Avaliação clínica, morfológica e molecular do carcinoma epidermoide oral com envolvimento mandibular / Clinical, morphological and molecular evaluation of oral squamous cell carcinoma with mandibular invasion

O carcinoma epidermóide (CEC) é a neoplasia maligna mais frequente em cavidade oral, representando aproximadamente 90% de todos os cânceres da boca. Esta neoplasia tem origem no epitélio de revestimento bucal, e a depender do subtipo e da sua localização, pode apresentar tendência marcante de invasão do tecido ósseo adjacente. Existe uma interação entre osso e neoplasia, e atenção principal deve ser dada aos reguladores relacionados à osteoclastogênese. As moléculas RANK/RANKL/OPG são fundamentais neste processo, e juntamente com a IL-6, promovem formação e ativação de osteoclastos, com consequente promoção da invasão mandibular. Neste sentido, o objetivo do estudo foi avaliar as características clínicas, radiográficas e histopatológicas dos pacientes submetidos a mandibulectomia para tratamento do carcinoma epidermóide oral, e correlacionar/associar com a expressão gênica na neoplasia de marcadores reguladores da invasão óssea. Foi realizado um estudo retrospectivo de pacientes tratados com algum tipo de mandibulectomia no A.C. Camargo Cancer Center durante o período de1990 a 2015. Um total de 220 casos foi selecionado, e destes, foram extraídos 40 casos, posteriormente submetidos as etapas de análise molecular. A análise molecular foi realizada por PCR quantitativa em tempo real (qPCR), por meio da mensuração de mRNA para os genes estudados, calibrados pelo gene endógeno GAPDH e por um pool de controle composto por RNA não neoplásico. Dos 220 pacientes incluídos na pesquisa, 161 eram do sexo masculino, a idade média foi de 60 anos, e 53,6% eram etilistas e 66,8% tabagistas. A invasão óssea da mandíbula foi observada através do laudo anatomopatológico em 77 pacientes, e foi associada à presença de margens comprometidas/exíguas e a localização da lesão (gengiva inferior, rebordo e região retro-molar). Ao final do estudo, 127 pacientes foram a óbito, e a média de sobrevida foi de 96,92 meses. Parâmetros clínicos, dos exames de imagem e histopatológicos foram significantemente associados com a sobrevida global, principalmente - tamanho do tumor, infiltração perineural, invasão óssea (imagem e anatomopatológico). Na análise multivariada, apenas invasão óssea e infiltração perineural aumentaram risco de óbito nos pacientes. Na análise molecular de expressão gênica foi possível avaliar que das 36 amostras do gene RANK, 16 eram downreguladas. Para RANKL, das 37 amostras, 28 estavam super expressas. Das 35 do gene da IL-6, 19 amostras eram downreguladas. E por fim, das 37 amostras do gene da OPG, 16 tinham expressão normal. A avaliação de associação entre a expressão dos genes analisados demostrou que o gene da IL-6 está associado a presença de invasão óssea, e que existe uma associação significativa entre as expressões do RANK, RANKL, OPG e IL-6. Maior sobrevida dos pacientes foi associada significativamente a uma expressão mais baixa de IL-6 (108,5 meses). Conclui-se que a invasão mandibular interfere na sobrevida global dos pacientes, e que este mecanismo de invasão óssea está associado com a expressão gênica dos genes RANK, RANKL, OPG e IL-6. A IL-6 downregulada nas células malignas está associada com maior sobrevida

