in nuclear extracts via binding to specific oligonucleotides. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: IL-1ß-stimulated IL-6 and IL-8 levels were both significantly inhibited (40%-60%) (P<.045) by 1,25D3, but not 20D3 (0%-15% inhibition, not statistically significant). Both 1,25D3 and 20D3 significantly and similarly inhibited IL-1ß-stimulated nuclear levels of p65 and phospho-cJun (P<.02). CONCLUSION: Reduction of the activation of NF-κB and AP-1 alone is not able to inhibit strongly the IL-1ß stimulated IL-6 and IL-8 gene expression. 1,25D3 but not 20D3 may affect some of the many other factors/processes/pathways that in turn regulate the expression of these genes. However, the results suggest that topical application of ligands of the vitamin D receptor may be useful in the local treatment of periodontitis while reducing adverse systemic effects.

Effect of glycated albumin and cranberry components on interleukin-6 and matrix metalloproteinase-3 production by human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Gingival fibroblasts have the potential to participate in periodontal inflammation and breakdown, producing interleukin (IL)-6 and matrix metalloproteinase (MMP)-3. Advanced glycation end products (AGEs), formed during diabetic hyperglycemia, might aggravate periodontal inflammation. The cranberry contains anti-inflammatory polyphenols, which inhibit proinflammatory activities of lipopolysaccharide (LPS)- and IL-1ß-stimulated human cells. Little is known of its effects on gingival fibroblast IL-6 or MMP-3 production stimulated by AGEs. The objectives were to determine cranberry effects on IL-6 and MMP-3 production by gingival fibroblasts exposed to the representative AGE, glycated human serum albumin (G-HSA), or LPS ± G-HSA. MATERIAL AND METHODS: Cranberry high molecular weight non-dialyzable material (NDM), was derived from cranberry juice. Normal human gingival fibroblasts were incubated with G-HSA or normal HSA or Porphyromonas gingivalis LPS (1 µg/mL) ± G-HSA, in the presence or absence of preincubation with NDM. IL-6 and MMP-3 were measured by enzyme-linked immunosorbent assay. Data were analyzed using one-way analysis of variance and Scheffe's F procedure. RESULTS: IL-6 production was stimulated by G-HSA or LPS (p < 0.01), which was inhibited in both cases by NDM (p < 0.002). [G-HSA+LPS] synergistically stimulated IL-6 production (p < 0.0001), which was inhibited by NDM. MMP-3 levels were not stimulated by G-HSA but were decreased by LPS (p < 0.02). [G-HSA+LPS] increased MMP-3 production significantly, vs. LPS (p = 0.0005). NDM inhibited MMP-3 levels in the presence of G-HSA or LPS, and in the presence of [G-HSA+LPS] (p < 0.0001). CONCLUSIONS: G-HSA ± LPS may have differential effects on IL-6 and MMP-3 production by human gingival fibroblasts, but both are inhibited by NDM. The study suggests that cranberry phenols may be useful in regulating the host response and perhaps treating periodontitis in patients with poorly controlled diabetes.

Re-evaluating the role of vitamin D in the periodontium.

The importance of vitamin D in maintaining skeletal health via the regulation of calcium has long been recognized as a critical function of this secosteroid. An abundance of literature shows an association between oral bone mineral density and some measure of systemic osteoporosis and suggests that osteoporosis/low bone mass may be a risk factor for periodontal disease. Recently, nonskeletal functions of vitamin D have gained notoriety for several reasons. Many cells that are not associated with calcium homeostasis have been demonstrated to possess membrane receptors for vitamin D. These include activated T and B lymphocytes, and skin, placenta, pancreas, prostate and colon cancer cells. In addition, vitamin D "insufficiency" is a worldwide epidemic and epidemiologic evidence has linked this condition to multiple chronic health problems, including cardiovascular and autoimmune diseases, hypertension and a variety of cancers. Interestingly, there is mounting evidence connecting diminished serum levels of vitamin D with increased gingival inflammation and supporting the concept of "continual vitamin D sufficiency" in maintaining periodontal health. The ability of vitamin D to regulate both the innate and the adaptive components of the host response may play an important role in this process. This review will examine the skeletal and nonskeletal functions of vitamin D, and explore its potential role in protecting the periodontium as well as in regulating periodontal wound healing.

Inhibition of interleukin 1ß-stimulated interleukin-6 production by cranberry components in human gingival epithelial cells: effects on nuclear factor κB and activator protein 1 activation pathways.

