Estrogen and extracellular matrix influence human gingival fibroblast proliferation and protein production

BACKGROUND: A paucity of information exists concerning how estrogen affects cellular function in the gingiva of women. In this study, the behavior of human gingival fibroblasts was examined in the presence of a potent estrogen, estradiol. METHODS: Quiescent, premenopausal gingival fibroblasts were incubated in the presence and absence of estradiol (1 nM) and/or raloxifene (100 nM). Cell number was determined and cell cycle analyzed using a flow cytometer. Collagen and non-collagen production in cell cultures grown on various extracellular matrices were determined using a radioactive microassay which measures collagenase-digestible and collagenase-resistant radiolabeled proteins. To ascertain if the gingiva contained specific estrogen-sensitive cell populations, a fluorescence-activated cell sorter was used to detect, sort, and enrich fibroblast populations responsive to estrogen. RESULTS: Cellular proliferation and the number of cells entering the S-phase of the cell cycle were significantly increased in mass cultures of fibroblasts stimulated by estradiol. Raloxifene did not antagonize the action of estradiol on cell proliferation. In regard to protein production, estradiol significantly reduced collagen production on plastic and collagen IV matrices; whereas non-collagen protein production on plastic and collagen I matrices was significantly reduced. Cell sorting of mass fibroblast populations revealed that, on average, 45% of the cells from the resident population selectively accumulated the estrogen probe. These sorted and estrogen-sensitive enriched cell populations proliferated in the presence of 1 nM estradiol, whereas the sorted, estrogen-deficient enriched fibroblast populations did not proliferate when incubated with 1 nM estradiol. CONCLUSIONS: These data indicate that estradiol can induce cellular proliferation while depressing protein production in cultures of human, premenopausal gingival fibroblasts. This cellular proliferation appears to be the result of a specific population of cells within the parent culture that responds to physiologic concentrations of estradiol.     

Acoustic energy affects human gingival fibroblast proliferation but leaves protein production unchanged

BACKGROUND, AIMS: Sonic toothbrushes are well-established in oral home care for plaque removal; however, the effects of low frequency acoustic (sonic) energy released from sonic toothbrushes to the cells of the periodontium have not been investigated. The purpose of this study was to evaluate the effects of sonic energy on human gingival fibroblast proliferation and protein production in cell culture. METHODS: Direct and indirect transfer calibration studies found the fundamental frequency of the Sonicare sonic toothbrush to be 261 hertz (Hz) with amplitudes ranging from 70 to 104 decibels (dB) in the human periodontium. Using an in vitro delivery system, which coupled a signal-wave generator with a bone transducer to mimic the energy delivered by the Sonicare toothbrush, the effects of signal, amplitude and duration were evaluated longitudinally using a gingival fibroblast cell culture model. 8 strains of fibroblasts isolated from healthy human gingiva were seeded at 30,000 cells/35 mm culture dish in minimum essential medium supplemented with 10% fetal bovine serum. To ascertain the relationship of the amplitude and the duration of sonic stimulation to cellular proliferation, gingival fibroblasts were subjected 2x daily to 261 Hz sound at various amplitudes (67-97 dB) for 0, 15, 30, 60, and 120 s on days 1, 3, 5, 7, and 10. RESULTS: It was found that either 30 or 120 s of sound exposure for 10 days of treatment had significant effects on cell proliferation in comparison to control cultures. Specifically, at day 10, 87 dB at 261 Hz for 30 s 2x daily resulted in a 25.5% increase in cell number (p<0.001), whereas 87 dB at 261 Hz for 120 s twice daily caused a 30.9% decrease in cell number (p<0.001) when compared to control cultures. When cells are stimulated under optimum acoustic conditions for 10 days, there was no difference between the treatment and control groups for collagen (p=0.897) or noncollagen (p=0.697) protein production. CONCLUSIONS: Sonic energy has been shown to both increase and decrease cellular proliferation depending on exposure time; however, during optimum sound-induced conditions for cellular proliferation, sonic energy had no effect on fibroblast protein production. These data suggest that sonic energy can affect the behavior of cells in culture. Further research into the mechanisms of these changes will provide important information for manipulating cellular behavior.     