Oral Squamous Cell Carcinoma (OSCC) is the most frequent malignant neoplasm in the oral cavity, representing 90% of all oral cancers. The tumor usually arises within the epithelium lining of the oral cavity, and in cases of certain subtype or location may reveal a tendency for invasion of the surrounding bone tissue. There is a relation between tumor and bone, and more attention should be given to the events related to osteoclastogenesis. The molecules RANK/RANKL/OPG are crucial in this process, and together with IL-6, promotes the formation and activation of osteoclasts, with influence on the mandibular invasion process. In this way, the aim of this study was to evaluate the clinical, demographic, imaging, and histopathology data of patients diagnosed with OSCC treated by mandibulectomy. Also, analyze the gene expression condition of the genes RANK, RANKL, OPG, and IL-6 on the tumor. A retrospective study was conducted on patients treated with mandibulectomy at the A.C. Camargo Cancer Center, between 1990 and 2015. Two hundred and twenty cases were retrieved, and 40 of them were selected for gene expression analysis. The mRNA expressions of the selected genes were determined by real-time polymerase chain reaction, extracted from the tumor, and calibrated by endogenous GAPDH and a control pool of non-neoplastic RNA. Among 220 patients, 161 were male, with a mean age of 60 years. 53.6% were alcohol users and 66.8% tobacco users. Mandible bone invasion was reported on 77 histopathology reports and associated with compromised margins and the site of the lesion (gingiva, alveolar ridge, and retromolar region). One hundred and twenty-seven patients died of the disease, and the mean overall survival rate was 96.92 months. Overall survival was associated with clinical, imaging, and pathological parameters, especially, tumor size, perineural growth, and bone invasion (both imaging and pathologic). Multivariate analysis revealed higher risk of death only to bone invasion and perineural growth. Gene expression analysis revealed - 16 downregulation of 36 RANK samples evaluated; 28 overexpression of 37 RANKL samples evaluated; 19 downregulation of 35 IL-6 analyzed, and 16 normal expression of 37 OPG samples evaluated. Association analysis revealed that IL-6 expression is associated with bone invasion, and there is also a significant association among the expression of RANK, RANKL, OPG, and IL-6. Higher overall survival rate was associated with an IL-6 down expression (108;5 months). In conclusion, mandibular invasion interferes with the patient's overall survivall rate, and the bone invasion mechanism is associated with gene expression of RANK, RANKL, OPG, and IL-6 on the tumor. IL-6 in downregulation expressed by malignant cells is associated with higher overall survivor rates

Advances in molecular mechanisms of bone invasion by oral cancer / 华西口腔医学杂志

Bone invasion by oral cancer is a common clinical problem, which affects the choice of treatment and predicts a poor prognosis. Unfortunately, the molecular mechanism of this phenomenon has not been fully elucidated. Current studies have revealed that oral cancer cells modulate the formation and function of osteoclasts through the expression of a series of signal molecules. Many signal pathways are involved in this process, of which receptor activator of nuclear factor-κB ligand/receptor activator of nuclear factor-κB/osteoprotegerin signaling pathway attracted much attention. In this review, we introduce recent progress in molecular mechanisms of bone invasion by oral cancer.

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.

Effects of advanced glycation end products on osteoclasts at different stages of differentiation / 南方医科大学学报

OBJECTIVE@#To explore the effect of advanced glycation end products (AGEs) on osteoclasts at different stages of differentiation.@*METHODS@#Raw264.7 cells cultured were induced for osteoclastogenesis using RANKL, and the stages of differentiation of the osteoclasts were determined with TRAP staining. The cells were then randomly divided into control group, early-stage AGEs intervention group and late-stage AGEs intervention group. The viability of the cells after AGEs treatment was assessed using CCK-8 method. The cells were examined after the induction for osteoclastogenesis using TRAP staining, and the expression levels of RANK, NFATC-1, TRAF-6, TRAP and CTSK mRNAs were tested with RT-PCR; the expressions of CTSK and RANK proteins were detected using Western boltting.@*RESULTS@#We defined the initial 3 days of induction as the early stage of differentiation and the time beyond 3 days as the late stage of differentiation of Raw264.7 cells. Intervention with AGEs at 100 mg/L produced no significant effects on the viability of the cells, but AGEs suppressed the cell proliferation at a concentration exceeding 100 mg/L. The number of osteolasts in the early- and late-stage intervention groups was greater than that in the control group, but the cell count differed significantly only between the early-stage intervention group and control group ( < 0.05). The gene expressions of RANK, NFATC-1, TRAF-6, TRAP and CTSK all increased after the application of AGEs in both the early and late stages of differentiation, but the changes were significant only in the early-stage intervention group ( < 0.05). The changes in CTSK and RANK protein expressions were consistent with their mRNA expressions.@*CONCLUSIONS@#AGEs can affect the differentiation of osteoclasts differently when applied at different stages, and intervention with AGEs at the early stage produces stronger effect to promote osteoclast differentiation than its application at a late stage.

