Impact of TLR9 and TLR7 gene polymorphisms on prognosis and survival of patients with oral squamous cell carcinoma.

Despite significant efforts in developing new diagnostic and therapeutic modalities, oral squamous cell carcinomas (OSCCs) still exhibit a high recurrence rate, a low five-year survival rate, and an increasing prevalence. Toll-like receptors (TLRs), which initiate and perpetuate immune mechanisms upon activation, have been linked to immune surveillance and the antitumor immune response. The aim of this study was to investigate the association between the polymorphisms of the TLR7 rs3853839 and TLR9 rs187084 genes and OSCC risk, clinicopathological features, and survival. Genotyping was assessed by real-time polymerase chain reaction (PCR) in 95 HPV negative OSCC patients and 107 age- and sex-matched healthy controls. Patients with lymph node metastases had higher frequencies of the TLR9 rs187084 CC variant genotype compared to the major TT genotype (P = 0.020) and to T-allele carriers (combined TT + CT genotypes, P = 0.015). A higher prevalence of advanced stage III was observed in patients with the TLR9 rs187084 variant CC genotype compared to TT (P = 0.047) and to T-allele carriers (TT + CT, P = 0.037). Kaplan-Meier analysis revealed a lower overall survival (OS) rate in patients with the TLR9 rs187084 variant CC genotype compared to the TT genotype (P = 0.010, log-rank test) and to T-allele carriers (TT + CT genotypes, P = 0.002), though it was not an independent predictor of OS. Both TLR9 rs187084 and TLR7 rs3853839 polymorphisms were associated with high alcohol consumption (P = 0.027 and P = 0.001, respectively). The investigated genetic variations were not associated with OSCC susceptibility. The variant CC genotype of the TLR9 rs187084 polymorphism might be a marker of poor survival and tumor progression in OSCC.

Resolution of peri-implant mucositis following standard treatment: A prospective split-mouth study.

BACKGROUND: Peri-implant mucositis (PIM) is a pathological precursor of peri-implantitis, but its pattern of conversion to peri-implantitis is unclear and complicated to diagnose clinically, while none of the available protocols yield complete disease resolution. The aim of this study was the evaluation of PIM responsiveness to standard anti-infective mechanical treatment (AIMT) at clinical and biomarker levels, and estimation of the diagnostic capacity of bone markers as surrogate endpoints and predictors. METHODS: Systemically healthy outpatients presenting one implant exhibiting clinical signs of inflammation confined within the soft tissue (PIM) and one healthy control (HC) implant at a non-adjacent position were included. Clinical parameters and peri-implant crevicular fluid samples were collected baseline and 6 months following mechanical therapy, to assess the levels of RANKL, OPG, and IGFBP2. PIM clustering was performed using machine learning algorithms. RESULTS: Overall, 38 patients met the inclusion criteria. Therapy resulted in the reduction of all clinical and biological indicators, but respective values remained significantly higher compared to HC. Clinical examination noted 30% disease resolution at the 6-month follow-up, while 43% showed no active bone resorption. OPG showed positive prognostic value for treatment outcome, while the clustering based on active bone resorption did not differ in terms of therapeutic effectiveness. CONCLUSION: AIMT is effective in reducing the clinical and biological indicators of PIM, but complete clinical resolution was achieved in only 30% of the cases. Around one third of PIM patients exhibited active bone resorption bellow clinical detectability that was not associated with disease progression and poor treatment responsiveness.

Study on the immunopathological effect of titanium particles in peri-implantitis granulation tissue: A case-control study.