BACKGROUND AND OBJECTIVE: In periodontitis, gingival epithelial cells can produce interleukin (IL)-6, a regulator of osteoclastic bone resorption, in response to IL-1ß. IL-1ß regulates cytokine expression via signaling pathways, including nuclear factor (NF)-κB and mitogen activated protein kinase (MAPK)/activator protein (AP)-1. Cranberry proanthocyanidins (PACs) inhibit IL-1ß-stimulated IL-6 production, but specific mechanisms are unclear. The objectives of this study were to determine effects of cranberry PACs on NF-κB and MAPK/AP-1 activation of IL-1ß-stimulated IL-6 production in gingival epithelial cells. MATERIAL AND METHODS: Cranberry high molecular weight non-dialyzable material (NDM), rich in PACs, was derived from cranberry juice. Human gingival epithelial cells [Smulow-Glickman (S-G)] were incubated with IL-1ß in the presence or absence of NDM or inhibitors of NF-κB, [nemo-binding domain (NBD) peptide] or AP-1 (SP600125), and IL-6 levels were measured by ELISA. Effects of NDM on IL-1ß-activated NF-κB and AP-1 and phosphorylated intermediates in both pathways were measured in cell extracts via binding to specific oligonucleotides and specific sandwich ELISAs, respectively. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: IL-1ß (≥ 0.1 nm) caused a time- and dose-dependent stimulation of S-G epithelial cell IL-6 production (p < 0.005). This was significantly decreased in a dose-dependent manner by NBD peptide or SP600125 [maximum inhibition ~30-40% (p < 0.02)], and together, the two inhibitors decreased IL-6 by ~80%, similar to the inhibition caused by NDM (p < 0.001). IL-1ß stimulated NF-κB and AP-1 activation (p < 0.003), which was inhibited by NDM (p < 0.0001). NDM did not significantly affect IL-1ß-stimulated levels of phosphorylated intermediates in the NF-κB pathway (IκBα) or the AP-1 pathway (c-Jun, ERK1/2). CONCLUSION: In S-G epithelial cells, IL-1ß appeared to upregulate IL-6 production via activation of both NF-κB and MAPK/AP-1 signaling pathways because cranberry NDM decreased nuclear levels of IL-1ß-activated NF-κB (p65) and AP-1 (phospho-c-Jun) and strongly inhibited IL-6 production. Lack of inhibition of phosphorylation of IκBα, c-Jun or ERK1/2 suggested that NDM might affect both pathways downstream from those points in S-G cells, such as ubiquitination and proteosomal degradation of IκBα, or inhibition of nuclear activity of c-Jun and/or ERK1/2. Defining these points of inhibition precisely may help identify molecular targets of cranberry polyphenols.

Inhibition of interleukin-17-stimulated interleukin-6 and -8 production by cranberry components in human gingival fibroblasts and epithelial cells.

BACKGROUND AND OBJECTIVE: Gingival epithelial cells and fibroblasts participate in periodontal inflammation and destruction, producing interleukin (IL)-6, a regulator of osteoclastic bone resorption, and the neutrophil chemoattractant IL-8. IL-17, a product of T-helper 17 cells, may play a role in periodontitis by stimulating cytokine production by gingival cells. The cranberry (Vaccinium macrocarpon) is rich in polyphenols, particularly proanthocyanidins, which have antioxidant and other beneficial properties. Cranberry components inhibit pro-inflammatory activities of lipopolysaccharide-stimulated human macrophages, gingival fibroblasts, and epithelial cells, but little is known of its effects on IL-17-stimulated cytokine production. The objectives were to determine the effects of IL-17 ± cranberry components on IL-6 and IL-8 production by human gingival epithelial cells and fibroblasts. MATERIAL AND METHODS: Cranberry high molecular weight non-dialyzable material (NDM), which is rich in proanthocyanidins, was derived from cranberry juice. Human gingival epithelial cells and normal human gingival fibroblasts were incubated with NDM (5-50 µg/mL), IL-17 (0.5-100 ng/mL), or NDM + IL-17 in serum-free medium for 6 d. IL-6 and IL-8 in culture supernatants were measured by ELISA. Membrane damage and viability were assessed by lactate dehydrogenase activity released into cell supernatants and activity of a mitochondrial enzyme, respectively. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: In both cell lines, IL-17 (≥ ~5-10 ng/mL) significantly stimulated production of IL-6 (p < 0.005) and IL-8 (p < 0.03). Non-toxic levels of NDM inhibited constitutive IL-6 and IL-8 production by epithelial cells (p ≤ 0.01) and fibroblasts (p ≤ 0.03) as well as IL-17-stimulated cytokine production by epithelial cells [IL-6 (maximum ~80% inhibition; p ≤ 0.0001); IL-8 (maximum ~70% inhibition; p ≤ 0.03)] and fibroblasts [IL-6 (maximum ~90% inhibition; p ≤ 0.0001); IL-8 (maximum ~80% inhibition; p ≤ 0.008)]. CONCLUSION: Cranberry NDM inhibition of constitutive and IL-17-stimulated IL-6 and IL-8 production by gingival fibroblasts and epithelial cells suggests that cranberry components could be useful as a host modulatory therapeutic agent to prevent or treat periodontitis.