Altered collagen metabolism in nifedipine-induced gingival overgrowth

Fibroblasts from nifedipine-induced fibrotic gingiva (NFG) have been characterized with respect to several cellular functions which could contribute to the characteristic clinical overgrowth of the gingiva: collagen synthesis and breakdown, glycosaminoglycan production, fibronectin synthesis, and proliferation. Histologic examination of NFG tissue revealed a hyperplastic epithelium with elongated, branched rete pegs. The connective tissue consisted of densely-packed collagen fibers and numerous enlarged fibroblasts, as well as regions of thinner, disorganized collagen fibers in the vicinity of scattered inflammatory and mast cells. Results of in vitro experiments showed that the fibroblast strains from the fibrotic gingiva (NFG) produced significantly greater amounts of collagen and lower levels of collagenase activity when compared to age- and sex-matched normal human gingival fibroblast strains. The NFG fibroblasts did not produce significantly greater amounts of fibronectin, and their level of glycosaminoglycan production was less than that of the normal fibroblasts. The NFG fibroblasts did not proliferate significantly more rapidly than the normal fibroblast strains. These findings therefore show that there are defects in the regulation of collagen production by NFG fibroblasts in vitro , and suggest that these alterations in collagen metabolism contribute to the over-deposition of collagen in this tissue, rahter than hyperproliferation of the fibroblasts or through the production of increased amounts of fibronectin and glycosaminoglycans.     

Inhibitory effect of NH4Cl on secretion of collagen in human gingival fibroblasts

Abstract – The general protein synthesis in human gingival fibroblasts as measured by ¹⁴C-proline incorporation was only moderately inhibited by 10 mM NH₄Cl during incubation for 36 h. The proportion secreted as noncollagen protein and recovered from the cellular fraction as collagen was not significantly affected, whereas a pronounced inhibitory effect on the secretion of collagen was evident after 24 h. This effect was dose dependent, with a significant inhibition of collagen secretion even at 2 mM ammonia. The applied concentrations of NH₄Cl had no significant effect on the hydroxylation of prolyl residues in collagen. Ammonia had no inhibitory effect on the secretion of fibronectin, another major secretory protein from fibroblasts. When comparing different lysosomotropic agents; NH₄Cl, chloroquine and methylamine, the most prominent effect was consistently found to be an inhibition of the secretion of collagen.     

Cigarette smoke condensate affects the collagen-degrading ability of human gingival fibroblasts

Background and Objective: Cigarette smoke condensate, the particulate matter of cigarette smoke, is composed of thousands of chemicals, including nicotine. Cigarette smoking is a risk factor for periodontal disease. This study investigated the influence of cigarette smoke condensate on the collagen‐degrading ability of human gingival fibroblasts and its mechanism. Material and Methods: Human gingival fibroblasts were exposed for 72 h to various concentrations of total particulate matter cigarette smoke condensate. Cell proliferation and cytotoxicity were evaluated using water‐soluble tetrazolium‐1 and lactate dehydrogenase, respectively. The collagen‐degrading ability of human gingival fibroblasts was evaluated in collagen‐coated six‐well plates. Conditioned media and membrane extracts were collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Results: Cell proliferation decreased and cytotoxicity increased in human gingival fibroblasts with increasing concentrations of cigarette smoke condensate. Cell proliferation decreased by more than 50% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 200 μg/mL, and cytotoxicity increased to more than 30% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 400 μg/mL. Cigarette smoke condensate increased the collagen‐degrading ability of human gingival fibroblasts, especially at a concentration of 100 μg/mL (1.5‐fold increase, p < 0.05) compared with the control. Cigarette smoke condensate increased the production of proMMP‐1, proMMP‐2, MMP‐14 and TIMP‐1, and decreased the production of TIMP‐2, in conditioned media. Furthermore, compared with the control group, cigarette smoke condensate increased the production of MMP‐2, MMP‐14 and TIMP‐2 in membrane extracts, especially at concentrations of 50–100 μg/mL. Conclusion: Cigarette smoke condensate affects human gingival fibroblast proliferation and is toxic at total particulate matter cigarette smoke condensate concentrations of ≥ 400 μg/mL. Cigarette smoke condensate can increase the collagen‐degrading ability of human gingival fibroblasts by altering the production and localization of MMPs and TIMPs.     

