Association between genetic polymorphisms in rank, rankl and opg and peri-implant diseases in patients from the Amazon Region

Abstract The present study evaluated polymorphisms in RANK, RANKL and OPG-encoding genes to assess whether they are associated with mucositis and peri-implantitis in a population from the Brazilian Amazon region. One hundred and fourteen patients with dental implants were included in the study. After clinical and radiographic examination, the sample was categorized into 4 groups, according to the peri-implant status: Healthy (n=71), Mucositis (n=30), Peri-implantitis (n=13) and Diseased (Mucositis + Peri-implantitis, n=43). Genomic DNA was extracted from buccal cells from saliva, and the genetic polymorphism in osteoprotegerin (OPG), Kappa nuclear factor activator receptor (RANKL) and nuclear kappa factor activator receptor (RANK) were genotyped by the real time PCR. Univariate and multivariate statistical analyses were performed to compare clinical variables among groups and to evaluate genotypes and alleles distributions and the established alpha was 5%. Age, peri-implant biotype, diabetes and presence of peri-implant biofilm were associated with mucositis (p<0.05) and peri-implantitis (p<0.05). Smoking, alcoholism, and periodontal biofilms were also associated with the presence of peri-implantitis (p<0.05). Univariate and multivariate analysis did not demonstrate an association of peri-implantitis or mucositis with any genetic polymorphism in RANK (rs3826620), RANKL (rs9594738) and OPG (rs2073618) (p>0.05). The studied genetic polymorphism in RANK, RANKL and OPG were not associated with mucositis and peri-implantitis in a Brazilian population from the Amazon region.

Resumo O presente estudo avaliou a associação da predisposição clínica e dos fatores genéticos com a presença de doenças peri-implantares. Cento e quatorze pacientes com implantes dentais instalados na Clínica de Especialização do Amazonas, Brazil, foram incluidos no estudo. Após exame clínico e radiográfico, a amostra foi categorizada em 4 grupos, de acordo com o Status peri-implantar: saúde (n=71), mucosite (n=30), peri-implantite (n=13) e doentes (mucosite + peri-implantite). DNA genômico foi extraído de células orais da saliva, e o polimorfismo genético em osteoprotegerina (OPG), ligante do receptor ativador do fator Kappa nuclear (RANKL) e receptor ativador do fator Kappa nuclear (RANK) foram genotipados por PCR em tempo real. O estudo se propôs a avaliar se os polimorfismos em RANK, RANKL e OPG estão envolvidos na patogênese da mucosite e da peri-implantite, e avaliar também a presença de fatores de risco moduladores da resposta em uma população brasileira. Idade, biotipo peri-implantar, diabetes e presença de biofilme peri-implantar foram associados a mucosite (p<0.05) e peri-implantite (p<0.05). Tabagismo, alcoolismo e biofilme periodontal também foram associados com a presença de peri-implantite (p<0.05). Análise univariada e multivariada não demonstraram associação de peri-implantite ou mucosite com os polimorfismos genéticos em RANK (rs3826620), RANKL (rs9594738) e OPG (rs2073618) (p>0.05). Os polimorfismos genéticos estudados não foram associados com mucosite e peri-implantite em uma população brasileira da região Amazônica.

Impact of history of periodontitis on gene expression of bone-related factors in young patients

Abstract Although dental implants and bone regenerative procedures are important approaches for the reestablishment of esthetics and function in young patients with a history of generalized aggressive periodontitis (GAP), no predictable outcomes have been reported, and the host osteo-immunoinflammatory response may play a relevant role in this context. In view of the lack of molecular investigations into the bone tissue condition of young patients with periodontitis, the aim of this study was to evaluate the gene expression of bone-related factors in this population. Bone biopsies were obtained from the posterior mandible in 16 individuals previously diagnosed with GAP and on periodontal support therapy and from 17 periodontally healthy (PH) patients. The gene expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OC), bone sialoprotein (BSP), and type I collagen (COL-I), important biomarkers of bone turnover, was evaluated by qRT-PCR. Lower TGF-β and OPG mRNA levels were observed in GAP patients compared to PH individuals (p ≤ 0.05). There were no between-group differences in levels of TNF-α, BSP, RANKL, OC, or COL-I mRNA (p>0.05). In young adults, a history of periodontal disease can negatively modulate the gene expression of important bone-related factors in alveolar bone tissue. These molecular outcomes may contribute to the future development of therapeutic approaches to benefit bone healing in young patients with history of periodontitis via modulation of osteo-immuno-inflammatory biomarkers.

