Diferencia en la expresión del ligando de receptor activador para el factor nuclear K B, en tejido tumoral frente a la infección por diferentes patógenos periodontales / Difference in receptor activator of nuclear factor K B expression in tumoral tissue by different periodontal pathogens

La enfermedad periodontal es una de las principales causas de pérdida dentaria. Clínicamente, esta patología, mediada por la desregulación del sistema inmune producto de una disbiosis ocurrida en el surco gingival, inicia con la inflamación de la encía y evoluciona con el daño irreversible de los tejidos que rodean el diente. El hueso alveolar es uno de los tejidos afectados esta patología, esto debido a la activación de osteoclastos por la sobreexpresión de la proteína RANKL en el huésped. El propósito de este trabajo es determinar el nivel de sobreexpresión de RANKL, en un modelo de células tumorales U2OS, frente a la infección con Porphyromonas gingivalis y Prevotella intermedia. Para identificar el nivel de RANKL, se definieron cuatro grupos: Un grupo control, no tratado; Grupo PG, tratado con P. gingivalis; Grupo PI, tratado con P. Intermedia; y un grupo PG+PI, tratado con ambas bacterias. El nivel relativo de la proteína RANKL fue determinado en el sobrenadante y en los extractos celulares de manera independiente, mediante la técnica Western blot. En sobrenadantes, el grupo PG mostró mayores niveles de RANKL comparados con PI (p < 0,05). En extractos celulares los niveles fueron mayores en el grupo PG+PI (p < 0,05). El grupo PI mostró los niveles más bajos de RANKL. La infección polimicrobiana resulta en una mayor expresión de RANKL en células tumorales U2OS, mientras que frente a la infección P. gingivalis, se observó mayor cantidad de RANKL soluble.

SUMMARY: Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by the deregulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed.

Effect of enhanced masticatory force on OPG, RANKL and MGF in alveolar bone of ovariectomized rats

Abstract Menopause induces oral bone loss, leading to various oral diseases. Mastication importantly affects bone metabolism in the jawbone. Objective: To analyze the effect of enhanced masticatory force on osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), and mechano-growth factor (MGF) in alveolar bone of ovariectomized rats and to study the mechanics mechanism of the alveolar bone of ovariectomized rats response to enhanced masticatory force. Methodology: Thirty Sprague Dawley rats were randomly divided into three groups: sham-operation group (fat around the removed ovary + normal hard diet), model group (ovariectomy + normal hard diet), and experimental group (ovariectomy + high hard diet). It was a 2-month experiment. Enzyme-linked immunosorbent assay (ELISA) detected serum estradiol (E2), osteocalcin (BGP) and alkaline phosphatase (ALP) in rats. Bone histomorphometric indices in the third molar region of maxilla were detected by micro-CT; protein expressions of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Western blot; and gene expression of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Quantitative Real-Time PCR. Results: Comparing with model group, serum E2 in experimental group increased but not significantly, serum BGP and serum ALP in experimental group decreased but not significantly, OPG in experimental group in alveolar bone increased significantly, RANKL in experimental group in alveolar bone decreased significantly, RANKL/OPG ratio in experimental group decreased significantly, MGF in experimental group in alveolar bone increased significantly, bone volume to total volume fraction increased significantly in experimental group, trabecular thickness increased significantly in experimental group, and trabecular separation decreased significantly in experimental group. Conclusion: Enhanced masticatory force affected the expression of OPG, RANKL, and MGF in alveolar bone of ovariectomized rats, improved the quality of jaw bone of ovariectomized rats, and delayed oral bone loss by ovariectomy.

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.

Effect of avocado/soybean unsaponifiables on periodontal repair in rats with arthritis and induced periodontitis

