Deep dental phenotyping and a novel FAM20A variant in patients with amelogenesis imperfecta type IG.

OBJECTIVES: To identify etiologic variants and perform deep dental phenotyping in patients with amelogenesis imperfecta (AI). METHODS: Three patients of two unrelated families were evaluated. Genetic variants were investigated by exome and Sanger sequencing. An unerupted permanent third molar (AI1) from Patient1 and a deciduous first molar (AI2) from Patient2, along with three tooth-type matched controls for each were characterized. RESULTS: All three patients harbored biallelic pathogenic variants in FAM20A, indicating AI1G. Of the four identified variants, one, c.1231C > T p.(Arg411Trp), was novel. Patient1 possessed the largest deletion, 7531 bp, ever identified in FAM20A. In addition to hypoplastic enamel, multiple impacted teeth, intrapulpal calcification, pericoronal radiolucencies, malocclusion, and periodontal infections were found in all three patients, gingival hyperplasia in Patient1 and Patient2, and alveolar bone exostosis in Patient3. Surface roughness was increased in AI1 but decreased in AI2. Decreased enamel mineral density, hardness, and elastic modulus were observed in AI1 enamel and dentin and AI2 dentin, along with decreased phosphorus, increased carbon, and increased calcium/phosphorus and carbon/oxygen ratios. Severely collapsed enamel rods and disorganized dentin-enamel junction were observed. CONCLUSIONS: We report a novel FAM20A variant and, for the first time, the defective mineral composition and physical/mechanical properties of AI1G teeth.

Management of hyperplastic tissue response following connective tissue grafting.

OBJECTIVE: While connective tissue graft shrinkage is a well-documented post-transplantation reaction, there is a literature gap concerning hyperplastic tissue response. Despite its infrequent occurrence, investigation is warranted due to its capacity to compromise esthetics, disrupt lip dynamics, and promote food retention. Moreover, efforts to mitigate hyperplastic tissue response often prove challenging, and there is a potential risk of exacerbating gingival tissue rebound. CLINICAL CONSIDERATIONS: This report presents a potential solution to managing tissue overgrowth after connective tissue grafting in five clinical cases. The patients underwent corrective surgery involving internal excision of excessive tissue while preserving the overlying mucosa. The surgical approach was tailored to excise hyperplastic tissue with minimal trauma, aiming to optimize esthetic outcomes. Subsequent follow-up assessments spanning 1-5 years demonstrated stable results, with no indications of relapse or recurrence of tissue overgrowth. CONCLUSIONS: Within the limitations of this case series, surgical internal excision holds promise as a viable treatment modality for addressing post-transplantation hyperplastic tissue response. CLINICAL SIGNIFICANCE: This case series addresses the challenge of uncontrolled tissue overgrowth following connective tissue grafting, a concern for which previous attempts have proven unsuccessful. Internal in-toto excision emerges as a promising approach for effectively eliminating overgrown tissue, offering potential advancements in the clinical management of this complication.

Enhancement of receptor-mediated calcium responses by phenytoin through the suppression of calcium excretion in human gingival fibroblasts.