Chronic Periodontitis and RANKL/OPG Ratio in Peri-Implant Mucosae Inflammation

Abstract tHistory of chronic periodontitis (CP) is a risk factor for oseointegration failure. The osteoclastogenesis system (RANK, RANKL and OPG) is critical for bone homeostatic control. We investigated the levels of OPG and RANKL in peri-implant tissues from volunteers with and without a history of CP and their association with mucosae inflammation. This is a single-blind case-contro study. Diagnosis of a history of CP and peri-implant examination was performed on 46 volunteers, divided into control (without history of CP, n=26) and CP group (with history of CP, n=20). Gingival biopsies were harvested during implant exposure. Quantitative PCR evaluated OPG/RANKL mRNA expressions. OPG and RANKL proteins were analyzed by western blot and immunohistochemistry assay. The chi-square test analyzed the significance of nominal variables between groups while continuous variables were analyzed by T-test or Mann-Whitney test, after Shapiro-Wilk test evaluation. The 2-ΔΔCT Livak method calculation evaluated the gene expression. Values of p<0.05 were considered statistically significant. Volunteers with CP history had 23 times higher chance of developing mucosae inflammation. High mucosae levels of RANKL (p=0.04) and RANKL/OPG (p=0.001) mRNA expressions were observed in CP group. CP volunteers showed increased RANKL protein levels in opposition to decreased OPG expression. Even without active periodontitis, volunteers with a history of CP had elevated gingival levels of RANKL/OPG and higher correlation with peri-implant mucosae inflammation and implant loss.

Resumo A história de periodontite crônica (CP) é um fator de risco para falhas na osseointegração. O sistema de osteoclastogênese (RANK, RANKL e OPG) é crucial para o controle da homeostase óssea. O objetivo deste estudo foi investigar os níveis de OPG e RANKL no tecido peri-implantar de voluntários com e sem histórico de CP e sua associação com inflamação da mucosa. Este é um estudo tipo caso-controle. O exame para diagnóstico de CP e na região peri-implantar foi realizado em 46 voluntários, divididos em controle (sem história CP, n=26) e grupo CP (com histórico de CP, n=20). Descartes gengivais foram obtidos durante a exposição do implante. PCR quantitativo avaliou a expressão do RNAm de OPG/RANKL. As proteínas OPG e RANKL foram analisadas por western blot e imunohistoquímica. O teste do qui-quadrado analisou a significância entre as variáveis nominais enquanto as variáveis contínuas foram analisadas pelo teste-t e Mann-Whitney, após o teste de Shapiro-wilk. O método do Livak 2--ΔΔCT avaliou a expressão gênica. Os voluntários com CP apresentaram 23 vezes mais chances de desenvolver inflamação da mucosa. Expressão elevada no RNAm de RANKL (p=0.04) e RANKL/OPG (p=0.001) foram observadas no grupo CP. Voluntários com CP mostraram aumento dos níveis da proteína RANKL em contraste com diminuída expressão de OPG. Mesmo sem periodontite ativa, voluntários com histórico de CP apresentaram elevado nível gengival de RANKL/OPG e alta correlação com inflamação peri-implantar e perda do implante.

