In vitro immunoexpression of extracellular matrix proteins in dental pulpal and gingival human fibroblasts

AIM: To compare the expression of extracellular matrix (ECM) components in human pulpal and gingival fibroblasts in vitro. METHODOLOGY: Cultured dental pulp fibroblasts and gingival mucosa fibroblasts were used. Tenascin (TN), fibronectin (FN), type I (col I) and III collagen (col III) and osteonectin (ONEC) were detected by immunofluorescence. Main morphological characteristics were also analysed by light microscopy (LM) and transmission electron microscopy. RESULTS: The results revealed different expression patterns of the proteins. TN and ONEC were only immunoexpressed by pulpal fibroblast cells, suggesting a role of these glycoproteins in formation of mineralized tissues. FN and col I were present in the cytoplasms of both cell types. No expression of col III was detected. Different morphological characteristics were visualized under LM, in which pulpal fibroblasts were spindle-shaped with a wide cytoplasm, while gingival fibroblast cells exhibited stellate/pyramidal configuration, with rounded nuclei. However, ultrastructurally, both cell lineages showed very well developed rough endoplasmatic reticulum and Golgi complex. CONCLUSIONS: Due to the immunodetection of TN and ONEC on pulpal fibroblasts, the present findings demonstrated that a pulpal fibroblast cell is similar to an osteoblastic cell rather than an undifferentiated mesenchymal cell, such as a gingival fibroblast cell. Functional differences between the two cell lines may then be suggested.     

Mechanical forces alter extracellular matrix synthesis by human periodontal ligament fibroblasts

Periodontal ligament fibroblasts (PDLFs) are a heterogeneous population of cells that are involved in the normal maintenance, repair and regeneration of both the ligament and adjacent hard tissues. Additionally, the ability of these cells to respond to mechanical stimulation suggests that they have a central role in mediating the osseous remodeling that underlies physiological and orthodontic tooth movement. To characterize their role further in this process, the current study evaluated the effect of tensional stress on the biosynthesis of extracellular matrix (ECM) proteins by human PDLFs. Cell strains were established from extracted human premolars and third molars. Cells exposed to 5% biaxial deformation (strain) at a frequency of 30 times/min for 24 hr exhibited statistically significant increases in type I collagen and fibronectin synthesis, and a statistically signficant decrease in tropoelastin production relative to unstretched controls. Cells exposed to 10% strain exhibited similar responses for fibronectin and tropoelastin while the amount of type I collagen synthesized by stretched cells did not differ from control levels. These results indicate that mechanical stimulation of PDLFs alters type I collagen, tropoelastin and fibronectin production and that these cells are differentially responsive to varying levels of mechanical stress. The ability of these cells to alter ECM protein synthesis in response to specific magnitudes of tensional stress may in part explain how PDLFs regulate ligament and hard tissue remodeling.     

Effect of phenytoin on collagen accumulation by human gingival fibroblasts exposed to TNF-alpha in vitro

OBJECTIVE: Tumor necrosis factor (TNF)-alpha is associated with chronic gingival inflammation and reported to induce gingival overgrowth (GO), while phenytoin (PHT) is also known to be a causative agent of GO. We examined the synergistic effect of PHT and TNF-alpha on collagen metabolism in human gingival fibroblasts (HGFs). MATERIALS AND METHODS: HGFs were cultured with TNF-alpha and PHT. Quantitative real-time RT-PCR was employed to determine the mRNA levels for collagen, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and integrin subunits. Cellular collagen endocytosis was determined using a flow-cytometry. RESULTS: The proliferation of HGFs was not affected by TNF-alpha or PHT individually, whereas both synergistically increased collagen accumulation in HGFs. Further, collagen mRNA expression was not increased by TNF-alpha or PHT, although together they markedly prevented cellular collagen endocytosis, associated with the suppression of alpha2beta1-integrin mRNA expression. The mRNA expression of MMP-1 and-2 was suppressed by PHT, while TIMP-1 mRNA expression was enhanced by both TNF-alpha and PHT. CONCLUSION: Our results suggest that TNF-alpha and PHT together cause impaired collagen metabolism by suppression of enzymatic degradation with MMPs/TIMP-1 and integrin-mediated endocytosis. These synergistic effects may also be involved in TNF-alpha- and PHT-induced collagen accumulation, leading to GO.     