Effects of cranberry components on human aggressive periodontitis gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Aggressive periodontitis (AgP) causes rapid periodontal breakdown involving AgP gingival fibroblast production of cytokines [i.e. interleukin (IL)-6, a bone metabolism regulator], and matrix metalloproteinase (MMP)-3. Lipopolysaccharide upregulates fibroblast IL-6 and MMP-3, via transcription factors (i.e. NF-κB). Cranberry (Vaccinium macrocarpon) inhibits lipopolysaccharide-stimulated macrophage and normal gingival fibroblast activities, but little is known of its effects on AgP fibroblasts. Objectives of this study are to use AgP fibroblasts, to determine cytotoxicity of cranberry components or periodontopathogen (Fusobacterium nucleatum, Porphyromonas gingivalis) lipopolysaccharide ± cranberry components, and effects of cranberry components on lipopolysaccharide-stimulated NF-κB activation and IL-6 and MMP-3 production. MATERIAL AND METHODS: AgP fibroblasts were incubated ≤ 6 d with high molecular weight non-dialyzable material (NDM) (derived from cranberry juice (1-500 µg/mL) or lipopolysaccharide (1 µg/mL) ± NDM. Membrane damage and viability were assessed by enzyme activity released into cell supernatants and activity of a mitochondrial enzyme, respectively. Secreted IL-6 and MMP-3 were measured by ELISA. NF-κB p65 was measured via binding to an oligonucleotide containing the NF-κB consensus site. Data were analyzed using analysis of variance and Scheffe's F procedure for post hoc comparisons. RESULTS: Short-term exposure to NDM, or lipopolysaccharide ± NDM caused no membrane damage. NDM (≤ 100 µg/mL) or lipopolysaccharide ± NDM had no effect on viability ≤ 7 d exposure. NDM (50 µg/mL) inhibited lipopolysaccharide-stimulated p65 (P ≤ 0.003) and constitutive or lipopolysaccharide-stimulated MMP-3 (P ≤ 0.02). NDM increased AgP fibroblast constitutive or lipopolysaccharide-stimulated IL-6 (P ≤ 0.0001), but inhibited normal human gingival fibroblast IL-6 (P ≤ 0.01). CONCLUSION: Lack of toxicity of low NDM concentrations, and its inhibition of NF-κB and MMP-3, suggest that cranberry components may regulate AgP fibroblast inflammatory responses. Distinct effects of NDM on AgP and gingival fibroblast production of IL-6 (which can have both positive and negative effects on bone metabolism) may reflect phenotypic differences in IL-6 regulation in the two cell types.

Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor ß.

BACKGROUND AND OBJECTIVE: Marijuana (Cannabis sativa) use may be associated with gingival enlargement, resembling that caused by phenytoin. Cannabidiol (CBD), a nonpsychotropic Cannabis derivative, is structurally similar to phenytoin. While there are many reports on effects of phenytoin on human gingival fibroblasts, there is no information on effects of Cannabis components on these cells. The objective of this study was to determine effects of CBD on human gingival fibroblast fibrogenic and matrix-degrading activities. MATERIAL AND METHODS: Fibroblasts were incubated with CBD in serum-free medium for 1-6 d. The effect of CBD on cell viability was determined by measuring activity of a mitochondrial enzyme. The fibrogenic molecule transforming growth factor ß and the extracellular matrix molecule fibronectin were measured by ELISA. Pro-MMP-1 and total MMP-2 were measured by ELISA. Activity of MMP-2 was determined via a colorimetric assay in which a detection enzyme is activated by active MMP-2. Data were analysed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: Cannabidiol had little or no significant effect on cell viability. Low CBD concentrations increased transforming growth factor ß production by as much as 40% (p < 0.001), while higher concentrations decreased it by as much as 40% (p < 0.0001). Cannabidiol increased fibronectin production by as much as approximately 100% (p < 0.001). Lower CBD concentrations increased MMP production, but the highest concentrations decreased production of both MMPs (p < 0.05) and decreased MMP-2 activity (p < 0.02). CONCLUSION: The data suggest that the CBD may promote fibrotic gingival enlargement by increasing gingival fibroblast production of transforming growth factor ß and fibronectin, while decreasing MMP production and activity.