Abstract Objective: This study aimed to evaluate the effect of avocado/soybean unsaponifiables (ASU) on periodontal repair in rats with induced periodontitis and arthritis. Methodology: Forty-five rats were submitted to periodontitis induction by insertion of ligatures into the upper second molars, maintained for 15 days. These animals were randomly allocated to 3 groups according to the presence of induced arthritis (ART) and the application of the ASU: Control (CTR) group-healthy animals, where saline solution was administered; ART-animals with induced arthritis, where saline solution was administered; ART/ASU-animals with induced arthritis, where ASU (0.6 mg/ kg) was administered. The drugs were administered daily by gavage and the animals were euthanized after 7, 15 and 30 days of the ligature removal. Bone resorption, inflammatory infiltrate composition and marker proteins expression of the differentiation and formation of osteoclasts (RANKL and TRAP) were assessed. Results: The ART/ASU group presented higher bone volume than the ART group at 7 and 30 days after the ligature removal. Furthermore, the ART group presented higher quantity of inflammatory cells and expression of TRAP and RANKL than the other groups. Conclusion: ASU administration improves the repair of periodontal tissues in an experimental periodontitis model in rats with induced arthritis.

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.

Effect of ProRoot MTA® and Biodentine® on osteoclastic differentiation and activity of mouse bone marrow macrophages

Abstract Objectives This investigation aimed to assess the differentiation inhibitory effects of ProRoot MTA® (PMTA) and Biodentine® (BIOD) on osteoclasts originated from murine bone marrow macrophages (BMMs) and compare these effects with those of alendronate (ALD). Materials and Methods Mouse BMMs were cultured to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of NF-κB (RANKL), treated with lipopolysaccharide. After application with PMTA, BIOD, or ALD, cell toxicities were examined using WST-1 assay kit, and RANKL-induced osteoclast differentiation and activities were determined by resorption pit formation assay and tartrate-resistant acid phosphate (TRAP) staining. The mRNA levels of osteoclast activity-related genes were detected with quantitative real time polymerase chain reaction. Expressions of molecular signaling pathways were assessed by western blot. All data were statistically analyzed with one-way ANOVA and Tukey's post-hoc test (p<0.05). Results Mouse BMMs applied with PMTA, BIOD, or ALD showed highly reduced levels of TRAP-positive osteoclasts. The BIOD treated specimens suppressed mRNA expressions of cathepsin K, TRAP, and c-Fos. Nonetheless, it showed a lower effect than PMTA or ALD applications. Compared with ALD, PMTA and BIOD decreased RANKL-mediated phosphorylation of ERK1/2 and IκBα. Conclusions PMTA and BIOD showed the inhibitory effect on osteoclast differentiation and activities similar to that of ALD through IκB phosphorylation and suppression of ERK signaling pathways.

Immunohistochemical expression of RANKL in oral giant cell lesions is predictive of aggressiveness

Abstract The aim of this study was to evaluate the immunohistochemical expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and of osteoprotegerin (OPG), important proteins correlated with osteoclastogenesis, in central giant cell lesions (CGCL) and peripheral giant cell lesions (PGCL) and to compare their expression with the histological and clinical parameters for quantification of multinucleated giant cells (MGC) and their nuclei, lesion size, and recurrences. Twenty cases of each lesion type were selected to quantify the number of MGCs and nuclei/mm2 of connective tissue. The immunoreactivity of RANKL and OPG was expressed as a percentage of the marked area in the stroma. Clinical data were collected from pathoanatomical and medical reports. No statistical differences were found for the number of MGCs (p = 0.24) between PGCL and CGCL, but the number of nuclei within the MGCs was higher in CGCL (p = 0.01). RANKL expression was higher in CGCL than in PGCL (p = 0.04) and all recurrent lesions showed higher RANKL and OPG expressions than nonrecurrent lesions. We report higher RANKL expression and a greater number of nuclei in CGCL, which may explain the difference in clinical behaviour between these lesions and their pathogenesis.

Alveolar bone healing in rats: micro-CT, immunohistochemical and molecular analysis