Effects of transforming growth factor-β and platelet-derived growth factor on human gingival fibroblasts grown in serum-containing and serum-free medium

Abstract. Both transforming growth factor-β (TGF-ß) and platelet-derived growth factor (PDGF) have been shown to affect cell proliferation in vitro. The hypothesis being tested was that the effects of the 2 cytokines would be modulated by the presence of serum in the medium. Gingival fibroblasts, obtained from periodontally healthy patients, were maintained in primary culture. Dose response experiments were performed for each growth factor in serum-free medium and in medium containing natural or heat-inactivated fetal bovine serum (10% FBS). Changes in cell numbers were quantified by crystal violet staining. The optimal concentrations of the individual factors (10 ng/ml TGF-ßI, 20 ng/ml PDGF-BB) were then used when the 2 factors were tested in various sequences. In serum-free medium or in medium with 10% natural serum, the response to PDGF-BB was dose-dependent up to 40 ng/ml; however, with 10% heat-inactivated serum, the maximal response was seen at 20 ng/ml. The largest increase in cell numbers was produced by the simultaneous exposure to the two cytokines, rather than a sequential presentation. The findings suggest that the 48-h growth response of human gingival fibroblasts to 10 ng/ml TGF-ß1 or 20 ng/ml PDGF-BB in serum-free medium was equivalent to growth obtained in medium containing heat-inactivated 10% FBS without added growth factors.     

Synthesis of sulfated glycosaminoglycans by human gingival fibroblasts from phenytoin-induced gingival overgrowth in vitro

Abstract – The in vitro synthesis of sulfated glycosaminoglycans (GAGs) was studied in gingival fibroblasts from two patients exhibiting phenytoin(PHT)-induced gingival overgrowth, i.e. pseudopockets, which required surgical excision, from one patient on PHT medication not exhibiting pseudopockets and from two normal controls. The results showed that the newly synthesized GAGs were distributed to the culture medium, to a pericellular pool and to the cell fraction. Gingival fibroblasts from the PHT-induced gingival overgrowth showed a significantly increased incorporation of ³⁵SO₄^2- into GAGs compared to the other strains, and this, increase was mainly confined to the dermatan sulfate fraction. These results are in accordance with our previous biochemical studies where increased amounts of GAGs were found in gingival biopsies from the PHT-induced lesion.     

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.     

Cytotoxic effects of gingival retraction cords on human gingival fibroblasts in vitro

The objective of this study was to determine the cytocompatibility of three different extracts of gingival retraction cords and to compare the cytotoxic effect of these materials on human gingival fibroblasts. Gingival retraction cords impregnated with aluminium sulphate (Gingi-Aid), dl-adrenaline HCl (Gingi-Pak) and non-drug-impregnated cord (Gingi-Plain) were eluted with culture medium for 10 min and 24 h. Cytotoxicity was judged using a tetrazolium bromide reduction assay. Our data demonstrated that gingival retraction cords applied alone almost completely inhibited cell viability (P < 0.05). In addition, the results also showed that the eluates from aluminium sulphate-impregnated cord, dl-adrenaline HCl-impregnated cord and non-drug-impregnated cord were cytotoxic to primary human gingival fibroblast cultures (P < 0.05). The cell viability of incubation of gingival fibroblasts containing 10-min eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 61, 21 and 70%, respectively. The cell viability of incubation of gingival fibroblasts containing 24 h eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 68, 58 and 72%, respectively. It was found that dl-adrenaline HCl-impregnated gingival retraction cord was the most toxic gingival retraction cord among the materials tested in all cultures (P < 0.05). The cytotoxicity decreased in an order of dl-adrenaline HCl-impregnated cord > aluminium sulphate-impregnated cord > non-drug-impregnated cord. The extent or degree of the cytotoxicity depended on the materials tested. Gingival retraction cords have significant potential for gingival toxicity. Careful management of gingiva retraction cords would lower the risk of potential gingival tissue damage during clinical application procedure and thus increase the success of prosthodontic procedures.     

Cytopathologic effects of arecoline on human gingival fibroblasts in vitro

Arecoline, a major betel nut alkaloid, has been detected in saliva obtained during betel nut chewing in concentrations up to 140 μg/ml, corresponding to 0.9 mM. Arecoline in the millimolar concentration range might participate in the initiation and/or progression of periodontal disease during the long-term effects of betel nut chewing. In this study, cell growth, cell proliferation, assessment of cytoplasmic enzyme lactate dehydrogenase (LDH) and collagen synthesis were used to investigate the effects of human gingival fibroblasts exposed to arecoline levels of 0–200 μg/ml. Control culture exhibited a normal monolayer of long spindle-shaped fibroblast morphology. Arecoline-treated human gingival fibroblasts showed a more rounded appearance and detached at the higher concentrations. At concentrations higher than 75 μg/ml, many cells had detached from the surface of the petri dish and numerous floating cells could be seen under the inverted microscope. At a concentrations higher than 25 μg/ml, arecoline inhibited cell growth, proliferation and collagen synthesis and increased LDH leakage in a dose-dependent manner (P<0.05). These results indicate that arecoline is a cytotoxic agent to human gingival fibroblasts. Repeated and long-term exposure to arecoline could impair gingival fibroblast function. Betel quid chewers might be more susceptible to destruction of the periodontium and less responsive to a regeneration procedures during periodontal therapy. Received: 31 August 1998 / Accepted: 3 November 1998     