Statins regulate interleukin-1ß-induced RANKL and osteoprotegerin production by human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Three-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase competitive inhibitors, or 'statins', are widely used for lowering cholesterol and thereby reducing the risk of a heart attack. Recent data suggest that statins influence metabolic bone activity by their actions on three molecules: RANKL; RANK; and osteoprotegerin (OPG), the soluble decoy receptor for RANKL. The purpose of this study was to evaluate OPG and RANKL production in resting and interleukin-1ß (IL-1ß)-activated human gingival fibroblasts (HGFs), and to determine the effect of statins on their production. MATERIAL AND METHODS: Fibroblasts were pre-incubated with atorvastatin or simvastatin for 24h in serum-free medium, and then incubated with IL-1ß for 6d. The concentration of OPG or RANKL in culture supernatants was measured by specific ELISA. Data were analyzed using analysis of variance and Scheffe's F procedure for post hoc comparison. RESULTS: IL-1ß (1×10(-8) m) stimulated a significant increase in the production of OPG on days 1, 3 and 6. There was a trend towards an increase in RANKL production as a result of stimulation with IL-1ß. Both statins, at multiple concentrations, significantly increased the constitutive RANKL/OPG ratio. Only atorvastatin at the highest concentration (5×10(-6) m) significantly increased the IL-1ß-stimulated RANKL/OPG ratio. CONCLUSION: IL-1ß significantly increased OPG production by HGFs. The statins differed minimally in their effects on OPG and RANKL production by resting and IL-1ß-activated HGFs. Both statins increased constitutive RANKL/OPG ratios, but generally not IL-1ß-stimulated ratios. Thus, statins may influence the production of RANKL and OPG by HGFs to favor bone catabolism, under noninflammatory conditions.

Effect of bisphosphonates on human gingival fibroblast production of mediators of osteoclastogenesis: RANKL, osteoprotegerin and interleukin-6.

BACKGROUND AND OBJECTIVE: Osteonecrosis of the jaw (ONJ) is associated with bisphosphonate (BP) therapy. BPs alter osteoblast production of mediators of osteoclastogenesis, including interleukin (IL)-6, RANKL and osteoprotegerin (OPG), a RANKL antagonist. This can inhibit bone turnover and lead to necrosis. There is little information on the contribution of gingival fibroblasts, near bone-resorption sites, to the IL-6/RANKL/OPG network, the effects of BPs, or fibroblast involvement in ONJ pathogenesis. Therefore, the objective of this study was to determine the effects of alendronate and pamidronate on the constitutive production, or the lipopolysaccharide (LPS)- or IL-1ß-stimulated production, of IL-6, RANKL and OPG by human gingival fibroblasts. MATERIAL AND METHODS: Human gingival fibroblasts were derived from explants obtained from healthy individuals with noninflamed gingiva. Cytotoxicity was determined by measuring the activity of a mitochondrial enzyme. Fibroblasts were pre-incubated or not with BPs (0.01 nm-1 µm), then incubated or not with LPS or IL-1ß. The concentrations of IL-6, OPG and RANKL were measured using ELISA. Data were analyzed using analysis of variance (ANOVA) and Scheffé's F procedure. RESULTS: LPS and BPs were not cytotoxic. The cells produced IL-6, OPG and RANKL, all of which were stimulated by IL-1ß or LPS (p ≤ 0.04). BPs generally increased the production of IL-6 and OPG (p ≤ 0.04) and decreased the production of RANKL (p ≤ 0.02). BPs generally further increased the production of LPS- or IL-1ß-stimulated IL-6 (p ≤ 0.04) and had no effect on, or further increased, the production of LPS- or IL-1ß-stimulated OPG (p ≤ 0.04). BPs decreased the production of LPS- or IL-1ß-stimulated RANKL (p ≤ 0.04) and decreased constitutive, LPS-stimulated and IL-1ß-stimulated RANKL/OPG ratios (p ≤ 0.02). CONCLUSION: The action of alendronate and pamidronate on human gingival fibroblasts, through altering the production of RANKL and OPG, appears to contribute to a microenvironment favoring the inhibition of bone resorption and ONJ.