BACKGROUND AND OBJECTIVES: Gingival overgrowth caused by phenytoin is proposed to be associated with Ca2+ signaling; however, the mechanisms that increase the intracellular Ca2+ concentration ([Ca2+ ]i ) are controversial. The current study aimed to elucidate the mechanism underlying the phenytoin-induced increase in [Ca2+ ]i in human gingival fibroblasts (HGFs). METHODS: Effects of 100 µM phenytoin on [Ca2+ ]i in HGFs were examined at the single-cell level using fluorescence images of fura-2 captured by an imaging system consisting of an EM-CCD camera coupled to an inverted fluorescence microscope at room temperature. RESULTS: Exposure of HGFs to 100 µM phenytoin induced a transient increase in [Ca2+ ]i in the absence of extracellular Ca2+ , indicating that the phenytoin-induced increase in [Ca2+ ]i does not require an influx of extracellular Ca2+ . In addition, phenytoin increased [Ca2+ ]i in HGFs depleted of intracellular Ca2+ stores by thapsigargin, indicating that neither Ca2+ release from stores nor inhibition of Ca2+ uptake is involved. Furthermore, the phenytoin-induced [Ca2+ ]i elevation was reduced to 18.8% in the absence of extracellular Na+ , and [Ca2+ ]i elevation upon removal of extracellular Na+ was reduced to 25.9% in the presence of phenytoin. These results imply that phenytoin increases [Ca2+ ]i of HGFs by suppressing the Na+ /Ca2+ exchanger. Suppression of intracellular Ca2+ excretion is thought to enhance the Ca2+ responses induced by various stimuli. Analysis at the single-cell level showed that stimulation with 1 µM ATP or 3 µM histamine increased [Ca2+ ]i in 20-50% of cells, and [Ca2+ ]i increased in many unresponsive cells in the presence of phenytoin. CONCLUSION: Our findings demonstrate that phenytoin induced increase in [Ca2+ ]i by the inhibition of Ca2+ efflux in HGFs. It was also found that phenytoin strongly enhanced small Ca2+ responses induced by stimulation with a low concentration of ATP or histamine by inhibiting Ca2+ efflux. These findings suggest a possibility that phenytoin causes drug-induced gingival overgrowth by interacting with inflammatory bioactive substances in the gingiva.

Cyclosporine A-induced gingival overgrowth in renal transplant patients accompanied by epithelial-to-mesenchymal transition.

OBJECTIVE: To investigate the association between the prevalence of cyclosporin A-induced gingival overgrowth and the expression of the epithelial-to-mesenchymal transition factors in the gingival tissues of renal transplant patients. BACKGROUND: Gingival overgrowth (GO) is a frequent complication in organ transplant patients treated with the immunosuppressant cyclosporin A (CsA). The epithelial-to-mesenchymal transition (EMT) is considered a factor contributing to CsA-induced GO. However, current knowledge on this topic is sparse. METHODS: Sixty-three renal transplant patients were divided into two groups according to the occurrence of GO: those with gingival overgrowth (GO+ group) and those without gingival overgrowth (GO- group). Data on age, sex, and use of immunosuppressant and calcium channel blocker medications, serum creatinine values, peak concentrations of blood CsA, and gingival hyperplasia scores were recorded to identify clinically pathogenic factors. Gingival tissues from five patients with CsA-induced GO and five healthy subjects were selected for histomorphological observation with hematoxylin-eosin staining, Masson staining, and immunohistochemical staining. The mRNA expression of EMT factors was detected with reverse transcription-quantitative PCR. RESULTS: The use of CsA significantly increased the prevalence of GO in renal transplant patients. The expression of α-SMA, SMAD4, and TGM2 was upregulated and that of E-cadherin was downregulated in the gingival tissues of patients with CsA-induced GO compared with those of the corresponding controls. CONCLUSION: Treatment with CsA is closely related to the occurrence of GO in renal transplant patients and EMT plays an important role in CsA-induced gingival tissue hyperplasia.

Drug-Induced Gingival Overgrowth-Molecular Aspects of Drug Actions.

Drug-induced gingival overgrowth (DIGO) is one of the side effects produced by therapeutic agents, most commonly phenytoin, nifedipine and cyclosporin A. However, the precise mechanism of DIGO is not entirely understood. A literature search of the MEDLINE/PubMed databases was conducted to identify the mechanisms involved in DIGO. The available information suggests that the pathogenesis of DIGO is multifactorial, but common pathogenic sequelae of events emerge, i.e., sodium and calcium channel antagonism or disturbed intracellular handling of calcium, which finally lead to reductions in intracellular folic acid levels. Disturbed cellular functions, mainly in keratinocytes and fibroblasts, result in increased collagen and glycosaminoglycans accumulation in the extracellular matrix. Dysregulation of collagenase activity, as well as integrins and membrane receptors, are key mechanisms of reduced degradation or excessive synthesis of connective tissue components. This manuscript describes the cellular and molecular factors involved in the epithelial-mesenchymal transition and extracellular matrix remodeling triggered by agents producing DIGO.

Phenytoin induces connective tissue growth factor (CTGF/CCN2) production through NADPH oxidase 4-mediated latent TGFß1 activation in human gingiva fibroblasts: Suppression by curcumin.