OBJECTIVES: To identify titanium particles (TPs) in biopsy specimens harvested from peri-implantitis lesions and secondarily to study the histopathological characteristics in peri-implantitis compared to periodontitis, in order to evaluate whether the presence of TPs could alter respective inflammatory patterns. MATERIAL AND METHODS: Biopsies containing granulation tissue were harvested during routine surgical treatment in 39 peri-implantitis cases and 35 periodontitis controls. Serial sections were obtained using titanium-free microtome blades. The first and last sections of the peri-implantitis specimens were used for identification of TPs by scanning electron microscopy coupled with dispersive X-ray spectrometry. Intermediate sections and periodontitis specimens were processed for descriptive histological study using haematoxylin-eosin staining and for immunohistochemical analysis using CD68, IL-6, Nf-kB and VEGF markers. RESULTS: TPs were identified in all peri-implantitis specimens as free metal bodies interspersed within granulation tissue. However, presence of macrophages or multinucleated giant cells engulfing the TPs were not identified in any specimen. Peri-implantitis granulations were characterized by a chronic inflammatory infiltrate rich in neutrophils. About half of peri-implantitis patients exhibited a subacute infiltrate characterized with lymphocytes interweaved with neutrophils and eosinophils. When compared to periodontitis, peri-implantitis tissues showed higher proportions of macrophages and a more intense neovascularization, based on significantly higher expression of CD68 and VEGF respectively. CONCLUSION: TPs were identified in all peri-implantitis specimens, but without evidencing any foreign body reaction suggestive for direct pathological effects of TPs. The peri-implantitis granulation tissue was characterized by intense neovascularization and presence of a chronic inflammatory infiltrate dominated by plasma cells, neutrophils and macrophages.

Underdiagnosis in Background of Emerging Public Health Challenges Related to Peri-Implant Diseases: An Interventional Split-Mouth Study.

Peri-implant diseases are an emerging public health problem, and it's considered that limitations of standard diagnostics play the role herein. The study objective was the estimation of pathological bone resorption at clinical and biological level in patients with peri-implant mucositis (PIM) and peri-implantitis (PI) before and 6 months after standard treatment and to compare them with healthy controls (HC). The split-mouth interventional study included 60 patients affected with PIM or PI. Patients that also presented at least one more HC were enrolled in the study and underwent standard non-surgical and surgical treatment, respectively. Standard clinical parameters and soluble levels of RANKL were measured in peri-implant crevicular fluid baseline and 6 months following treatment. Clinical parameters and RANKL significantly decreased following treatment in PIM and PI. However, bleeding on probing and probing depth remained significantly increased when compared to HC. RANKL answered requests for biomarker of peri-implant diseases, its baseline levels were significantly increased in PIM and PI, they decreased following treatment and reached HC in peri-implantitis, while in PIM RANKL remained significantly increased. Presence of pathological bone resorption in patients lacked its clinical signs, and respective persistence following treatment suggest the need for biomarker-supported diagnosis for timely diagnosis of peri-implantitis and appropriate orientation of respective management strategies.

Is the personalized approach the key to improve clinical diagnosis of peri-implant conditions? The role of bone markers.

BACKGROUND: Study objectives were 1) to estimate diagnostic capacity of clinical parameters, receptor activator of nuclear factor kappa-B (RANKL) and osteoprotegerin (OPG) to diagnose healthy peri-implant condition (HI), peri-implant mucositis (PIM) and peri-implantitis (PIMP) by assessing respective diagnostic accuracy, sensitivity, specificity and diagnostic ranges 2) to develop personalized diagnostic model (PDM) for implant monitoring. METHODS: Split-mouth study included 126 patients and 252 implants (HI = 126, PIM = 57, and PIMP = 69). RANKL and OPG concentrations were estimated in peri-implant crevicular fluid using enzyme-linked immunosorbent assay method and assessed with clinical parameters using routine statistics, while the diagnostic capacity of individual parameters and overall clinical diagnosis were estimated using classifying algorithms. PDM was developed using decision trees. RESULTS: Bleeding on probing (BOP), plaque index, and probing depth (PD) were confirmed reliable discriminants between peri-implant health and disease, while increase in PD (PD > 4 mm) and suppuration were good discriminants amongst PIM/PIMP. Bone turnover markers (BTMs) demonstrated presence of bone resorption in PIM; between comparable diagnostic ranges PIM/PIMP, PIMP was clinically distinguished from PIM in about 60% of patients while 40% remained diagnosed as false negatives. PDM demonstrated highest diagnostic capacity (accuracy: 96.27%, sensitivity: 95.00%, specificity: 100%) and defined HI: BOP ≤0.25%; PIM: BOP >0.25%, PD ≤4.5 mm; PIMP: BOP >0.25%, PD >4.5 mm and RANKL ≤19.9 pg/site; PIM: BOP >0.25%, PD >4.5 mm, and RANKL >19.9 pg/site. CONCLUSIONS: BTMs demonstrated capacity to substantially improve clinical diagnosis of peri-implant conditions. Considering lack of difference in BTMs between PIM/PIMP and cluster of PIM with exceeding BTMs, a more refined definition of peri-implant conditions according to biological characteristics is required for further BTMs validation and appropriate PIMP management.