Methamphetamine cytotoxicity and effect on LPS-stimulated IL-1beta production by human monocytes.

Methamphetamine (METH) abuse is associated with "METH mouth", characterized by rampant dental decay and destruction of periodontal bone and soft tissues. In periodontitis, monocyte/macrophages, stimulated by bacterial lipopolysaccharide (LPS), produce interleukin-1beta (IL-1beta), contributing to bone and soft tissue degradation. Effects of METH on monocyte/macrophages and its role in periodontitis are unknown. The objective of this study was to determine METH cytotoxicity and effects on constitutive and LPS-stimulated IL-1beta production in THP-1 human monocytes. METH significantly reduced cell viability, assessed by activity of a mitochondrial enzyme, by 20-40% after 24h, with recovery at longer periods. Brief exposure to METH caused <10% cytotoxicity (measured by an assay that detects membrane damage). LPS from E. coli or the periodontopathogen Fusobacterium nucleatum (F. n.) significantly increased IL-1beta production (measured by ELISA). Despite cytotoxicity of some METH concentrations, METH had no significant effect on constitutive IL-1beta production. However, METH generally increased LPS-stimulated IL-1beta levels, reaching statistical significance at 5x10(-5)M METH ( approximately 50% to >100% increase). The study suggests that METH potentiation of periodontopathogen LPS stimulation of IL-1beta in monocytes could contribute to periodontitis in METH abusers, consistent with other studies suggesting a role for increased IL-1beta in deleterious effects of METH.

Effect of myrrh oil on IL-1beta stimulation of NF-kappaB activation and PGE(2) production in human gingival fibroblasts and epithelial cells.

Anecdotal and scientific evidence suggest that myrrh oil (MO) has anti-inflammatory properties. Subtoxic MO levels decrease interleukin (IL)-1beta-stimulated production of the inflammatory cytokine IL-6 by human gingival fibroblasts, but not epithelial cells. IL-1beta upregulates IL-6 via PGE(2), and via NF-kappaB, a transcription factor for many inflammatory mediator genes. NF-kappaB is inhibited by sesquiterpene compounds (from plants other than myrrh). This study determined MO effect on IL-1beta-stimulated PGE(2) production and NF-kappaB activation in gingival fibroblasts and epithelial cells. Cells were preincubated with MO, exposed to IL-1beta, cytoplasmic and nuclear fractions were isolated, and activated NF-kappaB was measured using an ELISA-based assay. IL-1beta increased nuclear activated NF-kappaB levels in fibroblasts and epithelial cells [10- and 2.5-fold over controls, respectively (p=0.0001)], and these increases were not significantly affected by MO. PGE(2) was measured in cell supernatants by ELISA, after preincubation with MO and exposure to IL-1beta. MO inhibited IL-1beta-stimulated PGE(2) production by fibroblasts (p=0.001), but not epithelial cells. The data suggest that gingival epithelial cells and fibroblasts may differ in the magnitude of NF-kappaB activation after IL-1beta stimulation, and that MO inhibition of IL-1beta-stimulated IL-6 production in fibroblasts is due in part to inhibition of PGE(2), but not NF-kappaB activation. (Supported by NIDCR DE-0725.).

In vitro cytotoxic and anti-inflammatory effects of myrrh oil on human gingival fibroblasts and epithelial cells.

Limited scientific studies suggest that myrrh (Commiphora molmol) has antibacterial and anti-inflammatory activities. This study determined myrrh oil (MO) cytotoxicity to human gingival fibroblasts and epithelial cells and its effect, measured by ELISA, on interleukin (IL)-1beta-stimulated IL-6 and IL-8 production. Cell viability and cytotoxicity were determined by metabolic reduction of a tetrazolium salt to a formazan dye (MTT assay) and by release of lactate dehydrogenase (LDH) from membrane damaged (LDH release assay) cells, respectively. Based on the MTT assay, 24- and 48-h exposures to /=0.005%, maximally decreased viability of all cell lines. In the LDH release assay, exposure to /=0.0025% MO caused maximal cytotoxicity; /=0.0025% MO, probably reflective of loss of viability. At subtoxic MO levels (0.00001-0.001%), there was a significant reduction of IL-1beta-stimulated IL-6 and IL-8 production by fibroblasts, but not by epithelial cells.