NH4C1 and protein metabolism in human gingival fibroblasts

Abstract – The biologic effect of ammonia was studied in cultures of fibroblasts isolated from human gingiva. NH_CI in the range 2–20 mu was found to exhibit a concentration dependent growth inhibitory effect, with a delayed action wllich was most pronounced at low concentrations. Concomitant with the growth inhibitor)'effect a significant cellular accumulation of protein was evident. No effect on protein synthesis in general, as measured by G-protine incorporation, was found, whereas some inhibitory effect on collagen biosynthesis was indicated. Secretion of C-collagen and other labeled proteins was not affected. The only pronounced effect of ammonia on metabolism of the l4C-labeled proteins was an inhibition of the intracellular degradation of newly synthesized collagen, the lysosomes being suggested as the site for this degradation.     

粘膜固有层替代物体外培养的实验研究

目的：体外构建粘膜固有层的替代物,方法：将粘膜固有层的成纤维细胞,胶原、培养基等成分混合形成凝胶,并在体外培养,结果,复合的胶原细胞凝胶具有粘膜固有层的类似结构,收缩程度受细胞数量和胶原浓度的影响。结论：构建的胶原细胞凝胶可作为粘膜固有层的替代物。     

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.     

Production of inflammatory mediators and cytokines by human gingival fibroblasts following bacterial challenge

Bacteria can indirectly affect the course of periodontal diseases by activating host cells to produce and release inflammatory mediators and cytokines. These mediators and cytokines, manifest potent proinflammatory and catabolic activity and may play key roles in local amplification of the immune response as well as in periodontal tissue breakdown. This study tested the effect of Actinobacillus actinomycetemecomitans (Aa) and Campylobacter rectus (Cr) challenge on PGE₂, IL-1β, IL-6 and IL-8 production by human gingival fibroblasts (HGF). Contact-inhibited HGF were prepared and formalin-killed bacterial cells ( Aa JP2, ATCC 29523 & 33384 and Cr ATCC 33238) at 10⁶-10⁹ were added to the HGF. Culture supernatants were collected at varying time intervals and analyzed for cytokine and mediator content. All concentrations of Aa JP2 and Cr ATCC 33238 suppressed IL-1β production up to approximately 50% during the initial 3-12-h period. No bacterial concentration tested was able to increase IL-1β production above the maximum basal levels. Both bacterial species stimulated production of IL-6 and IL-8. Aa JP2 did not affect PGE₂ levels significantly, whereas Cr ATCC 33238 was stimulatory only at the highest concentration tested (10⁹). There were no significant differences among the three Aa strains with respect to IL-1β production. However, Aa ATCC 29523 and ATCC 33384 were less capable of stimulating IL-6 secretion and more efficient in stimulating. IL-8 production than Aa JP2. In general. Cr was the most potent enhancer of cyto-kine and mediator production by HGF. In conclusion, Aa and Cr are capable of amplifying the local immune response and promoting periodontal tissue inflammation by stimulating HGF to secrete mainly IL-6 and IL-8.     

Effects of nicotine on apoptosis in human gingival fibroblasts

Aim

Cigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants and it is a world widely known problem to health. Nicotine is the major compound of tobacco and known as the cause of gingivitis and periodontitis. It induces intracellular oxidative stress recognized as the important agent in the damage of biological molecules. The aim of this study is to clarify the cytotoxic pathway of nicotine in human gingival fibroblasts (HGFs).

Methods

Human gingival fibroblasts stimulated by nicotine were used as an in vitro model. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability and reactive oxygen species (ROS) generation was assessed with 2,7-dichlorofluoroscein diacetate (DCF-DA). Morphological change was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay, stained with 4,6-diamidino-2-phenylindole (DAPI). To delineate the roles of extracellular signal-regulated kinase (ERK), P38 and c-Jun N-terminal kinase (JNK), Western blot and caspase-3 (CASP3) activity assay were performed.

Results

Exposure of the human gingival fibroblasts to nicotine reduced cell viability by time and dose dependent and increased the generation of ROS. It also showed morphological evidence of increased apoptosis, resulted in transient activation of JNK and ERK concomitant with activation of P38, and stimulated apoptosis as evidenced by CASP3 activation and Poly ADP ribose polymerase (PARP) cleavage.

Conclusion

These results suggest that nicotine induces apoptosis through the ROS generation and CASP3 dependent pathways in HGFs.     

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.