OBJECTIVE AND BACKGROUND: Gingival overgrowth (GO) is a common side effect of some drugs such as anticonvulsants, immunosuppressant, and calcium channel blockers. Among them, the antiepileptic agent phenytoin is the most common agent related to this condition due to its high incidence. Transforming growth factor ß (TGFß) importantly contributes to the pathogenesis of GO. Connective tissue growth factor (CTGF or CCN2) is a key mediator of tissue fibrosis and is positively associated with the degree of fibrosis in GO. We previously showed that Src, c-jun N-terminal kinase, and Smad3 mediate TGFß1-induced CCN2 protein expression in human gingival fibroblasts (HGFs). This study investigates whether phenytoin can induce CCN2 synthesis through activated latent TGFß in HGFs and its mechanisms. METHODS: CCN2 synthesis, latent TGFß1 activation, and cellular reactive oxygen species (ROS) generation in HGFs were studied using western blot analysis, a TGFß1 Emax® ImmunoAssay System, and 2',7'-dichlorodihydrofluorescein diacetate (an oxidation-sensitive fluorescent probe), respectively. RESULTS: Phenytoin significantly stimulated CCN2 synthesis, latent TGFß1 activation, and ROS generation in HGFs. Addition of an TGFß-neutralizing antibody, TGFß receptor kinase inhibitor SB431542, and Smad3 inhibitor SIS3 completely inhibited phenytoin-induced CCN2 synthesis. General antioxidant N-acetylcysteine, NADPH oxidase (NOX) inhibitor diphenylene iodonium, and specific NOX4 inhibitor plumbagin almost completely suppressed phenytoin-induced total cellular ROS and latent TGFß1 activation. Curcumin dose-dependently decreased phenytoin-induced TGFß1 activation and CCN2 synthesis in HGFs. CONCLUSIONS: Our findings indicated that NOX4-derived ROS play pivotal roles in phenytoin-induced latent TGFß1 activation. Molecular targeting the phenytoin/NOX4/ROS/TGFß1 pathway may provide promising strategies for the prevention and treatment of GO. Curcumin-inhibited phenytoin-induced CCN2 synthesis is caused by the suppression of latent TGFß1 activation.

Cyclosporine-induced gingival overgrowth-Review.

Drug-induced gingival overgrowth (DIGO) is an undesirable effect resulting from the therapy of one of the three groups of drugs: phenytoin, calcium channel blockers, and cyclosporine A (CsA). It is caused by a fibrous overgrowth leading to gingivitis, periodontitis, and even tooth loss. Possible consequences include tooth decay worsening, pain and difficulty in eating, bleeding gums, and bad breath. The pathomechanism of the hypertrophy is unknown, but there is a correlation between insufficient oral hygiene and the severity of this phenomenon. The gender and age predilection of gingival hyperplasia as a result of CsA therapy is also noticeable. It is most common in children and adolescents of the male sex. The beneficial effect of the removal of tartar and local irritants in reducing the above symptoms has been demonstrated. One of the treatments for DIGO is conventional gingivectomy. The paper is a review article about cyclosporine-induced gingival hyperplasia.

Gingivectomy with high-power laser for correction of the gummy smile resulting from altered passive eruption-a case series.

To compare the efficacy between the surgical techniques of gingivectomy with high-power laser (HPL) and conventional gingivectomy for correction of a gummy smile (GS) due to altered passive eruption (APE). This was a case series of six female patients diagnosed with GS associated with APE. For the conventional gingivectomy procedure and for the one using the diode laser (808 nm, 2 W, in continuous mode), the six upper anterior teeth were divided into two groups (control (CG)-# 11, # 12, # 13, and test (TG)-# 21, # 22, # 23). Analyses of intraoperative bleeding and levels of pain and postoperative tissue repair were performed. The thermal pattern analysis was performed using infrared thermography. The level of significance was set at p < 0.05. There was no intraoperative bleeding in the TGs (p = 0.002). The CG showed tissue repair significantly better than the TG on the 14th postoperative day (p = 0.004). There were no statistically significant differences regarding the level of postoperative pain between the groups (p > 0.05). Regarding the thermographic analysis, there were also no statistically significant differences (p > 0.05). HPL gingivectomy was more effective, regarding the absence of intraoperative bleeding, while the conventional technique promoted better tissue repair. No significant differences were observed in the other parameters, possibly due to the minimal damage caused by gingivectomy, with either HPL or the conventional procedure, as there was no removal of bone tissue.