Effect of phenytoin and nifedipine on collagen gene expression in human gingival fibroblasts

Phenytoin (PHT), a widely used anticonvulsant, and nifedipine (NF), an anti-anginal drug, cause clinically similar gingival overgrowths in some patients. The aim of this work was to investigate their effects on collagen and protein synthesis and cellular proliferation in normal human gingival fibroblasts in vitro. Gingival fibroblasts were cultured from biopsies taken from three healthy individuals during operations on maxillary canines and incubated with various concentrations of NF (100 and 200 ng/ml) and PHT (5 and 10 micrograms/ml) for up to 7 days. The results showed that NF and PHT have a specific effect in reducing total protein and collagen synthesis but do not influence cell proliferation in healthy gingival fibroblasts in vitro. In addition the level of mRNA for type I collagen was decreased after incubation of the cells with the drugs for 1 or 2 days. The decrease in the level of type I collagen mRNA seemed to be specific since the level of type IV collagenase mRNA used as a reference RNA did not decrease.     

The effects of chlorhexidine digluconate on human fibroblasts in vitro.

The drug chlorhexidine has been widely utilized as a wound antiseptic and oral antimicrobial rinse. There have been numerous reports on its safety as an oral rinse, but its effects on wound healing have been contradictory. The present study utilized human fibroblasts derived from skin and oral tissues to test the effects of chlorhexidine on viability, growth, collagen gel contractions, and total protein synthesis. Cells were exposed for an hour to 0.005% and 0.002% chlorhexidine and for 30 seconds to 0.12% chlorhexidine. Our results indicate that a 0.002% concentration of the drug shows minimal cytotoxicity, but is able to suppress cell division almost completely. Collagen gel contraction, as a model of wound contraction, was also severely affected by all of the concentrations of chlorhexidine used. Total protein synthesis was suppressed by chlorhexidine in collagen gel culture. The data support the hypothesis that chlorhexidine is highly cytotoxic to cells in vitro, but various cell functions such as proliferation, collagen gel contraction, and protein synthesis are affected to different degrees by the drug.     

Influence of high glucose concentrations on glycosaminoglycan and collagen synthesis in cultured human gingival fibroblasts

Human gingival fibroblasts were used to study the effects of increasing concentrations of glucose on protein, collagen and glycosaminoglycan (GAG) synthesis. GAG-synthesis was measured as incorporation of 3H-glucosamine into pronase-resistant macromolecules and collagen synthesis was evaluated by 3H-proline incorporation into collagenase-sensitive protein. Incubation of the fibroblasts with glucose concentration ranging from 5 to 50 mM resulted in a dose-dependent reduction of collagen synthesis; labeled collagen in the culture medium was reduced to 60% of the control incubation (5mM glucose) when incubated with 50 mM glucose for 72 h. Cell-associated radioactivity was decreased to 80% under the same conditions. Although 3H-glycosamine incorporation into GAGs was reduced by increasing glucose concentrations (5 to 20 mM), protein synthesis and cell number were not influenced under the same conditions, as was also the case with distribution of macromolecules in the GAG fractions. The importance of these in vitro results to the incidence of chronic inflammatory periodontal disease in diabetic patients is discussed.     

水蛭素对牙龈成纤维细胞碱性成纤维细胞生长因子和转化生长因子-β1表达的影响

目的 观察水蛭素对人牙龈成纤维细胞（HGFs）碱性成纤维细胞生长因子（bFGF）及转化生长因子-β1（TGF-β1）表达变化的规律,探讨水蛭素影响牙龈改建的可能作用机制。方法 体外培养并鉴定HGFs,利用不同浓度的水蛭素分别作用于正常（对照组）和受长期机械外力作用后增生的HGFs（实验组）,通过实时定量聚合酶链反应法和免疫细胞化学法检测TGF-β1及bFGF的表达。结果 未加入水蛭素时,受长期机械外力作用后,实验组促进HGFs增殖胶原合成的TGF-β1表达升高,而抑制胶原合成的bFGF表达降低（P<0.05）。加入水蛭素干预增生的HGFs后,可正向调节bFGF表达,而负向调节TGF-β1的表达（P<0.05）。结论 外力作用干扰了HGFs胶原合成与降解之间的平衡,水蛭素可能通过调节这一平衡而促进牙龈改建过程。     

