Immunoexpression of α2-integrin and Hsp47 in hereditary gingival fibromatosis and gingival fibromatosis-associated dental abnormalities

Objective: The purpose of the present study was to investigate the expression of the α2-integrin subunit and heat shock protein 47 (Hsp47) in two families with isolated gingival fibromatosis (GF) form and one family with GF associated with dental abnormalities and normal gingiva (NG). Study Design: Immunohistochemistry was performed with antibodies against α2-integrin and Hsp47 in specimens from two unrelated families with hereditary gingival fibromatosis (Families 1 and 2) and from one family with a gingival fibromatosis-associated dental abnormality (Family 3); NG samples were used for comparison. The results were analysed statistically. Results: Immunoreactivity for α2-integrin and Hsp47 was observed in the nucleus of epithelial cells of both the basal and suprabasal layer and a more discreet signal was noted in connective tissue in all study samples. Hsp47 showed higher immunoreactivity in Family 2 compared with the other families (p≤0.05). Despite the markup α2-integrin was higher in Family 3 there was no statistically significant difference between the families studied (p≥0.05). Conclusions: Our results confirmed the heterogeneity of GF, such that similar patterns of expression of the condition may show differences in the expression of proteins such as Hsp47. Although no difference in α2-integrin expression was observed between GF and NG groups, future studies are necessary to determine the exact role of this protein in the various forms of GF and whether it contributes to GF pathogenesis. Key words:Gingival fibromatosis, integrin alpha2, heat shock protein Hsp47.     

Involvement of HMGB1 and RAGE in IL-1β-induced gingival inflammation

ObjectiveExtracellularly released high mobility group box 1 (HMGB1) protein behaves as a cytokine, promotes inflammation and participates in the pathogenesis of several disorders in peripheral organs. The role of HMGB1 and receptor for advanced glycation end products (RAGE) expressed in gingival inflammatory tissues was explored.MethodsReal time PCR was applied to assay HMGB1 and RAGE mRNA expression in gingival epithelial and fibroblast cells induced by interleukin-1β (IL-1β). A highly selective inhibitor of inducible nitric oxide (iNOS) was employed. ELISA was done for measurement of HMGB1 concentrations in cell culture media of gingival epithelial and fibroblast cells. Immunohistochemistry was performed to analyse the expression and sub-cellular localization of HMGB1, together with RAGE, in specimens obtained from patients with chronic inflammation.ResultsA time-dependent response of HMGB1 and RAGE expression in gingival cells to IL-1β induction was observed. IL-1β promotes HMGB1 production in human gingival epithelial cells in a nitric oxide-dependent manner. HMGB1 and RAGE appeared highly expressed in gingival inflammatory tissues.ConclusionThese results demonstrate that HMGB1 and RAGE are abundantly expressed in gingiva and promptly released during gingival inflammation. We suggest a role for HMGB1/RAGE/iNOS signalling on inflamed gingival epithelial cells.     

The induction expression of human β-defensins in gingival epithelial cells and fibroblasts

ObjectiveThe gingival epithelial cells and fibroblasts can produce antimicrobial peptides when stimulated by inflammatory cytokines. The purpose of the present study was to test whether gingival keratinocytes and gingival fibroblasts respond differently to inflammatory cytokine activation. This will enable us to understand the chronic inflammatory response in the process of periodontal disease.DesignGingival keratinocytes and fibroblasts were isolated and treated with different concentrations of IL-1β and quantitative real-time PCR was performed to evaluate the induced expressions of hBD-1, hBD-2 and hBD-3. The induced response was compared between the gingival epithelial cells and fibroblasts. The inhibitors of p38 protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) were applied to explore the molecular mechanism during the induction of hBDs in both cells.ResultsThe results showed that the hBDs expressions were found to be induced by different concentrations of IL-1β, but with several differences between gingival epithelial cells and fibroblasts. The hBDs mRNA expression in gingival fibroblasts was more sensitive compared with keratinocytes to different concentrations of IL-1β. The hBD-1 and hBD-3 expressions in these two cells were down-regulated by IL-1β and hBD-2 expression was up-regulated. The inflammatory cytokine IL-1β had dual effect on hBDs expression.ConclusionsThe gingival epithelial cells and fibroblasts respond differently to the inflammatory cytokine IL-1β which indicated different roles played by the two cells in the host defense. The dual effect of IL-1β on hBDs expression may contribute to the defensins down-regulation in periodontal disease.     