Mutations in KCNK4 that Affect Gating Cause a Recognizable Neurodevelopmental Syndrome.

Aberrant activation or inhibition of potassium (K+) currents across the plasma membrane of cells has been causally linked to altered neurotransmission, cardiac arrhythmias, endocrine dysfunction, and (more rarely) perturbed developmental processes. The K+ channel subfamily K member 4 (KCNK4), also known as TRAAK (TWIK-related arachidonic acid-stimulated K+ channel), belongs to the mechano-gated ion channels of the TRAAK/TREK subfamily of two-pore-domain (K2P) K+ channels. While K2P channels are well known to contribute to the resting membrane potential and cellular excitability, their involvement in pathophysiological processes remains largely uncharacterized. We report that de novo missense mutations in KCNK4 cause a recognizable syndrome with a distinctive facial gestalt, for which we propose the acronym FHEIG (facial dysmorphism, hypertrichosis, epilepsy, intellectual disability/developmental delay, and gingival overgrowth). Patch-clamp analyses documented a significant gain of function of the identified KCNK4 channel mutants basally and impaired sensitivity to mechanical stimulation and arachidonic acid. Co-expression experiments indicated a dominant behavior of the disease-causing mutations. Molecular dynamics simulations consistently indicated that mutations favor sealing of the lateral intramembrane fenestration that has been proposed to negatively control K+ flow by allowing lipid access to the central cavity of the channel. Overall, our findings illustrate the pleiotropic effect of dysregulated KCNK4 function and provide support to the hypothesis of a gating mechanism based on the lateral fenestrations of K2P channels.

Activated KCNQ1 channel promotes fibrogenic response in hereditary gingival fibromatosis via clustering and activation of Ras.

BACKGROUND AND OBJECTIVE: Activated potassium channels were found to be strongly correlated with gingival overgrowth (GO) phenotype as we reviewed syndromic hereditary gingival fibromatosis (HGF). Nevertheless, the functional roles of potassium channels in gingival fibrosis or gingival overgrowth remained uncovered. The aim of the present study was to explore the pathogenic role of aberrantly activated potassium channel in Hereditary Gingival Fibromatosis (HGF). METHODS: Gingival tissues were collected from 9 HGF patients and 15 normal controls. Expression of KCNQ1 was detected by immunohistochemistry. Gingival fibroblasts were isolated, and outward K+ currents were detected by whole-cell patch-clamp analysis, transmembrane potential was determined by flow cytometry. Normal human gingival fibroblasts (NHGFs) were transfected with KCNQ1 adenovirus or treated with KCNQ1 selective agonist ML277 and antagonist chromanol 293B. Accumulation of Extracellular Matrix (ECM) was measured by Western blotting and Sircol Soluble Collagen Assay. Content of secreted TGF-ß1 was measured by ELISA. Active RAS pull-down assay and cell immunofluorescence were utilized to verify RAS activation. RESULTS: KCNQ1 was upregulated in gingival tissues derived from HGF patients and HGF gingival fibroblasts presented increased outward K+ currents than NHGFs. Overexpression of KCNQ1, or KCNQ1 agonist ML277, promoted fibrotic responses of NHGFs. TGF-ß1 and KCNQ1 channels formed a positive feed-back loop. ML277 generated lateral clustering and activation of Ras on plasma membrane, followed by augmented MAPK/AP-1 signaling pathway output. JNK or ERK1/2 inhibitors suppressed ML277-induced AP-1 and ECM upregulation. CONCLUSION: Activation of KCNQ1 potassium channel promoted fibrogenic responses in NHGFs via Ras/MAPK/AP-1 signaling.

Antioxidants protect against gingival overgrowth induced by cyclosporine A.