Effect of tranilast on matrix metalloproteinase-1 secretion from human gingival fibroblasts in vitro

BACKGROUND: Some drugs such as phenytoin, calcium blockers, or cyclosporins are known to cause gingival fibrous hyperplasia, an unwanted side effect. Decreased collagen catabolism in overgrown gingival tissue has been proposed as one of the reasons causing the disease. The effect of tranilast, which suppresses collagen synthesis and cell proliferation, on matrix metalloproteinase (MMP-1) secretion from human gingival fibroblast, was studied in vitro. METHODS: Human gingival fibroblasts were cultured from specimens taken from healthy, periodontal, and overgrown gingival tissues. The effects of tranilast on cell proliferation and MMP-1 secretion from gingival fibroblast were assessed. Inhibitory effect of transforming growth factor (TGF)-beta secretion from gingival fibroblast by tranilast was also evaluated. RESULTS: Tranilast did not interfere with cell proliferation at the low concentrations. MMP-1 concentration significantly increased at the lower doses of tranilast up to about 2-fold compared to controls (P < 0.05). In contrast, higher doses of tranilast significantly decreased activity to 30% and 20%, respectively. MMP-1 secretion was inhibited significantly by phenytoin, nifedipine, and cyclosporin A and the depressed MMP-1 recovered to the control level with tranilast. The amount of secretion from normal and periodontitis gingival fibroblast specimens did not differ, but that from the overgrown gingiva was significantly less than the other types. Moreover, TGF-beta secretion was significantly inhibited by 300 microM of tranilast. CONCLUSIONS: Tranilast upregulates the expression of type 1 collagenase suppressed by gingival overgrowth-inducing drugs, and inhibits TGF-beta secretion from gingival fibroblasts. Therefore, tranilast could be considered as an agent for controlling gingival over-growth.     

Characterization of fibroblasts derived from human periodontal ligament and gingiva

Growth characteristics and macromolecular synthesis of fibroblasts derived from human periodontal ligament (PDLF) and gingiva (GF) have been compared in cell culture. Cells were isolated from explants and plated at 500,000 cells/100 mm culture dish (day 0) with daily changes of culture medium. DNA histograms were obtained by flow microfluorimetric analysis to confirm the growth state of the cell cultures. Human PDLF cultures became confluent at day 6 as determined by cell number and cell cycle analysis while GF were confluent by day 4. Initially, DNA content of logarithmically growing cells was significantly greater in GF cultures; however, when confluent, DNA content and cell number was greater in PDLF cultures. Total protein content in GF was slightly greater than PDLF until day 7 but this difference was not significant. Analysis of collagen and noncollagen protein synthesis revealed a greater trend in noncollagen protein synthesis in the GF cultures compared to PDLF cultures. Analysis of glycosaminoglycans in the culture medium of GF and PDLF revealed similar distributions of components. In the cellular fraction, GF had greater amounts of hyaluronic acid and heparin and lesser amounts of chondroitin sulfates A and C than PDLF cultures. The results indicate that the growth characteristics of PDLF and GF, although similar in many respects, do exhibit specific differences in proliferative rates and macromolecular synthesis. The differences observed in these parameters may be important during in vivo events, such as guided tissue regeneration, where significant functional differences are observed between gingival connective tissue and periodontal ligament connective tissue.     

Increased expression of collagen prolyl 4-hydroxylases in Chinese patients with hereditary gingival fibromatosis

OBJECTIVES: Hereditary gingival fibromatosis (HGF) is characterized by excess accumulation of interstitial collagen. However, until now, there has been controversy about the mechanism of collagen accumulation in HGF gingivae. The present study aimed to clarify the pathogenic mechanisms potentially involved. DESIGN: Gingival fibroblasts from three Chinese HGF patients and three healthy subjects were cultured. Cell proliferation was assessed by MTT assay. The mRNA levels of type I collagen, MMP-1, MMP-3, TIMP-1, prolyl 4-hydroxylase (P4H)alpha(I), alpha(II), alpha(III) and P4Hbeta were analyzed in gingival fibroblasts by RT-PCR. The protein production of type I collagen and P4H was examined respectively by ELISA and Western blot. RESULTS: In culture, HGF gingival fibroblasts showed similar growth characteristics to fibroblasts isolated from control gingivae. The mRNA and protein levels of type I collagen and P4Halpha in HGF fibroblasts were higher than those in controls. There were no detected differences in mRNA expression levels of MMP-1, MMP-3, TIMP-1, P4Halpha(II), alpha(III) and P4Hbeta between HGF and control fibroblasts. CONCLUSIONS: These data suggest that increased collagen post-translational modification by P4H may be one mechanism by which increased collagen accumulation occurs in some forms of HGF.     