Inter- and intra-observer agreement on Miller’s classification of gingival tissue recessions

Miller’s is the most commonly used classification of gingival tissue recessions, defined as the displacement of the soft tissue margin apical to the cemento-enamel junction. However, data on the reliability of this classification are missing so far, although reliability, which reflects the consistency of repeated measurements, is regarded as a prerequisite for judging the utility of a classification. The aim of the present study was to evaluate inter- and intra-observer agreement on Miller’s classification of gingival tissue recessions. Two hundred photographs (50 of each region: maxillary/mandibular anterior/posterior teeth) of gingival tissue recessions were evaluated twice by four observers with different degrees of experience in Miller’s classification, gingival phenotype, tooth shape, and identifiability of the cemento-enamel junction. The following inter- and intra-observer agreements were found: Miller’s classification, 0.72 and 0.73–0.95; gingival phenotype, 0.29 and 0.45–0.58; tooth shape, 0.39 and 0.44–0.59; and identifiability of the cemento-enamel junction, 0.21 and 0.30–0.59. A higher agreement was detected for anterior teeth. Further, gingival phenotype (thin-high scalloping) significantly correlated with tooth shape (long-narrow) (ρ = 0.662, p < 0.001). Miller’s classification of gingival tissue recessions was evaluated by four examiners using 200 clinical photographs and yielded substantial to almost perfect agreement, with higher agreement for anterior teeth. Although limited to photographic assessment, the present study offers the so far missing proof on the sufficient inter- and intra-observer agreement of this classification.     

TGF-β1 gene polymorphism in renal transplant patients with and without gingival overgrowth

Oral Diseases (2011) 17 , 414–419 Background: The incidence of gingival overgrowth among renal transplant patients treated with cyclosporine A ranges from 13% to 84.6%, and the overgrowth is not only esthetic but also a medical problem. We studied the determination of association between TGF‐β1 (TGFB1) gene polymorphism and gingival overgrowth in kidney transplant patients medicated with cyclosporin A. Methods: Eighty‐four kidney transplant patients with gingival overgrowth and 140 control transplant patients without overgrowth were enrolled into the case control study. TGFB1 polymorphism was determined using the PCR‐RFLP assay for +869T>C in codon 10 and +915G>C in codon 25 as well as TaqMan real‐time PCR assays for promoter ?800G>A and ?509C>T SNPs. Results: In kidney transplant patients suffering from gingival overgrowth, mean score of gingival overgrowth was 1.38 ± 0.60, whereas in control subjects it was 0.0. The patients with gingival overgrowth were characterized by similar distribution of TGFB1 genotypes and allele in comparison to subjects without gingival overgrowth. Among 16 potentially possible haplotypes of TGFB1 gene, only four were observed in the studied sample of kidney transplant patients: G_C_T_G, G_T_C_G, G_C_C_C, and A_C_T_G, with similar frequency in patients with and without gingival overgrowth. Conclusion: No association between the TGFB1 gene polymorphism and gingival overgrowth was revealed in kidney transplant patients administered cyclosporine A.     

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

Stein SH, Dean IN, Rawal SY, Tipton DA. Statins regulate interleukin‐1β ‐ induced RANKL and osteoprotegerin production by human gingival fibroblasts. J Periodont Res 2011; 46: 483–490. © 2011 John Wiley & Sons A/S 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‐1β (IL‐1β)‐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 24 h in serum‐free medium, and then incubated with IL‐1β for 6 d. 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‐1β (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‐1β. 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‐1β‐stimulated RANKL/OPG ratio. Conclusion: IL‐1β significantly increased OPG production by HGFs. The statins differed minimally in their effects on OPG and RANKL production by resting and IL‐1β‐activated HGFs. Both statins increased constitutive RANKL/OPG ratios, but generally not IL‐1β‐stimulated ratios. Thus, statins may influence the production of RANKL and OPG by HGFs to favor bone catabolism, under noninflammatory conditions.     