In vitro response of human gingival epithelioid S-G cells to minocycline.

Minocycline, a broad-spectrum antibiotic used in the treatment of acne and periodontal disease and to control inflammatory diseases such as rheumatoid arthritis, has recently been shown to induce a spectrum of adverse health effects. In the light of these contradictory data, this research was directed to provide basic information on the toxicology of minocycline, using in vitro cell culture models, and to evaluate its efficacy in periodontal therapies, particularly for wound healing. The human gingival epithelioid S-G cell line was used as the bioindicator. The greater toxicity of minocycline over doxycycline and tetracycline, related antimicrobial agents, probably correlated with its higher lipophilicity. The cytotoxicity of minocycline was unaffected by an S9 hepatic microsomal fraction, indicating that it is a direct-acting, rather than a metabolism-mediated, cytotoxicant. In comparative toxicity studies, much variation in the degree of sensitivity to minocycline was noted for different cell types. No correlation in the extent of sensitivity to minocycline and the physiologic state of the bioindicator cell (normal, transformed or malignant) was noted. The toxicity of minocycline to the S-G cells was dependent on its concentration and length of exposure. For a continuous 3-day exposure of the S-G cells to minocycline, the midpoint cytotoxicity (or, NR(50)) value, as quantified in the neutral red (NR) assay, was 204 microg/ml on day 1, 84 microg/ml on day 2, and 59 microg/ml on day 3. For a 1-h exposure of the S-G cells in phosphate buffered saline (PBS), the NR(50) value was 780 microg/ml minocycline. Although a 1-h exposure in PBS to 200 microg/ml minocycline exerted some toxicity, the S-G cells recovered on exposure to growth medium; irreversible, progressive damage occurred at 400 microg/ml minocycline and greater. Minocycline, at 50 microg/ml, enhanced attachment of the S-G cells to a gelatin-coated surface and cell migration towards an immobilized fibronectin gradient, both biologic parameters important in periodontal wound healing. Minocycline generally had little or no effect on production of the pro-inflammatory cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8), by non-activated S-G cells, the exception being stimulation of IL-6 at 48 h. IL-1beta, however, greatly stimulated IL-6 and IL-8 production, which was further increased by concurrent exposure to minocycline. This suggested that minocycline may enhance the ability of gingival epithelial cells to participate in the early, inflammatory phase of periodontal wound healing. The limitation of minocycline efficacy to a rather narrow window of concentration, centering about 50 microg/ml, and primarily for short-term exposures may possibly explain, in part, the contradictory clinical data on the health effects of this drug.

Cytotoxicity of mineral trioxide aggregate using human periodontal ligament fibroblasts.

The purpose of the present study was to compare the cytotoxicity of mineral trioxide aggregate (MTA) to other commonly used retrofilling materials, Super-EBA and amalgam. This was accomplished using a cell viability assay for mitochondrial dehydrogenase activity in human periodontal ligament fibroblasts after 24-hr exposure to extracts of varying concentrations of the test materials, in both freshly mixed and 24-hr set states. Methyl methacrylate 2% (vol/vol) served as the positive control, and complete culture medium served as the negative control. Differences in mean cell viability values were assessed by ANOVA (p < 0.05). In the freshly mixed state, the sequence of toxicity was amalgam > Super-EBA > MTA. In the 24-hr set state the sequence of toxicity at a low extract concentration was Super-EBA > MTA, amalgam, and Super-EBA > amalgam > MTA at a higher extract concentration. This study supports the use of MTA in the root-end environment.

Autocrine transforming growth factor beta stimulation of extracellular matrix production by fibroblasts from fibrotic human gingiva.