Abstract Alveolar bone healing after upper incisor extraction in rats is a classical model of preclinical studies. The underlying morphometric, cellular and molecular mechanism, however, remains imprecise in a unique study. Objectives The aim of this study was therefore to characterize the alveolar bone healing after upper incisor extraction in rats by micro computed tomographic (Micro-CT), immunohistochemical and real-time polymerase chain reaction (RT-PCR) analysis. Material and Methods Thirty animals (Rattus norvegicus, Albinus Wistar) were divided into three groups after upper incisors extraction at 7, 14, and 28 days. Micro-CT was evaluated based on the morphometric parameters. Subsequently, the histological analyses and immunostaining of osteoprotegerin (OPG), receptor activator of nuclear kappa B ligand (RANKL) and tartrate resistant acid phosphate (TRAP) was performed. In addition, RT-PCR analyses of OPG, RANKL, the runt-related transcription factor 2 (RUNX2), osteocalcin (OC), osteopontin (OPN), osterix (OST) and receptor activator of nuclear kappa B (RANK) were performed to determine the expression of these proteins in the alveolar bone healing. Results Micro-CT: The morphometric parameters of bone volume and trabecular thickness progressively increased over time. Consequently, a gradual decrease in trabecular separation, trabecular space and total bone porosity was observed. Immunohistochemical: There were no differences statistically significant between the positive labeling for OPG, RANKL and TRAP in the different periods. RT-PCR: At 28 days, there was a significant increase in OPG expression, while RANKL expression and the RANKL/OPG ratio both decreased over time. Conclusion Micro-CT showed the newly formed bone had favorable morphometric characteristics of quality and quantity. Beyond the RUNX2, OC, OPN, OST, and RANK proteins expressed in the alveolar bone healing, OPG and RANKL activity showed to be essential for activation of basic multicellular units during the alveolar bone healing.

Immunohistochemical and mRNA expression of RANK, RANKL, OPG, TLR2 and MyD88 during apical periodontitis progression in mice

Abstract Objective To evaluate and correlate, in the same research, the mRNA expression and the staining of RANK, RANKL, OPG, TLR2 and MyD88 by immunohistochemistry in the apical periodontitis (AP) progression in mice. Material and Methods AP was induced in the lower first molars of thirty-five C57BL/6 mice. They were assigned to four groups according to their euthanasia periods (G0, G7, G21 and G42). The jaws were removed and subjected to histotechnical processing, immunohistochemistry and real-time reverse transcription-PCR (qRT-PCR). Data were analyzed with parametric and nonparametric tests (α=0.05). Results An increase of positive immunoreactivity for RANK, RANKL, OPG, TLR2 and MyD88 was observed over time (p<0.05). The RANKL expression was different between the groups G0 and G42, G21 and G42 (p=0.006), with G42 presenting the higher expression in both comparations. The OPG expression was statistically different between the groups G0 and G7, G7 and G21 and G7 and G42 (p<0.001), with G7 presenting higher expression in all the time points. The TLR2 expression was different between the groups G0 and G42 (p=0.03), with G42 showing the higher expression. The MyD88 expression presented a statistical significant difference between groups G7, G21 and G42 compared with G0 (p=0.01), with G0 presenting the smallest expression in all the comparisons. The Tnfrsf11/Tnfrsf11b (RANKL/OPG) ratio increased with the AP progression (p=0.002). A moderate positive correlation between MyD88 and RANKL (r=0.42; p=0.03) and between MyD88 and TLR2 (r=0.48; p<0.0001) was observed. Conclusion The expression of the RANK, RANKL, OPG, MyD88 and TLR2 proteins as well as the ratio Tnfrsf11/Tnfrsf11b (RANKL/OPG) increased with AP progression. There was also a moderate positive correlation between the expression Myd88-Tnfrsf11 and Tlr2-Myd88, suggesting the relevance of Tlr2-Myd88 in bone loss due to bacterial infection.

Effects of avocado/soybean unsaponifiables (ASU) on the treatment of ligature-induced periodontitis in rats

Abstract The purpose of this study is to evaluate the effect of the avocado/soybean unsaponifiables (ASU) on the treatment of induced periodontitis in rats. Periodontitis was induced in 84 rats via ligature placement around the second upper molar, which was removed after 7 days, and scaling and root planning (SRP) was performed at this time. Subsequently, the rats were randomly allocated to four groups with 21 animals each: One SRP group in which saline solution was administered (SS), and three groups in which ASU was administered (0.6 g/kg/day), beginning either 7 days before the induction of periodontitis (SRP/ASU-7), on the day of periodontitis induction (SRP/ASU0), or on the day of treatment (SRP/ASU+7). ASU and SS were administered daily by gavage until the sacrifice of the animals (7, 15, and 30 days after SRP). The % bone in the furcation area was evaluated by histomorphometry and micro-CT. The expression of proteins (TRAP, RANKL, and alkaline phosphatase) and mRNA (IL-1β, TNF-α, IL-6, RANKL, and alkaline phosphatase) were evaluated by immunohistochemistry and qPCR. The SRP/ASU+7 group presented a higher percentage of bone fill in the furcation area and higher expression of alkaline phosphatase than in the SRP group (at 7 and 30 days, respectively). The SRP/ASU0 and SRP/ASU+7 groups presented lower expression levels of RANKL mRNA than the SRP and SRP/ASU-7 groups at 15 days. ASU administration on the day of the SRP treatment of the ligature-induced periodontitis promoted subtle beneficial effects on periodontal repair following the treatment of induced periodontitis within the experimental period of 7 days.