RANK/RANKL/OPG Expression in Rapid Maxillary Expansion

Abstract The aim of this study was to evaluate osteoclastogenesis signaling in midpalatal suture after rapid maxillary expansion (RME) in rats. Thirty male Wistar rats were randomly assigned to two groups with 15 animals each: control (C) and RME group. RME was performed by inserting a 1.5-mm-thick circular metal ring between the maxillary incisors. The animals were euthanized at 3, 7 and 10 days after RME. qRT-PCR was used to evaluate expression of Tnfsf11 (RANKL), Tnfrsf11a (RANK) and Tnfrsf11b (OPG). Data were submitted to statistical analysis using two-way ANOVA followed by Tukey test (a=0.05). There was an upregulation of RANK and RANKL genes at 7 and 10 days and an upregulation of the OPG gene at 3 and 7 days of healing. Interestingly, an increased in expression of all genes was observed over time in both RME and C groups. The RANKL/OPG ratio showed an increased signaling favoring bone resorption on RME compared to C at 3 and 7 days. Signaling against bone resorption was observed, as well as an upregulation of OPG gene expression in RME group, compared to C group at 10 days. The results of this study concluded that the RANK, RANK-L and OPG system participates in bone remodeling after RME.

Resumo O objetivo deste estudo foi avaliar a sinalização osteoclastogenese na sutura palatina após a expansão rápida da maxila (ERM) em ratos. Um total de 30 ratos Wistar machos foram divididos aleatoriamente em dois grupos com 15 animais cada: controle (C) e grupo ERM. ERM foi realizada através da inserção de um anel de metal circular de 1,5 mm de espessura entre os incisivos superiores. Os animais foram sacrificados aos 3, 7 e 10 dias após a RME. qRT-PCR foi utilizado para avaliar a expressão de Tnfsf11 (RANKL), Tnfrsf11a (RANK) e TNFRSF11b (OPG). Os dados foram submetidos à análise de variância de duas vias, seguido pelo teste de Tukey (a=0,05). Houve uma regulação positiva de genes RANK e RANKL aos 7 e 10 dias e uma regulação positiva do gene OPG aos 3 e 7 dias de tratamento. Curiosamente, foi observado um aumento na expressão de todos os genes ao longo do tempo nos grupos ERM e C. O RANKL/OPG mostrou um aumento na sinalização favorecendo a reabsorção óssea no ERM em comparação com o C nos períodos de 3 e 7 dias. Foi observada uma sinalização contra a reabsorção óssea, assim como, uma regulação favorável da expressão do gene OPG no grupo ERM, comparado ao grupo C aos 10 dias. Os resultados deste estudo permitem concluir que o sistema RANK, RANK-L e OPG participa de remodelação óssea após a ERM.

Hepatic Osteodystrophy: The Mechanism of Bone Loss in Hepatocellular Disease and the Effects of Pamidronate Treatment

OBJECTIVES: The present study was designed to evaluate the bone phenotypes and mechanisms involved in bone disorders associated with hepatic osteodystrophy. Hepatocellular disease was induced by carbon tetrachloride (CCl4). In addition, the effects of disodium pamidronate on bone tissue were evaluated. METHODS: The study included 4 groups of 15 mice: a) C = mice subjected to vehicle injections; b) C+P = mice subjected to vehicle and pamidronate injections; c) CCl4+V = mice subjected to CCl4 and vehicle injections; and d) CCl4+P = mice subjected to CCl4 and pamidronate injections. CCl4 or vehicle was administered for 8 weeks, while pamidronate or vehicle was injected at the end of the fourth week. Bone histomorphometry and biomechanical analysis were performed in tibiae, while femora were used for micro-computed tomography and gene expression. RESULTS: CCl4 mice exhibited decreased bone volume/trabecular volume and trabecular numbers, as well as increased trabecular separation, as determined by bone histomorphometry and micro-computed tomography, but these changes were not detected in the group treated with pamidronate. CCl4 mice showed increased numbers of osteoclasts and resorption surface. High serum levels of receptor activator of nuclear factor-κB ligand and the increased expression of tartrate-resistant acid phosphatase in the bones of CCl4 mice supported the enhancement of bone resorption in these mice. CONCLUSION: Taken together, these results suggest that bone resorption is the main mechanism of bone loss in chronic hepatocellular disease in mice.

Effect of triiodothyronine on the maxilla and masseter muscles of the rat stomatognathic system

The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9 percent) for 15 days. Thyroidectomy increased OPG (~40 percent) and OPN (~75 percent) mRNA expression, while T3 treatment reduced OPG (~40 percent) and OPN (~75 percent) in Tx, and both (~50 percent) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.

Effects of low-level laser therapy on human osteoblastic cells grown on titanium

The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.