Mechanism of azithromycin treatment on gingival overgrowth

Azithromycin is effective for the remission of cyclosporine A-induced gingival overgrowth (CIGO) in persons who have undergone renal transplant. To explain its mechanism in alleviating the clinical symptoms of these individuals, we examined the effect of azithromycin on cell proliferation and collagen turnover modified by cyclosporin A in human gingival fibroblasts from healthy persons and from persons who had undergone renal transplant. Cyclosporin A-induced proliferation of renal transplant fibroblasts and normal fibroblasts was inhibited by azithromycin. Azithromycin elevated the reduced metalloproteinase (MMP)-1 and MMP-2 activities in cyclosporine A-treated renal transplant fibroblasts and normal fibroblasts. In cyclosporine A-treated renal transplant fibroblasts, azithromycin blocked the accumulation of total collagen in culture media and the increase in type I collagen mRNA level, but recovered the reduced MMP-2 mRNA level to the control. These results suggest that azithromycin may improve CIGO by blocking cyclosporine A-induced cell proliferation and collagen synthesis, and by activating MMP-2 in gingival fibroblasts of persons with cyclosporine A-induced gingival overgrowth.     

Gingival fibroblast response to cyclosporin A and transforming growth factor β1

This study investigates a potential role for TGFβ₁ in the pathogenesis of cyclosporin A-induced gingival overgrowth (CsA-OG). TGFβ₁ was localized immunohistochemically in the connective tissue of both normal gingiva and CsA-OG. Intense staining for TGFβ₁ was detected at the tips of the dermal papillae of the overgrown gingiva. In addition, fibroblasts derived from healthy gingiva and fibroblasts derived from CsA-OG were cultured both as monolayers or embedded in a 3D-collagen gel. Fibroblast activity was monitored in terms of protein and collagen production in the presence of (i) 1 ng/ml TGFβ₁ (ii) 500 ng/ml CsA, or (iii) 500 ng/ml CsA and 1 ng/ml TGFβ₁. In monolayer culture TGFβ₁ significantly increased protein and collagen production in all cell strains (p<0.05); however, there was no difference in response between fibroblasts from overgrown and healthy tissue. The production of both protein and collagen was significantly lower in the presence of the combination of CsA and TGFβ₁ when compared with the maximal stimulation produced by TGFβ₁ alone. In gel, TGFβ₁ significantly elevated matrix production by all overgrown cell strains (p<0.05) but had little or no effect on the normal cell strains. The combination of CsA and TGFβ₁ in gel cultures reduced protein and collagen production by overgrown cell strains compared with TGFβ¹ alone. It is concluded that the cellular activity of gingival fibroblasts is dependant on culture conditions and that fibroblasts derived from overgrown gingival tissue are more responsive to TGFβ₁ than normal gingival fibroblasts when cultured in type I collagen gel.     

Copper stimulates human oral fibroblasts in vitro: a role in the pathogenesis of oral submucous fibrosis

Copper is implicated in the pathogenesis of several fibrotic disorders. Areca nut has been shown to have a high copper content and areca chewing is associated with oral submucous fibrosis (OSF). The effects of copper on human oral fibroblasts were investigated in vitro. Human oral fibroblasts were incubated with copper chloride (CuCl2) at concentrations ranging from 0.01 microM to 500 microM for 24 h, and in vitro cell proliferation was assayed by incorporation of tritiated-thymidine; soluble and non-soluble collagen synthesis was assayed using tritiated-proline. Addition of copper chloride at concentrations ranging from 0.1 microM to 50 microM increased the collagen synthesis by the oral fibroblasts compared with growth without copper (P<0.05). The addition of copper chloride neither increased the synthesis of non-collagenous proteins by the fibroblasts nor influenced their proliferation rate. We conclude that copper upregulates collagen production in oral fibroblasts. This appears to be concentration dependent, with peak collagen synthesis at 50 microM CuCl2. These in vitro results taken together with the recent findings of copper in oral biopsies from OSF subjects support the hypothesis that copper in areca nut acts as a mediator of OSF.     