Suppressor of cytokine signaling 1 expression during LPS-induced inflammation and bone loss in rats

Abstract This study aimed to characterize the dynamics of suppressor of cytokine signaling (SOCS1) expression in a rat model of lipopolysaccharide-induced periodontitis. Wistar rats in the experimental groups were injected three times/week with LPS from Escherichia coli on the palatal aspect of the first molars, and control animals were injected with vehicle (phosphate-buffered saline). Animals were sacrificed 7, 15, and 30 days after the first injection to analyze inflammation (stereometric analysis), bone loss (macroscopic analysis), gene expression (qRT-PCR), and protein expression/activation (Western blotting). The severity of inflammation and bone loss associated with LPS-induced periodontitis increased from day 7 to day 15, and it was sustained through day 30. Significant (p < 0.05) increases in SOCS1, RANKL, OPG, and IFN-γ gene expression were observed in the experimental group versus the control group at day 15. SOCS1 protein expression and STAT1 and NF-κB activation were increased throughout the 30-day experimental period. Gingival tissues affected by experimental periodontitis express SOCS1, indicating that this protein may potentially downregulate signaling events involved in inflammatory reactions and bone loss and thus may play a relevant role in the development and progression of periodontal disease.

Mecanismos intracelulares induzidos por leucotrienos durante a diferenciação osteogênica / Leukotriene-induced intracellular mechanisms during osteogenic differentiation

Os leucotrienos (LTs) são mediadores inflamatórios derivados da via 5- lipoxigenase (5-LO), com contribuição relevante na reabsorção óssea. Neste estudo investigamos o papel dos LTs na diferenciação osteogênica e o seu impacto na osteoclatogênese. Assim, foi avaliado o perfil ósseo dos camundongos 129/Sv (WT) e 5-LO Knockout (5-LO KO) por meio de microtomografia computadorizada, evidenciando maior densidade óssea vertebral e trabéculas mais espessas em machos 5-LO KO. Após isso, osteoblastos primários (OBL) foram isolados e cultivados para determinar a atividade de fosfatase alcalina (ALP) e o potencial de mineralização. Resultados mostraram que OBL KO possui maior atividade de ALP e mineralização, em todos os períodos quando comparados com WT. Em adição, o tratamento com os LTs B4 e D4 inibiu a deposição de cálcio. Os inibidores da síntese de LTs e os antagonistas do BLT1/2 foram efetivos em recuperar a formação dos nódulos mineralizados. A cinética do Alox5 apresentou um aumento da expressão nos períodos de maior diferenciação celular em OBL WT. Além disso, a expressão de OCN, MMPs 2 e 9 e RANKL foram aumentadas em células 5-LO KO em quase todos os períodos avaliados. Em geral, o estímulo com LTs, seus inibidores e antagonistas diminuiu a expressão de Sp7, Col1a1, Opg e MMP-9 e aumentou RANKL em células KO. A sinalização por meio de segundos mensageiros também foi avaliada. Células 5-LO KO apresentam menor concentração de cálcio intracelular (Ca2+i) em relação ao WT. No período de 14 dias, o estímulo com LTD4 inibiu a liberação Ca2+i independente da linhagem, em relação ao controle. Os níveis de cAMP foram menores em OBL 5- LO KO, em todos os grupos tratados ou controle. LTD4 diminuiu a concentração de cAMP, mas não LTB4, em OBL 5-LO KO. O estudo também quantificou a produção de LTB4 e outros eicosanoides em osteoblastos mostrando a sua capacidade de síntese. A análise proteômica revelou 89 proteínas com expressão diminuída em OBL 5-LO KO, de um total de 154, sendo a maioria relacionada ao citoesqueleto e ao metabolismo energético. Também foram identificadas 59 proteínas exclusivas em OBL 5-LO KO e 06 unicamente expressas em células WT, revelando as diferenças intrínsecas de cada animal. O perfil osteoclastogênico de camundongos WT vs. 5-LO KO mostrou diferenças significativas na análise fenotípica, TRAP e na expressão gênica de células derivadas da linhagem monocítica-macrofágica. Após o estímulo com M-CSF e RANKL, as células WT apresentaram osteoclastos gigantes multinucleados, porém, células 5-LO KO apresentaram uma população de células com formas e tamanhos variáveis, e menor grau de maturação. Em adição, os LTsexógenos não modularam a atividade da TRAP. O meio condicionado proveniente dos OBL WT e KO, retardaram o processo de formação dos osteoclastos. A análise da expressão gênica em osteoclastos mostrou diminuição da expressão de Alox5, Il- 1b, Il-6 e TNFa em células 5-LO KO. BLT1/2, CysLt1 e os marcadores da diferenciação Acp5, Ctsk e Nfact1 não apresentaram diferenças entre os animais. Em adição, o LTB4 diminuiu a expressão do Alox5 e a Il-1b foi aumentada em osteoclastos WT. Assim, os resultados demonstram que os LTs são capazes de modular o metabolismo ósseo, e a ausência do gene da 5-LO está relacionada ao maior perfil osteogênico.(AU)