Effect of dental caries on periodontal inflammatory status: A split-mouth study.

OBJECTIVE: This controlled split-mouth study aimed to estimate the effect of caries and related treatment on concentrations of interleukin (IL)-2, interferon (IFN)-γ, IL-12, IL-17A, IL-13, IL-10, IL-6, IL-5, IL-4, IL-22, tumor necrosis factor (TNF)-α, and IL1-ß in gingival crevicular fluid (GCF) of caries affected teeth before (B), 7 (7D) and 30 (30D) days post-treatment and to compare them with concentrations from healthy teeth. DESIGN: Study population included 81 systemically and periodontally healthy non-smokers exhibiting at least one shallow occlusal/ inter-proximal caries and one healthy tooth from the same morphologic group at the contralateral position. Following clinical exam, the GCF samples were collected baseline as well as 7D and 30D, while the biomarker measurement was performed using multiplex flowcytometry. RESULTS: Caries affected teeth exhibited significantly higher levels of IFN-γ, IL-1ß, IL-2, IL-4 and IL-6 when compared to healthy teeth. Post-treatment cytokines levels showed general trend of increase when compared to baseline, that was significant for IL-22 and IL-17 at 7D, while IFN-γ was significantly increased at 7D compared to the healthy teeth. At 30D, IFN-γ, TNF-α, IL-17 and IL-4 levels were significantly increased when compared to healthy teeth, while IL-2 levels were significantly higher than baseline levels. CONCLUSION: Considering significantly increased periodontal levels of inflammatory markers in caries affected teeth and in response to performed treatment, it seems that dental caries and related restorative treatment might contribute to periodontal inflammation via additive effects already in early-stage caries.

Association of Cytokine Gene Polymorphism with Peri-implantitis Risk.

PURPOSE: To investigate whether polymorphisms of cluster of differentiation 14 (CD14), tumor necrosis factor alpha (TNFα), interleukin (IL)6, IL10, and IL1ra genes are associated with the risk of peri-implantitis susceptibility in patients with dental implants in the Serbian population. MATERIALS AND METHODS: Isolated DNA from the blood was used for IL10-1082, TNFα-308, IL6-174, CD14-159, and interleukin 1 receptor antagonist (IL1ra) genotyping using polymerase chain reaction (PCR)-based methodology. Clinical parameters included: peri-implant pocket depth (PPD), Plaque Index (PI), Gingival Index (GI), bleeding on probing (BOP), and radiologic bone loss. RESULTS: The study included 98 patients with dental implants in function for at least 1 year, divided into peri-implantitis (34) and healthy peri-implant tissue (64) groups. The percentage distribution of smokers was significantly different between patients who developed peri-implantitis and patients with healthy peri-implant tissue (71% vs 42%, respectively) and associated with increased peri-implantitis risk (OR: 3.289, 95% CI: 1.352 to 8.001; P = .007). A positive history of periodontitis was more frequent in the peri-implantitis group (62%) than in the healthy peri-implant tissue (20%) group and associated with increased peri-implantitis risk (OR: 6.337, 95% CI: 2.522 to 15.927; P = .0001). Frequencies of CD14-159, TNFα-308, IL10-1082, and IL6-174 genotypes were significantly different between patients with and without peri-implantitis. However, logistic regression revealed only TNFα-308 polymorphic GA/AA genotypes (OR: 8.890, 95% CI: 2.15 to 36.7; P = .003) and smoking (OR: 6.2, 95% CI: 1.44 to 26.7; P = .014) as independent factors associated with increased peri-implantitis risk, while CD14-159 polymorphic CT/TT genotypes were associated with decreased risk for peri-implantitis (OR: 0.059, 95% CI: 0.009 to 0.355; P = .002). CONCLUSION: The findings suggest that smoking and the presence of TNFα-308 GA/AA genotypes may increase the risk for peri-implantitis, while CD14-159 polymorphic CT/TT genotypes decrease the risk. The results also indicate significant association of CD14-159, TNFα-308, and IL6-174 genotypes and clinical parameters in the Serbian population. However, future studies in larger patient groups are necessary to confirm these observations.