An innovative approach for gingival re-contouring using a provisional restoration and positive pressure – A case report

Increase in awareness among patients about the cosmetic options in dentistry make the clinician to be competent enough to meet the needs of the patients. The appearance of the gingival tissues surrounding the teeth plays a critical role in anterior aesthetics and also gingival perspective is concerned with the soft tissue envelope surrounding the teeth. The gingival texture, shape, tooth to tooth progression and its relation to the extra oral tissues are interdependent on many factors. To improve the contour of gingiva we can have either surgical or nonsurgical approaches like application of pressure. In this case positive pressure was applied on soft tissue in an attempt to create the illusion of the restoration emerging from the tissue and the formation of “pseudo” interdental papillae. This article describes a technique of using a uniquely designed provisional restoration for the application of circumferential pressure for contouring gingiva.     

Vitamin C influences antioxidative,anti-inflammatory and wound healing markers in smokers’ gingival fibroblasts in vitro

BackgroundSaudi Arabia has an overall smoking rate of 15.9%. The link between smoking and periodontal disease has been studied extensively. It is possible for human gingival fibroblasts to accumulate nicotine intracellularly over a period of four hours. Additionally, unmetabolized nicotine is released into the environment. Tobacco presence can impair tissue inflammation, wound healing, and organ development. To counterbalance tobacco toxins, vitamin C has been added to a variety of products.AimThis study aims to analyze the RNA expression of antioxidant, anti-inflammatory, and wound healing proteins in human gingival fibroblasts from smokers and nonsmokers using polymerase chain reaction.Materials and MethodshGFs were extracted from clinically healthy periodontium sites of adult male subjects. Both heavy cigarette smokers and never-smokers participated as subjects. Cells were cultured and subcultured in supplemented growth medium. Vitamin C was inducted in the medium at the experimental 6th passage. RNA expression analysis (qRT-PCR) was performed to analyze adhesion, proliferation, and extracellular matrix expression.ResultsThe results revealed marked expression of a wound healing gene (VEGF-A) in never-smokers (p value = 0.016). GPX3 and SOD3 represent antioxidants that are highly expressed in treated never-smoker cells. SOD2 significantly increased (p value = 0.016) in smokers after vitamin C exposure. The anti-inflammatory markers IL-6 and IL-8 were lower among smokers than among nonsmokers (p < 0.0001).ConclusionTobacco smoking suppressed gingival fibroblasts' abilities to regenerate, heal, combat inflammation, and resist free radicals. Vitamin C at cellular levels was beneficial and should be considered in the treatment component of smokers in the dental clinic.     

Periodontal ligament and gingival fibroblasts participate in the production of TGF-β, interleukin (IL)-8 and IL-10

The aim of this study was to quantify and compare the production of transforming growth factor beta (TGF-β), interleukin (IL)-8 and IL-10 by human cultured periodontal ligament and gingival fibroblasts both obtained from the same donors challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Fibroblasts were exposed to 0.1-10 μg/mL of LPS from P. gingivalis and after 24 h the supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA). TGF-β protein production was upregulated in a concentration-dependent manner, mainly in gingival fibroblasts, which was statistically significant when challenged by 10 μg/mL LPS. Additionally, at this concentration, gingival fibroblasts had almost a two-fold increase in the amount of TGF-β when compared to periodontal ligament fibroblasts. Both periodontal ligament and gingival fibroblasts showed an increase in IL-8 production when challenged with 1 μg/mL and 10 μg/mL LPS. IL-10 production remained unaffected when challenged by any of the LPS concentrations tested in either periodontal ligament or gingival fibroblasts. Our results demonstrate that periodontal ligament and gingival fibroblasts when challenged by LPS from P. gingivalis with 24 h may play a critical role in producing TGF-β and IL-8 but not IL-10.     