Periodontal disease, peri-implant disease and levels of salivary biomarkers IL-1ß, IL-10, RANK, OPG, MMP-2, TGF-ß and TNF-α: follow-up over 5 years

Abstract Objective The aim of this study was to evaluate the levels of salivary biomarkers IL-1β, IL-10, RANK, OPG, MMP-2, TG-β and TNF-α in individuals with diagnosis of peri-implant mucositis in the absence or presence of periodontal and peri-implant maintenance therapy (TMPP) over 5 years. Material and Methods Eighty individuals diagnosed with peri-implant mucositis were divided into two groups: one group that underwent periodontal and peri-implant regularly maintenance therapy, called GTP (n=39), and a second group that received no regular maintenance GNTP (n=41). Each participant underwent a complete periodontal and peri-implant clinical examination. Collection of saliva samples and radiographic examination to evaluate peri-implant bone levels were conducted at two times: initial examination (T1) and after 5 years (T2). The salivary samples were evaluated through ELISA for the following markers: IL-1β, IL-10, RANK, OPG, MMP-2, TGF and TNF-α. Results A higher incidence of peri-implantitis was observed in the GNTP group (43.9%) than in the GTP group (18%) (p=0.000). All individuals (n=12) who presented peri-implant mucositis and had resolution at T2 were in the GTP group. After 5 years, there was an increase in the incidence of periodontitis in the GNTP group compared to the GTP group (p=0.001). The results of the study revealed an increase in the salivary concentration of TNF-α in the GNTP group compared to the GTP group. The other salivary biomarkers that were evaluated did not show statistically significant differences between the two groups. Conclusions The salivary concentration of TNF-α was increased in individuals with worse periodontal and peri-implant clinical condition and in those with a higher incidence of peri-implantitis, especially in the GNTP group. Longitudinal studies in larger populations are needed to confirm these findings and elucidate the role of this biomarker in peri-implant disease.

Gliclazide reduced oxidative stress, inflammation, and bone loss in an experimental periodontal disease model

Abstract Objective The aim of this study was to evaluate the effects of gliclazide on oxidative stress, inflammation, and bone loss in an experimental periodontal disease model. Material and Methods Male albino Wistar rats were divided into no ligature, ligature, and ligature with 1, 5, and 10 mg/kg gliclazide groups. Maxillae were fixed and scanned using micro-computed tomography to quantify linear and bone volume/tissue volume (BV/TV) and volumetric bone loss. Histopathological, immunohistochemical and immunofluorescence analyses were conducted to examine matrix metalloproteinase-2 (MMP-2), cyclooxygenase 2 (COX-2), cathepsin K, members of the receptor activator of the nuclear factor kappa-Β ligand (RANKL), receptor activator of nuclear factor kappa-Β (RANK), osteoprotegerin (OPG) pathway, macrophage migration inhibitory factor (MIF), superoxide dismutase-1 (SOD-1), glutathione peroxidase-1 (GPx-1), NFKB p 50 (Cytoplasm), NFKB p50 NLS (nuclear localization signal), PI3 kinase and AKT staining. Myeloperoxidase activity, malondialdehyde and glutathione levels, while interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels were evaluated by spectroscopic ultraviolet-visible analysis. A quantitative reverse transcription polymerase chain reaction was used to quantify the gene expression of the nuclear factor kappa B p50 subunit (NF-κB p50), phosphoinositide 3-kinase (PI3k), protein kinase B (AKT), and F4/80. Results Micro-computed tomography showed that the 1 mg/kg gliclazide treatment reduced linear bone loss compared to the ligature, 5 mg/kg gliclazide, and 10 mg/kg gliclazide treatments. All concentrations of gliclazide increased bone volume/tissue volume (BV/TV) compared to the ligature group. Treatment with 1 mg/kg gliclazide reduced myeloperoxidase activity, malondialdehyde, IL-1β, and TNF-α levels (p≤0.05), and resulted in weak staining for COX-2, cathepsin k, MMP-2, RANK, RANKL, SOD-1, GPx-1,MIF and PI3k. In addition, down-regulation of NF-κB p50, PI3k, AKT, and F4/80 were observed, and OPG staining was strong after the 1 mg/kg gliclazide treatment. Conclusions This treatment decreased neutrophil and macrophage migration, decreased the inflammatory response, and decreased bone loss in rats with ligature-induced periodontitis.