CD14 and TNFα single nucleotide polymorphisms are candidates for genetic biomarkers of peri-implantitis.

OBJECTIVES: This study aims to investigate whether CD14-159 C/T and TNFα -308 A/G single nucleotide polymorphisms are associated with peri-implantitis and to evaluate their effects on bone resorption by correlating with local levels of receptor activator nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). MATERIAL AND METHODS: Study population included 369 Southeastern Europe Caucasians (180 with peri-implantitis and 189 with healthy peri-implant tissues). Genotyping was performed using polymerase chain reaction from the periphery blood samples, while RANKL and OPG were evaluated in peri-implant crevicular fluid specimens using enzyme-linked immunosorbent assay. RESULTS: Analysis of CD14-159 C/T polymorphism showed that genotype of CC nucleic acid combination was associated with peri-implantitis demonstrating a fivefold increased risk in these carriers. Furthermore, for TNFα -308 A/G polymorphisms, it was evidenced that AG genotype was associated with peri-implantitis and a fivefold increased risk in these carriers. Peri-implantitis patients with CC genotype at CD14-159 exhibited significantly higher concentrations of RANKL and relative ratio RANKL/OPG when compared to patients with CT genotype, while concentration of biomarkers between different genotypes at TNFα -308 remained insignificant. CONCLUSION: Within the limitations of the study, we can conclude that CD14-159 C/T and TNFα -308 A/G polymorphisms are associated with peri-implantitis and may present biomarkers for peri-implantitis. CLINICAL RELEVANCE: Investigated genetic markers might serve as precious parameters in clinical practice in course of treatment planning and prognosis, since preventive and treatment approach could be positively shifted and adjusted depending on present genotype.

Estimation of bone loss biomarkers as a diagnostic tool for peri-implantitis.

BACKGROUND: The aims of this study are to estimate the profile of bone loss biomarkers in peri-implant tissues and to identify potential prognostic biomarkers of peri-implantitis. METHODS: Peri-implant crevicular fluid samples collected from 164 participants (52 patients with peri-implantitis, 54 with mucositis, and 58 with healthy peri-implant tissues) were analyzed using enzyme-linked immunosorbent assays to evaluate concentrations of the receptor activator of nuclear factor-κB (RANK), soluble RANK ligand (sRANKL), osteoprotegerin (OPG), cathepsin-K, and sclerostin. RESULTS: Concentrations of RANK, sRANKL, OPG, and sclerostin were significantly increased in patients with peri-implantitis compared with patients with healthy peri-implant tissues. Comparisons between peri-implantitis and mucositis demonstrated significantly higher values of sclerostin in peri-implantitis samples. Comparisons between mucositis and healthy peri-implant tissues showed significantly increased levels of RANK and cathepsin-K in mucositis. CONCLUSION: These results are suggestive of a role of sRANKL, OPG, and sclerostin as prognostic biomarkers in peri-implantitis.

Receptor activator of nuclear factor kappa B (RANK) as a determinant of peri-implantitis.