Leukotrienes (LTs) are inflammatory mediators derived from the 5-lipoxygenase (5-LO) pathway, with a relevant contribution in bone resorption. In this study we investigated the role of LTs in osteogenic differentiation and its impact on osteoclastogenesis.Thus, the bone profile of the 129/Sv (WT) and 5-LO Knockout mice (5-LO KO) was evaluated by computerized microtomography, showing higher vertebral bone density and thicker trabeculae in 5-LO KO males. After that, primary osteoblasts (OBL) were isolated and cultured to determine alkaline phosphatase activity (ALP) and mineralization potential. Results showed that OBL KO has higher ALP activity and mineralization, in all periods when compared with WT. In addition, the treatment with LTB4 and LTD4 inhibited calcium deposition. Inhibitors of LT synthesis and BLT1/2 antagonists were effective to recover the mineralized nodules formation. The kinetics of Alox5 showed an increase in expression during cellular differentiation period in WT OBL. In addition, expression of OCN, MMPs 2 and 9 and RANKL were increased in 5- LO KO cells in almost all evaluated periods. In general, the stimulation with LTs, their inhibitors and antagonists decreased the expression of Sp7, Col1a1, Opg and MMP- 9. But it increased the RANKL expression in KO cells. The second messengers signaling was also evaluated. 5-LO KO cells showed lower concentration levels of intracellular calcium (Ca2+ i) when compared to WT cells. In the 14-day period, the LTD4 treatment inhibited the Ca2+i independent of the murine lineage, relative to the control. cAMP levels were lower in OBL 5-LO KO, in all treated or control groups. LTD4 decreased the concentration of cAMP, but not LTB4, in KO cells. The study also quantified the production of LTB4 and other eicosanoids in osteoblasts showing their ability to synthesize those metabolites. The proteomic analysis revealed 89 downregulated proteins in OBL KO, out of a total of 154, most of them related to cytoskeleton and energy metabolism. Also 59 identified proteins were unique in OBL 5-LO KO and 06 exclusively expressed in WT cells, revealing the intrinsic differences of each strain. The osteoclastogenic profile of WT vs. 5-LO KO showed significant differences in phenotypic analysis, TRAP and in the gene expression of cells derived from the monocyte-macrophage-lineage. After M-CSF and RANKL stimulation, WT cells showed multinucleated giant osteoclasts. However, 5-LO KO cells presented a population of cells with variable shapes and sizes, and a lower maturation stage. In addition, exogenous LTs did not modulate TRAP activity. The conditioned medium from OBL WT and 5-LO KO delayed the formation process of osteoclasts. Gene expression analysis in osteoclasts showed decreased expression of Alox 5, Il-1b, Il-6 and TNFα in 5-LO KO cells. BLT1/2, CysLt1 and the osteoclast differentiation markers Acp5, Ctsk and Nfact1 showed no differences between the strains. In addition, LTB4 decreased the expression of Alox5, and IL-1b was increased in WT osteoclasts. Thus, the results demonstrate that the LTs are able to modulate the bone metabolism, and the absence of the 5-LO gene is related to the greater osteogenic profile.(AU)