Effects of the cathepsin K inhibitor with mineral trioxide aggregate cements on osteoclastic activity

OBJECTIVES: Root resorption is an unexpected complication after replantation procedures. Combining anti-osteoclastic medicaments with retrograde root filling materials may avert this resorptive activity. The purpose of this study was to assess effects of a cathepsin K inhibitor with calcium silicate-based cements on osteoclastic activity. METHODS: MC3T3-E1 cells were cultured for biocompatibility analyses. RAW 264.7 cells were cultured in the presence of the receptor activator of nuclear factor-kappa B and lipopolysaccharide, followed by treatment with Biodentine (BIOD) or ProRoot MTA with or without medicaments (Odanacatib [ODN], a cathepsin inhibitor and alendronate, a bisphosphonate). After drug treatment, the cell counting kit-8 assay and Alizarin red staining were performed to evaluate biocompatibility in MC3T3-E1 cells. Reverse-transcription polymerase chain reaction, tartrate-resistant acid phosphatase (TRAP) staining and enzyme-linked immunosorbent assays were performed in RAW 264.7 cells to determine the expression levels of inflammatory cytokines, interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2). Data were analyzed by one-way analysis of variance and Tukey's post hoc test (p < 0.05). RESULTS: Biocompatibility results showed that there were no significant differences among any of the groups. RAW 264.7 cells treated with BIOD and ODN showed the lowest levels of TNF-α and PGE2. Treatments with BIOD + ODN were more potent suppressors of inflammatory cytokine expression (p < 0.05). CONCLUSION: The cathepsin K inhibitor with calcium silicate-based cement inhibits osteoclastic activity. This may have clinical application in preventing inflammatory root resorption in replanted teeth.

Tetrabromobisphenol A Promotes the Osteoclastogenesis of RAW264.7 Cells Induced by Receptor Activator of NF-kappa B Ligand In Vitro

BACKGROUND: Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame-retardants, is a representative persistent organic pollutants group. Studies on TBBPA toxicity have been conducted using various target cells; however, few studies have investigated TBBPA toxicity in bone cells. Therefore, this study investigated the in vitro effects of TBBPA on osteoclasts, a cell type involved in bone metabolism. METHODS: RAW264.7 cells were cultured in medium containing 50 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL) and varying concentrations of TBBPA. To evaluate the effects of TBBPA on the differentiation and function of osteoclasts, osteoclast-specific gene expression, tartrate-resistant acid phosphatase (TRAP) activity, bone resorbing activity, mitochondrial membrane potential (MMP) and mitochondrial superoxide were measured. RESULTS: The presence of 20 μM TBBPA significantly increased TRAP activity in RANKL-stimulated RAW264.7 cells, the bone resorbing activity of osteoclasts, and the gene expression of Akt2, nuclear factor of activated T-cells cytoplasmic 1, and chloride channel voltage-sensitive 7. However, TBBPA treatment caused no change in the expression of carbonic anhydrase II, cathepsin K, osteopetrosis-associated transmembrane protein 1, Src, extracellular signal-related kinase, GAB2, c-Fos, or matrix metalloproteinase 9. Furthermore, 20 μM TBBPA caused a significant decrease in MMP and a significant increase in mitochondrial superoxide production. CONCLUSION: This study suggests that TBBPA promotes osteoclast differentiation and activity. The mechanism of TBBPA-stimulated osteoclastogenesis might include increased expression of several genes involved in osteoclast differentiation and reactive oxygen species production.

Effect of risperidone on proliferation and apoptosis of MC3T3-E1 cells

This aim of this study was to assess the molecular mechanism of osteoporosis in schizophrenia patients with risperidone use. Here, we investigated the effects of risperidone on cellular proliferation and apoptosis of a preosteoblast cell line, MC3T3-E1. Cell viability and apoptotic rate of MC3T3-E1 were detected by cell counting kit-8 and flow cytometry at a serial dose of risperidone and at different time points, respectively. Bone transformation relevant gene serum osteocalcin (BGP), collagen 1, tumor necrosis factor-α (TNF-α), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels were determined by real-time PCR (qPCR). Their protein expression patterns were evaluated using western blot. The results revealed that risperidone dramatically inhibited MC3T3-E1 cell proliferation in a dose-dependent manner. It also significantly induced MC3T3-E1 cell apoptosis. TNF-α gene and protein levels were greatly enhanced after risperidone treatment. In contrast, BGP, collagen 1, OPG, and RANKL gene and protein levels were markedly downregulated. Our study indicated that risperidone suppressed MC3T3-E1 cell proliferation and induced apoptosis. It also regulated BGP gene and protein expression.