Hereditary gingival fibromatosis (HGF) is a fibrotic enlargement of the gingiva. HGF gingiva contains large amounts of collagen and other extracellular matrix (ECM) molecules. In vitro, HGF fibroblasts produce greater amounts of the ECM components fibronectin (FN) and type 1 collagen than normal human gingival (GN) fibroblasts. Transforming growth factor beta (TGF beta) is a cytokine important in regulating tissue repair and regeneration after injury, and stimulating fibroblast proliferation and the production of FN and collagens. The objective of this study was to determine whether HGF fibroblasts produce TGF beta and, with the use of neutralizing antibodies to TGF beta isoforms, if their increased expression of FN and type 1 collagen is under autocrine TGF beta control. The HGF strains produced greater amounts of TGF beta1 and TGF beta2 (P < or = 0.003) as well as FN (P < or = 0.04) and type 1 collagen (P < or = 0.03) (measured by specific ELISA) than the GN strains. Treatment of HGF fibroblasts with anti-TGF beta1, beta2, or beta3, as well as a combination of all 3 antibodies, decreased their FN production by up to 60% (P < or = 0.04), and was able to decrease FN production by HGF fibroblasts to the levels of the GN fibroblasts. When used alone, the neutralizing antibodies decreased type 1 collagen production by the HGF fibroblasts by up to 40% (P = 0.014), and treatment with all 3 antibodies caused decreases of up to 55% (P = 0.0005). The results suggest that autocrine stimulation by the increased amounts of TGF beta isoforms made by HGF fibroblasts contributes to their increased production of FN and type 1 collagen.

Increased proliferation, collagen, and fibronectin production by hereditary gingival fibromatosis fibroblasts.

HEREDITARY GINGIVAL FIBROMATOSIS (HGF) is a fibrotic enlargement of the gingiva. HGF gingiva contains large amounts of interstitial collagen and other extracellular matrix (ECM) molecules. Increased proliferation and elevated production of the ECM molecules type I collagen and fibronectin (FN) could contribute to the clinical increased bulk of HGF gingiva. Fibroblast strains from HGF gingiva and normal human gingival fibroblast strains (GN) were used in this in vitro study. Fibroblast proliferation was determined by ELISA which measured the incorporation of 5-bromo-2'-deoxyuridine into DNA. The results showed that HGF fibroblast strains proliferated more rapidly than GN fibroblasts (68% to 488% increase, depending on the strains) (P < or = 0.01), the only exception being one HGF strain versus one normal strain. All HGF strains produced greater amounts of FN (measured by ELISA) than all of the normal fibroblast strains (23% to 49% increase, depending on the strain) (P < or = 0.04). Similarly, all HGF strains made significantly greater (P < or = 0.3) amounts of type I collagen (also measured by ELISA) than all of the normal strains (55% to 235% increase, depending on the strain). The results show that, in vitro, HGF fibroblasts display several phenotypic characteristics of activated fibroblasts: increased proliferative rates as well as increased production of FN and type I collagen, consistent with in vitro studies of fibroblasts derived from other types of fibrotic tissue. These results suggest that the increased proliferation of HGF fibroblasts and their increased production of extracellular matrix molecules such as collagen and FN may contribute to the clinical gingival enlargement characteristics of HGF.

Effects of nicotine on proliferation and extracellular matrix production of human gingival fibroblasts in vitro.

Normal function of gingival fibroblasts is essential for maintenance of the gingival extracellular matrix (ECM), but under inflammatory conditions in gingival tissue which may occur with tobacco use, they can also act in its destruction. The purpose of this study was to determine the effects of nicotine, a major component of tobacco, on gingival fibroblast proliferation, the production of fibronectin (FN), and the production and breakdown of type I collagen to elucidate its role in periodontal destruction associated with its use. A human gingival fibroblast strain derived from a healthy individual with non-inflamed gingiva was used in this study. Nicotine at concentrations > 0.075% caused cell death, and at 0.075% and 0.05% it caused transient vacuolization of the fibroblasts. At concentrations of 0.001% to 0.075%, nicotine significantly inhibited proliferation (P < or = 0.03), measured by the incorporation of [3H]-thymidine into DNA. The production of FN and type I collagen was significantly inhibited by nicotine at > or = 0.05% (P < or = 0.001), measured using specific ELISAs. On the other hand, nicotine at > or = 0.025% significantly increased collagenase activity (P < or = 0.008), using [3H]-gly and [14C]-pro-labeled type I collagen gels as substrate. The results show that, in vitro, nicotine inhibits the growth of gingival fibroblasts and their production of FN and collagen, while also promoting collagen breakdown. This suggests that nicotine itself may augment the destruction of the gingival ECM occurring during periodontal inflammation associated with smokeless tobacco use.

Role of saliva and salivary components as modulators of bleaching agent toxicity to human gingival fibroblasts in vitro.