BACKGROUND/AIM: Peri-implantitis presents inflammatory process that affects soft and hard supporting tissues of osseointegrated implant based on inflammatory osteoclastogenesis. The aim of this study was to investigate whether receptor activator of nuclear factor kappa B (RANK) concentrations in peri-implant crevicular fluid could be associated with clinical parameters that reflect inflammatory nature of peri-implantitis. METHODS: The study included 67 patients, 22 with diagnosed peri-implantitis, 22 persons with healthy peri-implant tissues and 23 patients with periodontitis. Clinical parameters from each patient were recorded and samples of peri-implant/gingival crevicular fluid were collected for the enzyme-linked immunosorbent assay (ELISA) analysis. RESULTS: RANK concentration was significantly increased in samples from the patients with peri-implantitis when compared to healthy implants (p < 0.0001), where the average levels were 9 times higher. At the same time RANK concentration was significantly higher in peri-implantitis than in periodontitis sites (p < 0.0001). In implant patients pocket depths and bleeding on probing values were positively associated with high RANK concentrations (p < 0.0001). CONCLUSION: These results revealed association of increased RANK concentration in samples of peri-implant/gingival crevicular fluid with peri-implant inflammation and suggests that RANK could be a pathologic determinant of peri-implantitis, thereby a potential parameter in assessment of peri-implant tissue inflammation and a potential target in designing treatment strategies.

Bone loss biomarkers associated with peri-implantitis. A cross-sectional study.

AIM: To investigate the levels of biomarkers associated with osteoclastogenesis in patients suffering peri-implantitis and to compare them with levels in healthy peri-implant sites and severe chronic periodontitis. MATERIAL AND METHODS: Peri-implant/gingival crevicular fluid samples and clinical parameters including: bleeding on probing, modified Plaque Index (PlI), pocket depth and clinical attachment level were collected from 70 patients (23 with peri-implantitis, 25 with healthy peri-implant tissues and 22 with severe chronic periodontitis). The concentrations of sRANKL, RANK and OPG were evaluated using enzyme-linked immunosorbent assays; they were compared between the groups and correlated with the clinical findings. RESULTS: sRANKL (P = 0.01), RANK (P = 0.01) and OPG (P = 0.03) concentrations were significantly higher in peri-implantitis sites when compared to those in healthy implant sites, although differences in the sRANKL/OPG ratio were not statistically significant. In these sites all three markers were significantly correlated with the clinical parameters, with exception of OPG/PI correlation that remained insignificant (P = 0.121). When comparing peri-implantitis and periodontitis findings, RANK was significantly higher in peri-implantitis sites whereas, sRANKL (P = 0.03) and sRANKL/OPG ratio (P = 0.004) were significantly higher in periodontitis sites. Among periodontitis and healthy implant sites the same differences have been observed for both sRANKL (P = 0.000) and sRANKL/OPG ratio (P = 0.000), furthermore RANK was higher in periodontitis sites as well (P = 0.010). CONCLUSION: The findings of this preliminary study on a relatively small sample size suggest that the PICF levels of biomarkers sRANKL, RANK, and OPG are associated with peri-implant tissue destruction and the pattern of these biomarkers differed when compared to periodontitis.

Calcium and magnesium content in hard tissues of rats under condition of subchronic lead intoxication.

Lead manifests toxic effects in almost all organs and tissues, especially in: the nervous system, hematopoietic system, kidney and liver. This metal has a special affinity for deposition in hard tissue, i.e., bones and teeth. It is generally believed that the main mechanism of its toxicity relies on its interaction with bioelements, especially with Ca and Mg. This article analyses the influence of Pb poisoning on Ca and Mg content in hard tissues, (mandible, femur, teeth and skull) of female and young rats. Experiments were carried out on 60 female rats, AO breed, and on 80 of their young rats (offspring). Female rats were divided into three groups: the first one was a control group, the second one received 100 mg/kg Pb2+ kg b.wt. per day in drinking water, the third one received 30 mg/kg Pb(2+) kg b.wt. per day in drinking water. Young rats (offspring) were divided into the same respective three groups. Lead, calcium and magnesium content in hard tissues (mandible, femur, teeth-incisors and skull) was determined by flame atomic absorption spectrophotometry in mineralized samples. There was a statistically significant Pb deposition in all analyzed female and young rat hard tissues. Ca and Mg contents were significantly reduced in all female and young rat hard tissues. These results show that Pb poisoning causes a significant reduction in Ca and Mg content in animal hard tissues, which is probably the consequence of competitive antagonism between Pb and Ca and Mg.