Determination of the optimal diabetes duration for bone regeneration experiments in an alloxan-induced diabetic rabbit calvarial defect model

PURPOSE: The purpose of this study was to evaluate the optimal diabetes duration for bone regeneration experiments in an alloxan monohydrate (ALX)–induced diabetic rabbit calvarial defect model by evaluating the association between diabetes duration and bone healing capacity. METHODS: Twenty-four New Zealand white rabbits were used. Twenty-two rabbits were injected with 100 mg/kg of ALX to induce experimental diabetes. These rabbits were divided into 4 groups, including a control group and groups with diabetes durations of 1 week (group 1), 2 weeks (group 2), and 4 weeks (group 3). Calvarial defects were created at 1, 2, and 4 weeks after ALX injection and in the control rabbits. Cone-beam computed tomography (CBCT) scanning was performed on the day of surgery and at 2 and 4 weeks after surgery. The rabbits were sacrificed 4 weeks after surgery, followed by histological and immunofluorescence analysis. RESULTS: The diabetic state of all diabetic rabbits was well-maintained throughout the experiment. Reconstructed 3-dimensional CBCT imaging showed more rapid and prominent bone regeneration in the control group than in the experimental groups. Histological staining showed notable bone regeneration in the control group, in contrast to scarce bone formation in the experimental groups. The appearance and immunoreactivity of receptor activator of nuclear factor-kappa B and osteoprotegerin did not show notable differences among the groups. CONCLUSION: ALX administration at 100 mg/kg successfully induced experimental diabetes in rabbits. The effect of diabetes on bone healing was evident when the interval between diabetes induction and the intervention was ≥1 week.

Establish mouse osteoblast -osteoclast cell co-culture system in a Transwell chamber / 中国骨伤

<p><b>OBJECTIVE</b>To establish osteoblast-osteoclast cell co-culture system in a Transwell chamber, and detect cell viability of osteoblasts and osteoclasts in system.</p><p><b>METHODS</b>Osteoblast MC3T3-E1 and mouse monocytes RAW264.7 were cultivated in vitro. RANKL-induced mouse RAW264.7 monocytes differentiated into mature osteoclasts, osteoblast-osteoclast cell co-culture system was established in Transwell chamber. Cell activity of osteoblasts and osteoclasts were detected by CCK-8 experimenting, Alizarin Red staining, TRAP staining. The expression of OPG, ALP, RANKL, TGF-b1 gene and RANKL protein in osteoblast MC3T3-E1 were detected by PCR, Western-Blot methods. Also, the expression of RANK, NF-κB in gene and protein level in osteoclast were measured through the same method respectively.</p><p><b>RESULTS</b>The co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell were established in Transwell chamber. Co-culture system affected cell division activities of osteoblasts and osteoclasts. Differentiation of osteoblasts were increased, while differentiation of osteoclast division were slight decreased under microscope observation. OPG (0.65±0.08) and ALP (0.16±0.01) gene expression of co-culture system were less than single culture OPG(1.00±0.08) and ALP (1.01±0.16); TGF-b1(4.42±0.21) and RANKL(4.12±1.04) of osteoblasts in co-culture system were higher than TGF-b1(1.00±0.10) and RANKL(1.00±0.09) under single culture. However, gene expression of RANK(0.63±0.06) and NF-κB(0.64±0.08) in co-culture system were decreased than RANK(1.00±0.08) and NF-κB(1.00±0.09), in single culture, and had significant differences. Similarly, protein expression of OPG(0.43±0.05) and NF-κB(0.59±0.05) of co-culture system were less than OPG(0.84±0.06) and NF-κB(1.13±0.03) of single culture. While RANKL protein expression (0.54±0.03)of co-culture system was more than single culture RANKL(0.31±0.03), and had statistically differences, which was in agreement of the trend of gene expression change.</p><p><b>CONCLUSIONS</b>Co-culture system of mouse MC3T3-E1 cells and RAW264.7 cell was viable in Transwell chamber, and the activity of osteoblasts is higher than osteoclasts in co-culture system.</p>