OBJECTIVE: We investigated the importance of reactive oxygen species (ROS) on developing gingival overgrowth (GO) and then introduced the antioxidant strategy to prevent, or even reduce GO. BACKGROUND: Gingival overgrowth is a common side effect of the patients receiving cyclosporine A (CsA), an immune suppressant. Although it has been broadly investigated, the exact pathogenesis of the induced GO is still uncertain. METHODS: We cultured human primary gingival fibroblasts and used animal model of GO to investigate the ameliorative effects of antioxidants on CsA-induced GO. To examine the CsA-induced oxidative stress, associated genes and protein expression, and the overgrown gingiva of rats by using immunocytochemistry, confocal laser scanning microscopy, real-time PCR, ELISA, gelatin zymography, gingival morphological, and immunohistochemical analysis. RESULTS: We found for the first time that ROS was responsible for the CsA-induced oxidative stress and TGF-ß1 expression in human primary gingival fibroblasts, as well as the GO of rats. The antioxidants (oxidative scavenger of vitamin E and an antioxidative enzyme inducer of hemin) ameliorated CsA-induced pathological and morphological alterations of GO without affected the CsA-suppressed il-2 expression in rats. CsA-induced oxidative stress, HO-1, TGF-ß1, and type II EMT were also rescued by antioxidants treatment. CONCLUSIONS: We concluded that CsA repetitively stimulating the production of ROS is the cause of CsA-GO which is ameliorated by treating antioxidants, including vitamin E and sulforaphane. Furthermore, the immunosuppressive effect of CsA is not interfered by antioxidant treatments in rats. This finding may thus help the clinician devise better prevention strategies in patients susceptible to GO.

Influence of different anti-hypertensive drugs on gingival overgrowth: A cross-sectional study in a Turkish population.

OBJECTIVE: The purpose of this study was to evaluate the occurrence rate of drug-induced gingival overgrowth (DIGO) in patients treated with angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and calcium channel blockers (CCBs) such as amlodipine, lercanidipine, and benidipine, as well as to assess the relationship of those mentioned above with medication variables and oral hygiene. METHODS: Sociodemographic details, DIGO, and clinical periodontal parameters were obtained from one hundred and thirty-one patients receiving ACE inhibitors, ARBs, and CCBs for a period of at least 2 years. RESULTS: The occurrence rate of DIGO was 19.6% in patients using CCB, 12.5% in the ARB group, and 7.5% in the ACE inhibitor group. In a subgroup analysis of CCBs, DIGO was found to be 31.8% in the amlodipine group, 13.3% in the lercanidipine group, and 7.1% in the benidipine group. While there was a significant relationship between amlodipine drug dosage and DIGO, no association was found between the duration of therapy and DIGO in all CCB subgroups. CONCLUSION: There was no difference between the groups in terms of DIGO. Duration of therapy and drug dosage did not affect the severity of DIGO in both ACE inhibitors and ARB groups.

Hereditary gingival fibromatosis in children: a systematic review of the literature.

OBJECTIVES: Hereditary gingival fibromatosis (HGF) is an uncommon, inherited condition with slow and progressive fibrous hyperplasia of the gingiva. Due to its association with mastication, speech, and occlusion problems, early diagnosis is important. We sought to summarize the available data regarding the epidemiology, clinical characteristics, and outcomes of children with HGF (< 18 years). METHODS: A systematic literature review of the MEDLINE and Cochrane Library databases was conducted with respect to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement (end-of-search date: March 1, 2019). RESULTS: A total of 99 articles reporting on 146 patients were included. The mean age was 10.82 ± 3.93 years, and generalized gingival enlargement was seen in 97.16% (95% CI 92.69 to 99.14). Jaw, gingival, and teeth abnormalities; poor oral hygiene; eating; or speech difficulties were typical HGF-induced, while 60.90% had extraoral manifestations (95% CI 52.41 to 68.78). The disease was most commonly inherited in an autosomal dominant manner (88.41%, 95% CI 78.5 to 94.26), and about one-third of the patients had syndromic HGF (33.85%, 95% CI 23.50 to 46.00). Gingivectomy was performed in the majority of cases (91.15%, 95% CI 84.31 to 95.29), and recurrence was seen in 33.85% (95% CI 23.50 to 46.00). CONCLUSION: HGF should be suspected in children with nodularity and gingival fibrosis, teeth abnormalities, or jaw distortion. Family history can help to establish the diagnosis. CLINICAL RELEVANCE: More cases should focus on longer-term follow-up after gingivectomy as disease recurrence is not uncommon.