Este estudo teve como objetivo investigar o efeito do laser diodo de gálio-alumínio-arsênio (GaAlAs) em células osteoblásticas humanas cultivadas sobre discos de Ti. Para tanto, células osteoblásticas foram obtidas por digestão enzimática de osso alveolar humano e cultivadas sobre discos de Ti por 17 dias. As células foram submetidas à irradiação no 3º e 7º dias na dose de 3 J/cm2 e comprimento de onda de 780 nm e células não irradiadas foram usadas como controle. A irradiação não alterou a proliferação celular, atividade de ALP e formação de matriz mineralizada. Microscopia por epifluorescência indicou que após 24 h da aplicação do laser, as culturas irradiadas apresentaram áreas sem células, que mais tarde foram repovoadas por células em fase de proliferação e menos diferenciadas. O laser aumentou a expressão gênica relativa da ALP, OC, BSP e BMP-7 e reduziu a de RUNX2, OPN e OPG. Os resultados indicam que a terapia com laser modula de forma complexa as respostas celulares, estimulando a diferenciação osteoblástica. Assim, é possível sugerir possíveis benefícios do laser na osseointegração de implantes de Ti apesar do efeito deletério às células imediatamente após a irradiação.

Hip fracture risk and different gene polymorphisms in the turkish population

BACKGROUND: We aimed to discuss the risk assessments for both patients with hip fractures due to fall-related, low energy traumas and non-fractured control patients by examining bone mineral density and genetic data, two features associated with femoral strength and hip fracture risk. METHODS: Twenty-one osteoporotic patients with proximal femur fractures and non-fractured, osteoporotic, age- and gender-matched controls were included in the study. Bone mineral density measurements were performed with a Lunar DXA. The COL1A1, ESR, VDR, IL-6, and OPG genes were amplified, and labeling of specific gene sequences was performed in a multiplex polymerase chain reaction using the osteo/check PCR kit from the whole blood of all subjects. RESULTS: The bone mineral density (trochanteric and total bone mineral density values) of the fracture group was significantly decreased relative to the control group. We were not able to conduct statistical tests for the polymorphisms of the COL1A1, ESR, and VDR genes because our results were expressed in terms of frequency. Although they were not significant, we did examine differences in the IL-6 and OPG genes polymorphisms between the two groups. We concluded that increasing the number of cases will allow us to evaluate racial differences in femoral hip fracture risk by genotypes.

Expression and Regulation of Osteoprotegerin in Adipose Tissue

PURPOSE: Osteoprotegerin (OPG), a potent inhibitor of osteoclastic bone resorption, has a variety of biological functions that include anti-inflammatory effects. Adipocytes and osteoblasts share a common origin, and the formation of new blood vessels often precedes adipogenesis in developing adipose tissue microvasculature. We examined whether OPG is secreted from adipocytes, therefore contributing to the prevention of neovascularization and protecting the vessels from intimal inflammation and medial calcification. MATERIALS AND METHODS: The mRNA expression of OPG and receptor activator of NF-kappaB ligand (RANKL) was measured in differentiated 3T3L1 adipocytes and adipose tissues. RESULTS: OPG mRNA expression increased with the differentiation of 3T3L1 adipocytes, while RANKL expression was not significantly altered. OPG mRNA was expressed at higher levels in white adipose tissue than in brown adipose tissue and was most abundant in the epididymal portion. In differentiated 3T3L1 adipocytes, Rosiglitazone and insulin reduced the OPG/RANKL expression ratio in a dose- and time- dependent manner. In contrast, tumor necrosis factor-alpha (TNF-alpha) increased the expression of both OPG and RANKL in a time-dependent manner. The OPG/RANKL ratio was at a maximum two hours after TNF-alpha treatment and then returned to control levels. Furthermore, OPG was abundantly secreted into the media after transfection of OPG cDNA with Phi C31 integrase into 3T3L1 cells. CONCLUSION: Our results indicate that OPG mRNA is expressed and regulated in the adipose tissue. Considering the role of OPG in obesity-associated inflammatory changes in adipose tissue and vessels, we speculate that OPG may have both a protective function against inflammation and anti-angiogenic effects on adipose tissue.