Evolution of Periodontal Disease: Immune Response and RANK/RANKL/OPG System

Abstract The aim of this study was to evaluate markers of bone loss and immune response present in evolution of periodontal disease. One hundred and two Wistar rats were divided into three animals groups: PD0, without ligation and PD15 days and PD60 days, submitted to ligation placement with a sterile 3-0 silk cord in the cervical region of the upper first molar on both sides. Samples were obtained from the gingival tissue for histomorphometric analysis, immunohistochemical analysis of RANK, RANKL, OPG, characterization of the inflammatory infiltrate, quantification of nitric oxide, MCP-1, RANTES, IP10 chemokines, and expression of the TGF-b1, VEG, and bFGF. The number of inflammatory cells in gingival tissue was higher in PD60 samples. The collagen content and the area occupied by birefringent collagen fibers were lower for PD60. Differential leukocyte counting showed that there was a significantly higher polymorphonuclear influx in group PD15, while PD60 showed a greater number of lymphocytes. PD60 showed higher RANTES, IP-10, MCP-1 gene transcripts, as well as a higher nitric oxide concentration. Clinical evaluation revealed that the PD60 group presented an increase in furcal area. In conclusion, in this animal model the increase of RANK/RANKL and HGF markers is related to a specific immune response, and probably contributed to the evolution of periodontal disease. Investigating the effect of these biomarkers can help in targeted therapy for bone resorption, since blocking these can inhibit bone loss.

Resumo Este estudo avaliou marcadores de perda óssea e da resposta imune presentes na evolução da doença periodontal. Cento e dois ratos Wistar foram divididos em três grupos de animais: PD0, sem ligadura e PD15 dias e PD60 dias, submetidos a colocação de ligadura com um fio de seda estéril 3-0 na região cervical do primeiro molar superior em ambos os lados. Foram obtidas amostras de tecido gengival para análise histomorfométrica, análises imunohistoquímicas de RANK, RANKL, OPG, caracterização do infiltrado inflamatório, quantificação de óxido nítrico, expressão de quimiocinas MCP-1, RANTES, IP10 e do TGF-b1, VEGF e bFGF . O número de células inflamatórias no tecido gengival foi maior nas amostras PD60. O teor de colágeno na área ocupada pelas fibras de colágeno birrefringentes foram menores para PD60. A contagem diferencial de leucócitos mostrou que houve um influxo polimorfonuclear significativamente maior no grupo PD15, enquanto que PD60 mostrou número maior de linfócitos. PD60 apresentou transcritos de genes RANTES, IP-10, MCP-1 mais elevados, bem como uma maior concentração de óxido nítrico. A avaliação clínica revelou que o grupo PD60 apresentou aumento da área óssea exposta na região da furca. Em conclusão, neste modelo animal o aumento dos marcadores RANK/RANKL e HGF está relacionado a uma resposta imunológica específica e provavelmente contribuiu para a evolução da doença periodontal. Investigar o efeito destes biomarcadores pode ajudar na terapia dirigida para a reabsorção óssea, uma vez que bloquear estes pode inibir a perda óssea.

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.

Cervical pessary in prevention of preterm birth: A case series / Philippine Journal of Obstetrics and Gynecology

Preterm birth defined as birth between 20-37 weeks age of gestation, poses major concerns as it causes serious health problems. Across 184 countries, the rate of preterm birth ranges from 5% to 18% of babies born and the Philippines ranks 8th out of 184 countries for the number of babies born prematurely, and ranks 17th for the total number of deaths due to complications from preterm birth. Management of incompetent cervix as one of the causes of preterm birth is cerclage. However, pessary insertion is an alternative especially in cases where cerclage may not be employed. To date, there have been no local published reports on effectiveness of pessary in prevention of preterm birth. Hence this study aims to report on cases supporting the use of pessary in preterm birth. This is a case series of three patients with short functional cervical lengths (