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

Rawal SY, Dabbous MKh, Tipton DA. Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor β. J Periodont Res 2012; 47: 320–329. © 2011 John Wiley & Sons A/S 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 β 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 β 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 β and fibronectin, while decreasing MMP production and activity.     

Evaluation of milk fat globule-epidermal growth factor–factor VIII and IL-1β levels in gingival crevicular fluid and saliva in periodontal disease and health

Odontology - The aim of this study is to determine the levels of MFG-E8 and interleukin (IL)-1β in saliva and gingival crevicular fluid (GCF) associated with periodontal health and disease....     

The effectiveness of dentifrices without and with sodium lauryl sulfate on plaque,gingivitis and gingival abrasion—a randomized clinical trial

Objectives

The aim of this study was to compare the efficacy of a dentifrice without sodium lauryl sulfate (SLS) to a dentifrice with SLS in young adults aged 18–34 years on gingivitis.

Material and methods

One hundred twenty participants (non-dental students) with a moderate gingival inflammation (bleeding on probing at 40–70 % of test sites) were included in this randomized controlled double blind clinical trial. According to randomization, participants had to brush their teeth either with dentifrice without SLS or with SLS for 8 weeks. The primary outcome was bleeding on marginal probing (BOMP). The secondary outcomes were plaque scores and gingival abrasion scores (GA) as well as a visual analogue scale (VAS) score at exit survey. Baseline and end differences were analysed by univariate analysis of covariance (ANCOVA) test, between group differences by independent t test and within groups by paired sample t test.

Results

BOMP improved within groups from on average 0.80 at baseline to 0.60 in the group without SLS and to 0.56 in the group with SLS. No statistical difference for BOMP, plaque and gingival abrasion was found between both groups. VAS scores for taste, freshness and foaming effect were significantly in favour of the SLS-containing dentifrice.

Conclusion

The test dentifrice without SLS was as effective as a regular SLS dentifrice on gingival bleeding scores and plaque scores. There was no significant difference in the incidence of gingival abrasion.

Clinical relevance

In patients diagnosed with gingivitis, a dentifrice without SLS seems to be equally effective compared to a dentifrice with SLS and did not demonstrate any significant difference in gingival abrasion. In patient with recurrent aphthous ulcers, the absence of SLS may even be beneficial. However, participants indicate that they appreciate the foaming effect of a dentifrice with SLS more.

     

Biological roles of KGF,CTGF and TGF-β in cyclosporine-A- and phenytoin- induced gingival overgrowth: A comparative experimental animal study

ObjectiveTo identify the possible biological roles of keratinocyte growth factor (KGF), connective tissue growth factor (CTGF) and transforming growth factor-β (TGF-β) in cyclosporine-A (CsA) and phenytoin (PNT)-induced gingival overgrowth (GO) and to correlate them with each other.MethodsSixty adult male albino rats were selected and divided into 3 equal groups. Group I rats received no treatment. Group II rats were administrated CsA for 7 weeks. Group III were administrated PNT for the same period. Rats were euthanized at the end of the experiment and routine tissue processing was carried out. The obtained specimens were stained with H&E, KGF, CTGF and TGF-β antibodies.ResultsOne-way MANOVA test for KGF, CTGF and TGF-β revealed an overall significant difference between the different groups (P < 0.001). LSD post hoc test for multiple comparisons revealed a significant difference between each two groups. Two-tailed Pearson correlation for group II revealed non-significant weak positive correlations between KGF & CTGF and between CTGF & TGF-β. Non-significant weak negative correlation was found between KGF & TGF-β. Meanwhile, group III revealed non-significant weak positive correlation between KGF & TGF-β and between CTGF & TGF-β. Significant moderate positive correlation was found between KGF & CTGF.ConclusionThe findings of the present study indicated that KGF, CTGF and TGF-β have biological roles in progression of CsA- and PNT- induced GO. KGF plays a greater role in CsA- induced GO than in PNT- induced GO. Meanwhile, CTGF and TGF-β play a role in PNT- induced GO greater than in CsA- induced GO.     