Effect of nicotine-treated epithelial cells on the proliferation and collagen production of gingival fibroblasts

BACKGROUND, AIMS: Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. METHOD: In the present study, we developed an in vitro model to study the interactions between nicotine-treated epithelial cells (EC) and gingival fibroblasts (GF) derived from the same patient. EC were treated with nicotine concentrations varying from 1 microg/ml to 500 microg/ml and their effect on different functions of GF was studied. The proliferation of GF was evaluated by the incorporation of 3H-thymidine. A dose-dependent inhibition was observed with nicotine concentrations > or =100 microg/ml. Similar results were observed when studying the total protein synthesis of GF by incorporation of 3H-proline into non-dialyzable material. RESULTS: When collagen production was evaluated by 3H-proline incorporation into collagenase-sensitive protein, a dose-dependent reduction was observed: the degree of inhibition varied from 25% with 50 microg/ml nicotine, to almost 60% with 500 microg/ml. Interestingly, the production of non-collagenous proteins decreased by almost 50% only when EC were treated with the highest concentration of nicotine. CONCLUSIONS: The results suggest that epithelial cells, acting as mechanical barrier, can reduce but not completely eliminate the deleterious effect of nicotine on gingival fibroblasts.     

Role of the c-myc proto-oncogene in the proliferation of hereditary gingival fibromatosis fibroblasts

BACKGROUND: Hereditary gingival fibromatosis (HGF) is a fibrotic gingival enlargement. In previous work, HGF fibroblasts grew faster and produced more collagen and fibronectin (FN) than normal gingival (GN) fibroblasts. HGF FN and collagen production, but not proliferation, were under autocrine transforming growth factor (TGF)-beta control, suggesting other means of activation of HGF proliferation. Elevated/prolonged expression of the proto-oncogene c-myc is implicated in disregulation of cell growth. The objectives of this study were to: 1) determine if c-myc expression is abnormal in quiescent and serum-stimulated HGF and GN fibroblasts and 2) determine the relationship between c-myc expression and fibroblast proliferation using a c-myc antisense oligonucleotide (ODN). METHODS: Proliferation was determined by enzyme-linked immunosorbent assay (ELISA), measuring incorporation of bromodeoxyuridine into DNA. Expression of c-myc was determined by quantitative polymerase chain reaction (PCR), using incorporation of fluorescent dCTP and detection via electrophoresis. RESULTS: Proliferation was minimal until 24 hours or more after serum stimulation, when HGF proliferation was greater than GN (P < or = 0.02). All cells expressed c-myc mRNA at quiescence and > or = 1 hour after serum stimulation. Expression of c-myc in quiescent HGF fibroblasts was elevated, and it peaked and remained higher after serum stimulation than in GN cells. Proliferation of an HGF cell line was inhibited by 4 microM c-myc antisense ODN (14% decrease; P < or = 0.006) and 8 microM c-myc antisense ODN (approximately 80% decrease; P < or = 0.0001), but generally not by c-myc sense ODN. This effect was reversed by hybridizing the c-myc antisense and sense ODNs (P = 0.007). CONCLUSION: Data suggest that elevated proliferation of an HGF fibroblast cell line is related to elevated c-myc expression.     