Effects of paeonol on the function of bone marrow-derived macrophage from Porphyromonas gingivalis-induced mice / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This work aims to examine the effects of paeonol treatment on the ability of bone marrow-derived macrophage (BMM) to excrete inflammatory factors and to differentiate into osteoclasts upon induction with Porphyromonas gingivalis (P. gingivalis). This work also aims to investigate the underlying mechanisms of these abilities.</p><p><b>METHODS</b>BMM culture was treated with different paeonol concentrations at for 1 h and then stimulated with P. gingivalis for 24 h before programmed death-ligand 1 (PD-L1) was quantified with flow cytometry. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). The BMM culture was treated with the receptor activator for nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), and then with paeonol for 1 h prior to induction with P. gingivalis. Then, osteoclast formation was assessed using tartrate resistant acid phosphatase (TRAP) staining. The osteoclast-related proteins TRAP and receptor activator of nuclear factor-κB (RANK) were quantified by Western blotting.</p><p><b>RESULTS</b>Paeonol was nontoxic to BMM within a range of 10-50 μmol·L⁻¹. Flow cytometry showed that paeonol inhibited PD-L1 expression in P. gingivalis-induced BMM in a dose-dependent manner. ELISA indicated that paeonol dose-dependently inhibited the excretion of TNF-α, IL-1β, and IL-6 by P. gingivalis-induced BMM (P<0.01). TRAP staining revealed that paenol treatment inhibited the differentiation of P. gingivalis-induced BMM into osteoclasts. Western blot results suggested that paeonol decreased the expression of TRAP and RANK in BMM.</p><p><b>CONCLUSIONS</b>Paeonol dose-dependently inhibited the excretion of the inflammatory factors TNF-α, IL-1β, and IL-6 by P. gingivalis-induced BMM in a dose-dependent manner. Moreover, paenol treatment prevented the differentiation of P. gingivalis-induced BMM differentiation into osteoclasts.
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Cervical pessary in prevention of preterm birth: A case series / Philippine Journal of Obstetrics and Gynecology

Preterm birth defined as birth between 20-37 weeks age of gestation, poses major concerns as it causes serious health problems. Across 184 countries, the rate of preterm birth ranges from 5% to 18% of babies born and the Philippines ranks 8th out of 184 countries for the number of babies born prematurely, and ranks 17th for the total number of deaths due to complications from preterm birth. Management of incompetent cervix as one of the causes of preterm birth is cerclage. However, pessary insertion is an alternative especially in cases where cerclage may not be employed. To date, there have been no local published reports on effectiveness of pessary in prevention of preterm birth. Hence this study aims to report on cases supporting the use of pessary in preterm birth. This is a case series of three patients with short functional cervical lengths (

Different engagement of TLR2 and TLR4 in Porphyromonas gingivalis vs. ligature-induced periodontal bone loss

Abstract This study was conducted to investigate the roles of different Toll-like receptor (TLR) signaling in Porphyromonas gingivalis (P. gingivalis)-induced and ligature-induced experimental periodontal bone resorption in mice. Wild-type (WT), TLR2 knockout (KO), TLR4KO, and TLR2&4 KO mice with C57/BL6 background were divided into three groups: control, P. gingivalis infection, and ligation. Live P. gingivalis or silk ligatures were placed in the sulcus around maxillary second molars over a 2-week period. Images were captured by digital stereomicroscopy, and the bone resorption area was measured with ImageJ software. The protein expression level of gingival RANKL was measured by ELISA. The gingival mRNA levels of RANKL, IL-1β, TNF-α, and IL-10 were detected by RT-qPCR. The results showed that P. gingivalis induced significant periodontal bone resorption in WT mice and TLR2 KO mice but not in TLR4 KO mice or TLR2&4 KO mice. For all four types of mice, ligation induced significant bone loss compared with that in control groups, and this bone loss was significantly higher than that in the P. gingivalis infection group. RANKL protein expression was significantly increased in the ligation group compared with that in the control group for all four types of mice, and in the P. gingivalis infection group of WT, TLR2 KO, and TLR4 KO mice. Expression patterns of RANKL, IL-1β, TNF-α, and IL-10 mRNA were different in the P. gingivalis infection group and the ligation group in different types of mice. In summary, P. gingivalis-induced periodontal bone resorption is TLR4-dependent, whereas ligation-induced periodontal bone resorption is neither TLR2- nor TLR4-dependent.