Prioritization of predisposing factors of gingival hyperplasia during orthodontic treatment: the role of amount of biofilm.

BACKGROUND: The mechanism of gingival growth that may occur during fixed orthodontic treatment is not yet fully understood and the amount of dental plaque is often incriminated. The objective of this study was to evaluate the prevalence of gingival growth during multi-attachment orthodontic treatment and to prioritize its predicting factors, especially the quantity of biofilm. METHODS: This comprehensive cross-sectional descriptive study was conducted on orthodontic patients aged 9 to 30 years, in good health, treated by a fixed appliance. Periodontal clinical parameters such as plaque index, gingival index, probing pocket depth, periodontal phenotype and gingival enhancement index were recorded. Likewise, the brushing habits and the date of the last scaling were noted. The orthodontic parameters studied were the duration of the treatment, the type of bracket, the alloys used for the arches and the type of ligatures. Descriptive statistics were carried out, and variables presenting p value < 0.25 were included in a multivariate analysis to calculate the Odds Ratio (OR) of gingival enlargement". RESULTS: A total of 193 patients were included (16.38 ± 4.89 years). Gingival growth occurred for 49.7% of patients included. The predisposing factors for this pathology during fixed orthodontic treatment were conventional metal brackets (p = 0.021), mouth breathing (p = 0.040), male gender (p = 0.035), thick periodontal phenotype (p = 0.043), elastomeric ligations (p = 0.007), duration of treatment (p = 0.022) and presence of plaque (p = 0.004). After achievement of the logistic regression, only two factors remained related to gingival enlargement: metallic brackets (OR: 3.5, 95% CI: 1.1-10.55) and duration of treatment (OR: 2.03, 95% CI: 1.01-4.08). The amount of plaque would not be directly related to the development of gingival increase during orthodontic treatment. CONCLUSIONS: Among the predisposing factors that underlie gingival growth during multi-attachment therapy, the amount of plaque is not found. The qualitative assessment of the plaque and its evolution during treatment could clarify the role of the biofilm in the occurrence of gingival overgrowth.

Elevated Interleukin-17A expression in amlodipine-induced gingival overgrowth.

BACKGROUND AND OBJECTIVES: Amlodipine, a calcium channel blocker derivative, is frequently used by patients with high blood pressure. Studies reported that it can induce gingival overgrowth. However, the underlying mechanism is not fully described yet. Interleukin-17A (IL-17A) is known as a proinflammatory cytokine, but current studies indicate that it has a role in fibrotic disorders and epithelial-mesenchymal transition (EMT). The aim of this study was to figure out the possible role of IL-17A in amlodipine-induced gingival overgrowth. MATERIALS AND METHODS: Twenty-nine (29) individuals participated in the study, and they were assigned into 3 groups based on medical status and clinical periodontal examination; 9 patients with amlodipine-induced gingival overgrowth, 11 patients with inflammatory gingival overgrowth, and 9 healthy individuals as a control group. Clinical periodontal parameters including plaque index (PI), gingival index (GI), and gingival overgrowth index (GOI) were recorded. Blood and gingival crevicular fluid (GCF) samples were obtained. Gingival tissues were taken by appropriate periodontal surgery following initial periodontal therapy. To detect IL-17A on tissue samples, immunohistochemistry (IHC) was performed. Quantitative analysis was done, and the expression level of IL-17A was given as the percent positively stained cells. Enzyme-linked immunosorbent assay (ELISA) kits were used to analyze IL-17A in serum and GCF samples. RESULTS: All recorded clinical parameters were significantly higher in gingival overgrowth groups compared with control. Evaluation of inflammation on tissue sections did not show any significant change within the groups. Immunohistochemistry findings showed that IL-17A expression was increased in amlodipine samples (81.90%) compared with control samples (42.35%) (P < .001). There was an increase in the inflammatory group (66.08%) which is significantly less than the amlodipine group (P < .05). IL-17A levels in serum and GCF samples were not different within the study groups. CONCLUSION: In this study, elevated IL-17A expression regardless of inflammation shows that amlodipine might cause an increase of IL-17A in gingival tissues. This increase might induce fibrotic changes and EMT in gingival overgrowth tissues. The association of IL-17A with fibrosis and EMT in gingival tissues requires further investigation.