Cellular viability and genetic expression of human gingival fibroblasts to zirconia with enamel matrix derivative (Emdogain?)

PURPOSE

The objective of this study was to investigate the biologic effects of enamel matrix derivative (EMD) with different concentrations on cell viability and the genetic expression of human gingival fibroblasts (HGF) to zirconia surfaces.

MATERIALS AND METHODS

Immortalized human gingival fibroblasts (HGF) were cultured (1) without EMD, (2) with EMD 25 µg/mL, and (3) with EMD 100 µg/mL on zirconia discs. MTT assay was performed to evaluate the cell proliferation activity and SEM was carried out to examine the cellular morphology and attachment. The mRNA expression of collagen type I, osteopontin, fibronectin, and TGF-β1 was evaluated with the real-time polymerase chain reaction (RT-PCR).

RESULTS

From MTT assay, HGF showed more proliferation in EMD 25 µg/mL group than control and EMD 100 µg/mL group (P<.05). HGFs showed more flattened cellular morphology on the experimental groups than on the control group after 4h culture and more cellular attachments were observed on EMD 25 µg/mL group and EMD 100 µg/mL group after 24h culture. After 48h of culture, cellular attachment was similar in all groups. The mRNA expression of type I collagen increased in a concentration dependent manner. The genetic expression of osteopontin, fibronectin, and TGF-β1 was increased at EMD 100 µg/mL. However, the mRNA expression of proteins associated with cellular attachment was decreased at EMD 25 µg/mL.

CONCLUSION

Through this short term culture of HGF on zirconium discs, we conclude that EMD affects the proliferation, attachment, and cell morphology of HGF cells. Also, EMD stimulates production of extracellular matrix collagen, osteopontin, and TGF-β1 in high concentration levels.

CLINICAL RELEVANCE

With the use of EMD, protective barrier between attached gingiva and transmucosal zirconia abutment may be enhanced leading to final esthetic results with implants.     

Identification and consequences of distinct Löe-Silness gingival index examiner styles for the clinical assessment of gingivitis

BACKGROUND: In clinical studies, gingivitis is most frequently assessed by the L?e-Silness gingival index (GI). The objective of this work was to develop an understanding of how clinicians experienced with GI differ with respect to how they apply GI and to assess the impact of different examination styles on statistical outcomes and magnitude of treatment differences. METHODS: A method was developed to mathematically relate the average GI score and degree of bleeding observed for a subject. Graphical analyses were used to profile examiner styles with respect to using the GI index. A prospective single-center, examiner-blind study comparing the effects of a staggered prophylaxis on gingivitis was then conducted, where a difference in gingivitis was created between two balanced groups by providing subjects a prophylaxis at two staggered time points. Subjects were assigned to one of two cohorts; within each cohort, group 1 subjects received a dental prophylaxis following the baseline examination and group 2 subjects received a dental prophylaxis 8 weeks later. Five to 7 days after the group 2 prophylaxis, all subjects were examined for GI. Twelve experienced clinicians participated. RESULTS: Retrospective analyses indicated the presence of distinct examiner styles which are based on the frequency that a given GI score (0, 1, 2, or 3) is measured by a clinician. In the prospective study, all 12 examiners observed statistically significant differences between the prophylaxis treatment groups at the final visit for both mean number of bleeding sites and mean GI; the magnitude ranged from 21.5% to 84.6% for mean number of bleeding sites and 9.4% to 39.2% for mean GI. There were 4 distinct styles employed by these experienced clinicians. CONCLUSIONS: Varying examiner styles impact the structure of resulting data. Importantly, the implementation of arbitrary thresholds (e.g., 20%) regarding percent treatment differences between groups as a guideline for judging the clinical significance is scientifically unsupported. A more scientific criterion in the field of gingivitis clinical testing would be the independent demonstration of statistical superiority compared to a negative control and/or a demonstration of similar or superior efficacy to clinically proven positive controls. In addition, interexaminer calibration is a mechanism that can be utilized to minimize the impact of different examiner styles in clinical settings involving more than one examiner.