Low-level laser therapy associated to a resistance training protocol on bone tissue in diabetic rats

ABSTRACT Objective The present study aimed to evaluate the in vivo response of a resistance training and low-level laser therapy (LLLT) on tibias and femurs of rats with diabetes mellitus (DM). Materials and methods Forty male Wistar rats were randomly distributed into four experimental groups: control group (CG), diabetic group (DG), diabetic trained group (TG) and diabetic trained and laser irradiated group (TLG). DM was induced by streptozotocin (STZ) and after two weeks laser and resistance training started, performed for 24 sessions, during eight weeks. At the end of the experiment, animals were euthanized and tibias and femurs were removed for analysis. Histological, histomorphometrical, immunohistochemistry and mechanical analyses were performed. Results Trained groups, with or without laser irradiation, showed increased cortical area, bone density and biomechanical properties. The immunohistochemical analysis revealed that TG and TLG demonstrated an increased RUNX2 expression. RANK-L immunoexpression was similar for all experimental groups. Conclusion In conclusion, it can be suggested that the resistance exercise program stimulated bone metabolism, culminating in increased cortical tibial area, bone mineral content, bone mineral density and biomechanical properties. Furthermore, the association of physical exercises and LLLT produced higher values for bone mineral content and stiffness. Consequently, these data highlight the potential of physical exercise in the management of bone loss due to DM and the possible extra osteogenic stimulus offered by lasertherapy. Further long-term studies should be carried out to provide additional information.

Relationship between hypertension and periapical lesion: an in vitro and in vivo study

Abstract The aim of this study was to compare potential aspects of periapical lesion formation in hypertensive and normotensive conditions using hypertensive (BPH/2J) and wild-type control (BPN/3J) mice. The mandibular first molars of both strains had their dental pulp exposed. At day 21 the mice were euthanized and right mandibular molars were used to evaluate the size and phenotype of apical periodontitis by microCT. Proteins were extracted from periapical lesion on the left side and the expressions of IL1α, IL1β and TNFα were analyzed by ELISA. Bone marrow stem cells were isolated from adult mice femurs from 2 strains and osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (TRAP) in vitro. The amount of differentiated osteoclastic cells was nearly double in hypertensive mice when compared to the normotensive strain (p < 0.03). Periapical lesion size did not differ between hypertensive and normotensive strains (p > 0.7). IL1α, IL1β and TNFα cytokines expressions were similar for both systemic conditions (p > 0.05). Despite the fact that no differences could be observed in periapical lesion size and cytokines expressions on the systemic conditions tested, hypertension showed an elevated number of osteoclast differentiation.

Avaliação das citocinas TNF-α, RANKL e OPG e do número de osteoclastos no reparo de defeito ósseo em calvária de ratos diabéticos tratados com matriz óssea desmineralizada / Evaluation of cytokines TNF-α, RANKL and OPG and the number of osteoclasts on repair of bone defects in skulls of diabetic rats treated with demineralized bone matrix