Up-regulation of Notch1 inhibits proliferation and differentiation of osteoclast in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed to explore the effect of the up-regulation of Notch1 on osteoclastogenesis induced to osteoclasts by receptor activator for nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factors (MCSF) in vitro.</p><p><b>METHODS</b>The bone marrow stem cells (BMSCs) of Rosa(-notch1) mice were cultured and induced to osteoclasts by RANKL and MCSF. The BMSCs were transfected with the Ad-Cre-green fluorescent protein (GFP) virus or Ad-GFP virus. Total RNA from cells was extracted, and the gene expression levels of Notch1, Notch2, Notch3, Notch4, Deltal, Delta3, Delta4, Jagged1, Hes1, and tartrate resistant acid phosphatase (TRAP) were detected at the defined stage by reverse transcription-polymerase chain reaction (RT-PCR). Osteoclast formation was analyzed by TRAP assay.</p><p><b>RESULTS</b>The number of TRAP-positive multinuclear cells of the experimental group significantly decreased compared with that of the control group. The mRNA expression levels of Notch1, Notch3, Jagged1, Delta3, and Hesl of the experimental group were significantly higher than those of the control group, whereas the TRAP mRNA expression of the experimental group was significantly lower than that of the control group (P<0.05).</p><p><b>CONCLUSION</b>Up-regulation of Notch1 inhibit osteoclastogenesis of BMSCs induced by RANKL and MCSF in vitro.</p>

Effects of laser solid forming of porous titanium on proliferation of osteoblast and RANKL/OPG expression / 中南大学学报(医学版)

To evaluate the effect of laser solid forming (LSF) of porous titanium on receptor activator of NF-κB ligand (RANKL)/osteoprorotegerin (OPG) expression and osteoblast cells growth.
 Methods: The DMEM and sterile saline were used for porous titanium extract. The osteoblast cells were cultured in the extract while equal amount of  DMEM and sterile saline were added to the control group. The growth of the cells were observed under an inverted phase contrast microscope. MTT was used to detect the growth inhibitory rates. The adhesion capacity of osteoblasts were measured. The growth in the material surface was examined by the electron microscope, and the expressions of RANKL and OPG were determined by Westen blot.
 Results: At the first day, the osteoblast proliferation rate was significantly different (P0.05); at each time point, the osteoblast proliferation rate were significantly different between the two groups (P0.05). The laser solid forming of porous titanium showed well bone compatibility.
 Conclusion: The porous titanium did not affect osteoblast proliferation due to its well bone compatibility. It did not affect the OPG/RANKL/RANK-axis system of bone metabolism, exibiting a wide applicable prospect for tissue engineering.

Cytocompatibility and biocompatibility of nanostructured carbonated hydroxyapatite spheres for bone repair

ABSTRACT Objective The aim of this study was to investigate the in vitro and in vivo biological responses to nanostructured carbonated hydroxyapatite/calcium alginate (CHA) microspheres used for alveolar bone repair, compared to sintered hydroxyapatite (HA). Material and Methods The maxillary central incisors of 45 Wistar rats were extracted, and the dental sockets were filled with HA, CHA, and blood clot (control group) (n=5/period/group). After 7, 21 and 42 days, the samples of bone with the biomaterials were obtained for histological and histomorphometric analysis, and the plasma levels of RANKL and OPG were determined via immunoassay. Statistical analysis was performed by Two-Way ANOVA with post-hoc Tukey test at 95% level of significance. Results The CHA and HA microspheres were cytocompatible with both human and murine cells on an in vitro assay. Histological analysis showed the time-dependent increase of newly formed bone in control group characterized by an intense osteoblast activity. In HA and CHA groups, the presence of a slight granulation reaction around the spheres was observed after seven days, which was reduced by the 42nd day. A considerable amount of newly formed bone was observed surrounding the CHA spheres and the biomaterials particles at 42-day time point compared with HA. Histomorphometric analysis showed a significant increase of newly formed bone in CHA group compared with HA after 21 and 42 days from surgery, moreover, CHA showed almost 2-fold greater biosorption than HA at 42 days (two-way ANOVA, p<0.05) indicating greater biosorption. An increase in the RANKL/OPG ratio was observed in the CHA group on the 7th day. Conclusion CHA spheres were osteoconductive and presented earlier biosorption, inducing early increases in the levels of proteins involved in resorption.