Galectin-8 mediates fibrogenesis induced by cyclosporine in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: During cyclosporine-induced gingival overgrowth, the homeostatic balance of gingival connective tissue is disrupted leading to fibrosis. Galectins are glycan-binding proteins that can modulate a variety of cellular processes including fibrosis in several organs. Here, we study the role of galectin-8 (Gal-8) in the response of gingival connective tissue cells to cyclosporine. METHODS: We used human gingival fibroblasts and mouse NIH3T3 cells treated with recombinant Gal-8 and/or cyclosporine for analyzing specific mRNA and protein levels through immunoblot, real-time polymerase chain reaction, ELISA and immunofluorescence, pull-down with Gal-8-Sepharose for Gal-8-to-cell surface glycoprotein interactions, short hairpin RNA for Gal-8 silencing and Student's t test and ANOVA for statistical analysis. RESULTS: Galectin-8 stimulated type I collagen and fibronectin protein levels and potentiated CTGF protein levels in TGF-ß1-stimulated human gingival fibroblasts. Gal-8 interacted with α5ß1-integrin and type II TGF-ß receptor. Gal-8 stimulated fibronectin protein and mRNA levels, and this response was dependent on FAK activity but not Smad2/3 signaling. Cyclosporine and tumor necrosis factor alpha (TNF-α) increased Gal-8 protein levels. Finally, silencing of galectin-8 in NIH3T3 cells abolished cyclosporine-induced fibronectin protein levels. CONCLUSION: Taken together, these results reveal for the first time Gal-8 as a fibrogenic stimulus exerted through ß1-integrin/FAK pathways in human gingival fibroblasts, which can be triggered by cyclosporine. Further studies should explore the involvement of Gal-8 in human gingival tissues and its role in drug-induced gingival overgrowth.

Symptom-orientated oral hygiene for children after heart transplantation: Effectiveness of a standardized prophylactic program.

Children with CHD, especially heart-transplanted patients, are predisposed to have caries lesions, gingivitis and other oral findings like gingival hyperplasia. The aim of the study was the implementation of a specific oral hygiene program in these patients and its effect on the improvement of oral health, especially gingival overgrowth. For this, we used a newly developed systematic GHI to evaluate and describe this gingival alteration. Thirty-three children, aged 6 to 15 years with cardiac transplants (9 girls, 24 boys), were examined and introduced into a specific oral hygiene program. Each child showed evidence of gingival hyperplasia. They were randomly divided into three groups with the following oral care measurements: Group ZZ tooth brushing, Group ZZS tooth brushing and mouth rinsing, Group ZZSS tooth brushing, mouth rinsing and the use of an additional single and sulcus toothbrush. A significant decline of all oral health parameters could be proven in all groups. Gingival hyperplasia (GHI) improved as well as plaque accumulation (QHI). The children who used in addition to toothbrushing rinsing solutions and/or additional miniature toothbrushes showed better parameters of the gingival hygiene indexes from the baseline examination until the end of the study. The results show that any infant with cardiac transplant has to be introduced into an individualized oral hygiene program underlining the need of comprehensive dental care in cooperation with pediatric cardiology.

miR-200a inhibits proliferation rate in drug-induced gingival overgrowth through targeting ZEB2.