Regulation of human gingival fibroblast growth and synthetic activity by cyclosporine-A in vitro

Gingival overgrowth is an adverse side-effect seen in a proportion of patients taking cyclosporin-A which indicates that cyclosporine-A may modulate the activities of cells other than T lymphocytes. Therefore, the effect of cyclosporine on human gingival fibroblasts has been studied in vitro. Cyclosporine-A was found to stimulate DNA synthesis and the proliferative activity of these cells with maximal stimulation noted at a concentration of 10(-9) g/ml. Although this stimulation was most noticeable in the presence of 10% fetal calf serum, proliferation still occurred in serum-free medium. In the presence of lipopolysaccharide, at a concentration which normally inhibits gingival fibroblast proliferation, cyclosporine retained its capacity to stimulate proliferative activity. Fibroblasts isolated from overgrown gingival tissue responded to a greater extent than those isolated from a healthy site from the same individual. This stimulatory effect was not restricted to gingival fibroblasts, since human foreskin fibroblasts responded in a similar fashion. Cyclosporine-A did not significantly alter protein or proteoglycan production by these cells. These responses are considered to reflect the in vivo response of gingival overgrowth in patients taking cyclosporine-A. The reversal of lipopolysaccharide inhibition of gingival fibroblast proliferation by cyclosporine-A may explain, in part, why gingival overgrowth is most prominent in areas of heavy dental plaque accumulation.     

Effects of cytokines on gingival fibroblasts in vitro are modulated by the extracellular matrix

Fibroblast function in gingival tissue is thought to be regulated by the local cellular environment – both the extracellular matrix and soluble factors. In an attempt to artificially re-create this situation fibroblasts have been cultured within 3-dimensional collagen gels in an environment more physiologically comparable to connective tissue. Using such a model we investigated the effects of the extracellular matrix on gingival fibroblast growth and synthetic activity and on the cellular responsiveness to 4 soluble factors – epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF- β ₁) and interleukin-1 β (IL-1 β ). Fibroblasts cultured within collagen gels showed similar growth rates, an increased production of collagen but reduced levels of hyaluronan synthesis in comparison to cells in monolayer culture. Cellular responsiveness to soluble mediators was also modulated by the collagen matrix, with a generalised reduction in response by cells embedded within the matrix. The stimulatory effects of EGF and PDGF on cell growth in monolayer over a 14-day period were only found during the initial stages of culture within gels. Similarly the stimulation of matrix production by cells induced by TGF- β ₁, on plastic was reduced or even negated when cells were cultured in collagen gels. On plastic IL-1 β significantly stimulated cell growth but had no effect on either collagen or hyaluronan production by fibroblasts. In gel cultures, this cytokine had no effect on cell proliferation, but significantly inhibited both collagen and hyaluronan synthesis. These findings further illustrate the usefulness of fibroblast-populated collagen gels as a model system for studying the modulatory effects of soluble factors and extracellular matrix molecules on fibroblast function in vitro .     

Tetracycline inhibition identifies the cellular origin of interstitial collagenases in human periodontal diseases in vivo

Mammalian interstitial collagenases (E.C.3.4.24.7) are considered as key initiators of collagen degradation in periodontal diseases. However, the cellular sources of collagenases present in gingival crevicular fluid have not been completely clarified. Resident fibroblasts and epithelial cells as well as infiltrating neutrophils and monocyte/macrophages are potential sources of the enzymes. We have recently found significant differences in tetracycline inhibition between human neutrophil and fibroblast interstitial collagenases. To address the cellular source of collagenase present in gingival crevicular fluid in 2 distinct periodontal diseases, we studied the tetracycline inhibition of collagenase in gingival crevicular fluid of patients with localized juvenile periodontitis and adult periodontitis. Gingival crevicular fluid samples were collected from deep (greater than 5 mm) periodontal pockets and assayed for collagenase in the presence of 0-1000 microM doxycycline as well as a chemically modified tetracycline devoid of antimicrobial activity (4-de-dimethylaminotetracycline). The drug concentration required to inhibit 50% of collagenase activity (IC50) in localized juvenile periodontitis gingival crevicular fluid was 280 microM for doxycycline and 470 microM for 4-de-dimethylaminotetracycline. Significantly lower values, 10-20 microM, were obtained for collagenase in gingival crevicular fluid of patients with adult periodontitis. We propose that systemic tetracycline levels are efficient inhibitors of collagenase in gingival crevicular fluid in affected sites of patients with adult periodontitis but not of patients with localized juvenile periodontitis and that the fibroblast type interstitial collagenase is the predominant collagenase type in gingival crevicular fluid in affected sites of patients with localized juvenile periodontitis and the neutrophil collagenase in adult periodontitis gingival crevicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)