Mild oxygenating agents generating H2O2 are used for effective at-home tooth bleaching, but can cause gingival ulcers in some patients. There are concerns about the possible pathological effects of relatively long-term exposure of oral tissues to bleaching agents. Previous work in our laboratory showed that a bleaching agent, which generates approximately 3% H2O2 from carbamide peroxide, was toxic to human gingival fibroblasts in vitro, but that the toxicity was abolished by treatment with the H2O2-destroying enzyme catalase. The purpose of the present study was to determine if whole saliva, the salivary enzyme lactoperoxidase (LP) (which, like catalase, removes H2O2), or salivary mucin protected fibroblasts from bleaching agent toxicity. The cells were exposed to 0.05% agent with or without saliva, LP, mucin or catalase (as a positive control based on our previous study) and assessed for effect on viability/morphology (by microscopic observation), proliferation (by [3H]-thymidine incorporation), and the production of fibronectin (FN) and type I collagen (by ELISA). While the bleaching agent at 0.05% caused cell death, the cells appeared viable and morphologically normal when treated with the bleaching agent and LP (> or = 0.1 microM), saliva LP, and catalase from agent inhibition of proliferation (P < or = 0.04) and FN production (P < or = 0.01). Mucin had statistically insignificant or no protective effect as assessed by the above parameters. Treatment with saliva, LP, mucin, and catalase gave complete or partial protection from agent-inhibition of collagen production (P < or = 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of mast cell-macrophage interactions on the production of collagenolytic enzymes by metastatic tumor cells and tumor-derived and stromal fibroblasts.

Histological examination of the metastatic rat mammary adenocarcinoma line MTLn3 showed that macrophages and mast cells were frequently localized at the tumor periphery in the stromal tissues adjacent to the zones of tumor invasion. The interactions of these host cells with tumor cells and tumor-associated fibroblasts could be important in stimulating the production of extracellular matrix-degrading enzymes that facilitate tumor invasion and metastatic spread. Therefore, we examined the effects of isolated, activated macrophages and mast cells on the secretion of collagenolytic activities by normal fibroblasts, metastatic mammary adenocarcinoma cells and tumor-associated fibroblasts. Medium from activated macrophages or degranulated mast cells stimulated significant increases in production of collagenolytic activities by normal and tumor-associated fibroblasts and MTLn3 tumor cells. Medium from activated macrophages that had been pretreated with medium from degranulated mast cells, however, were less stimulatory to fibroblasts and tumor cell production of collagenolytic activities than medium from degranulated mast cells alone. We also examined the effects of two cytokines, interleukin-1 alpha and tumor necrosis factor-alpha on activated macrophage- and degranulated mast cell-stimulation of fibroblast and tumor cell collagenolytic activities. The two cytokines alone or in combination stimulated increased production of collagenolytic activities by fibroblasts and tumor cells. Addition of the cytokines to degranulated mast cell products resulted in secretion of higher collagenolytic enzyme activities by normal fibroblasts (but not by tumor-derived fibroblasts or tumor cells) than with degranulated mast cell product-treatment of either target cell alone. Cytokines used in combination with macrophage-conditioned medium were less effective in stimulating fibroblast and tumor cell collagenase activities than cytokines alone. Thus normal infiltrating host cells such as macrophages and mast cells can have profound effects on the production of degradative enzymes by tumor cells and tumor-associated stromal fibroblasts.

Effects of a bleaching agent on human gingival fibroblasts.

Mild oxygenating agents generating low concentrations of hydrogen peroxide (H2O2) are effective alternatives to heat-activated 30% H2O2 in bleaching discolored, vital teeth. There are concerns about possible pathological effects of long-term exposure to bleaching agents, and irritation and ulceration of the gingiva and other oral soft tissues can occur. The objective of this study was to determine the effect of one of these agents on gingival fibroblasts in vitro. Microscopic examination revealed that concentrations of 0.05% to 0.025% of the agent appeared to kill most of the cells. At concentrations of 0.025% to 0.017% some morphological changes were noted; the cells appeared normal at concentrations of < or = 0.0125%. The agent significantly (P < or = 0.002) decreased proliferation (measured by incorporation of [3H]-thymidine into cellular DNA) at concentrations as low as 0.006%. The agent also had a dose-dependent effect on fibronectin production, measured by ELISA, causing significant (P < or = 0.03) decreases at concentrations as low as 0.017%. The agent significantly decreased the production of types I (P < or = 0.01) and III (P < or = 0.04) collagens (measured by ELISA) at concentrations as low as 0.0125%. Type V collagen was not detected under any conditions. Catalase, which catalizes the breakdown of H2O2, abolished toxic effects of a 0.05% solution. The results show that in vitro, the agent is toxic to human gingival fibroblasts, inhibiting several cellular functions.(ABSTRACT TRUNCATED AT 250 WORDS)