BACKGROUND/PURPOSE: Gingival overgrowth can occur as a result of poor oral hygiene or a side effect of taking certain medications, such as cyclosporine A (CsA). It has been shown that this immunosuppressant drug induces epithelial-to-mesenchymal transition (EMT) in the gingival epithelium but the associated molecular mechanism remains to be elucidated. METHODS: We first assessed the relative expression of microRNA-200a (miR-200a) in response to the CsA treatment using qRT-PCR. Next, luciferase reporter assay was applied to examine whether miR-200a was able to regulate ZEB2 and Western blot was utilized to measure the expression of ZEB2 in normal human gingival fibroblasts (HGFs). To confirm the significance of miR-200a and ZEB2 in the CsA-induced gingival overgrowth, miR-200a inhibitor and shRNA mediated knockdown of ZEB2 were used and cell proliferation in HGFs was assessed by MTT assay. RESULTS: The expression of miR-200a was dose-dependently downregulated following the CsA treatment. Luciferase reporter assay confirmed that ZEB2 was a direct downstream target regulated by miR-200a and ZEB2 was indeed increased after the administration of CsA. We demonstrated that knockdown of ZEB2 hampered the CsA-induced HGFs proliferation and the elevated cell proliferation due to inhibition of miR-200a was reversed by repression of ZEB2. CONCLUSION: Our results showed that insufficient miR-200a in HGFs caused by CsA administration may lead to gingival enlargement mediated by the upregulation of ZEB2. This finding supported that CsA-induced EMT contributed to the adverse effect of using CsA and miR-200a may serve as an upstream target to prevent the overgrowth of the gingiva.

Follicular dendritic cell-secreted protein gene expression is upregulated and spread in nifedipine-induced gingival overgrowth.

Follicular dendritic cell-secreted protein (FDC-SP) is secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium (JE). Its expression could be controlled during inflammatory process of gingiva; however, responsible mechanism for gingival overgrowth and involvement of FDC-SP in clinical condition is still unclear. We hypothesized that JE-specific genes are associated with the initiation of drug-induced gingival enlargement (DIGE) called gingival overgrowth, and investigated the changes of JE-specific gene's expression and their localization in overgrown gingiva from the patients. Immunohistochemical analysis revealed that the FDC-SP localization was spread in overgrown gingival tissues. FDC-SP mRNA levels in GE1 and Ca9-22 cells were increased by time-dependent nifedipine treatments, similar to other JE-specific genes, such as Amelotin (Amtn) and Lamininß3 subunit (Lamß3), whereas type 4 collagen (Col4) mRNA levels were decreased. Immunocytochemical analysis showed that FDC-SP, AMTN, and Lamß3 protein levels were increased in GE1 and Ca9-22 cells. Transient transfection analyses were performed using luciferase constructs including various lengths of human FDC-SP gene promoter, nifedipine increased luciferase activities of -345 and -948FDC-SP constructs. These results raise the possibility that the nifedipine-induced FDC-SP may be related to the mechanism responsible for gingival overgrowth does not occur at edentulous jaw ridges.

Role of Cyclosporine in Gingival Hyperplasia: An In Vitro Study on Gingival Fibroblasts.

BACKGROUND: Gingival hyperplasia could occur after the administration of cyclosporine A. Up to 90% of the patients submitted to immunosuppressant drugs have been reported to suffer from this side effect. The role of fibroblasts in gingival hyperplasia has been widely discussed by literature, showing contrasting results. In order to demonstrate the effect of cyclosporine A on the extracellular matrix component of fibroblasts, we investigated the gene expression profile of human fibroblasts after cyclosporine A administration. MATERIALS AND METHODS: Primary gingival fibroblasts were stimulated with 1000 ng/mL cyclosporine A solution for 16 h. Gene expression levels of 57 genes belonging to the "Extracellular Matrix and Adhesion Molecules" pathway were analyzed using real-time PCR in treated cells, compared to untreated cells used as control. RESULTS: Expression levels of different genes were significantly de-regulated. The gene CDH1, which codes for the cell adhesion protein E-cadherin, showed up-regulation. Almost all the extracellular matrix metalloproteases showed down-regulation (MMP8, MMP11, MMP15, MMP16, MMP24, MMP26). The administration of cyclosporine A was followed by down-regulation of other genes: COL7A1, the transmembrane receptors ITGB2 and ITGB4, and the basement membrane constituents LAMA2 and LAMB1. CONCLUSION: Data collected demonstrate that cyclosporine inhibits the secretion of matrix proteases, contributing to the accumulation of extracellular matrix components in the gingival connective tissue, causing gingival overgrowth. Patients affected by gingival overgrowth caused by cyclosporine A need to be further investigated in order to determine the role of this drug on fibroblasts.