Neste trabalho, foi avaliado a participação dos osteoclastos bem como a ação das citocinas RANKL, OPG e TNF-α durante a formação e remodelação óssea em defeitos ósseos de tamanho crítico em ratos normoglicêmicos e diabéticos tratados ou não com a MAOD. Para isso, foram utilizados 250 ratos machos Wistar. Trinta ratos foram utilizados para coleta dos fêmures e tíbias, os quais foram processados para obtenção da MAOD. Os demais 220 ratos foram divididos em Grupo Não Diabétido (CTL, n=110) e Grupo Diabético (DIAB, n= 110) induzido pela aplicação de uma dose única de 47 mg/Kg de massa corporal de estreptozotocina. Um defeito transósseo de 8 mm de diâmetro foi realizado nos ossos parietais dos ratos, sendo que, nos subgrupos CTL MAOD e DIAB MAOD, os defeitos foram preenchidos com MAOD e nos grupos CTL COAG e DIAB COAG apenas com coágulo sanguíneo. Após 0, 7, 14, 21 e 42 dias, as calotas cranianas foram coletadas para determinação da densidade de volume, número de osteoclastos/mm2 na área do defeito, quantificação por imunoistoquimica e expressão do RNAm para as proteínas RANKL, OPG e TNF-α. Os resultados para volume do tecido ósseo neoformado foi maior nos grupos CTL COAG e CTL MAOD, bem como no grupo DIAB MAOD quando comparado com DIAB COAG (CTL MAOD > CTL COAG e DIAB MAOD > DIAB COAG). O número de osteoclastos nos grupos CTL aumentaram significantemente (3,69 osteoclasto/mm2), enquanto que nos grupos MAOD aumentaram gradualmente até os 42 dias (2,8 osteoclasto/mm2). Os resultados para imunomarcação mostraram que a MAOD promove 1,28 vezes maior expressão de OPG, bem como de TNF-α tanto no grupo CTL (1,59 vezes) como no DIAB (1,76 vezes). Os resultados para expressão do RNAm para OPG mostrou que a média dos valores do grupo COAG comparado com a do grupo MAOD foi 1,91 vezes maior no grupo COAG. Já os valores para expressão de RANKL permaneceram constantes no grupo DIAB MAOD, com aumento significativo de 2,57 vezes aos 42 dias, sendo 4,3 vezes maior, quando comparado com a média dos outros grupos no mesmo período. Conclui-se que nos animais normoglicemicos, o tratamento com a MAOD aumenta a expressão de OPG, RANKL e TNF-α, assim como a atividade osteoclástica, promovendo reabsorção da MAOD e formação de tecido ósseo, enquanto que nos animais diabéticos, a atividade osteoclástica foi reduzida, sem alteração nos níveis de OPG e RANKL, reduzindo a reabsorção da MAOD e consequentemente da formação óssea.(AU)

Participation of osteoclasts was evaluated in reabsorption process of demineralized allogenic bone matrix (DABM) as well as the activity of cytokines RANKL, OPG and TNF- α during formation and bone remodeling in critial size defect of normoglycemic and diabetic rats treated or not with DABM. Therefore, 250 male Wistar rats were used. Thirty rats had femurs and tibias collected and processed to obtain DABM. 220 rats were divided into control group (CTL, n=110) and diabetic group (DIAB, n= 110) injected by a single dose of 47 mg/Kg of body weight streptozotocin. Were made 8mm bone defect on skulls of rats, in subgroups CTL DABM and DIAB DABM, defects were filled with DABM and subgroups CTL CLOT and DIAB CLOT were filled with blood clot. After 0, 7, 14, 21 and 42 days, the skulls were collected to determine the volume density, number of osteoclasts/mm2 into defects area, quantification by immunohistochemistry and RNAm expression of RANKL, OPG and TNF-α cytokines. The results of volume density of newly formed bone was higher in CTL CLOT and CTL DABM, as well as in DIAB DABM compared to DIAB CLOT (CTL DABM > CTL CLOT and DIAB DABM > DIAB CLOT). The number of osteoclasts in CTL groups increased to 3,69 osteoclasts/mm2, while in subgroups treated with DABM gradually increased up until 42 days (2,8 osteoclasts/mm2). Immunohistochemistry showed that DABM promotes an increase of 1.28-fold of OPG expression, as well as TNF-a expression in CTL group (1.59-fold) and DIAB group (1.76-fold). The results of RNAm expression of OPG showed that the average values of the CLOT subgroup compared to the average values of DABM subgroup was 1.91- fold higher in CLOT subgroup. The values of RANKL RNAm expression increase 2.57-fold at 42 days, being 4.3-fold higher than the average os the other groups in the same period. In conclusion, in the normoglicemic animals (CTL group), the treatment with DABM increase the expression of OPG, RANKL and TNF-α as the activity of osteoclasts, leading to DABM resorption and bone tissue formation, while in diabetic animals, the osteoclast activity was reduced, without changes in the leves of OPG and RANKL, decreasing DABM resorption and bone formation.(AU)

Static compression regulates OPG expression in periodontal ligament cells via the CAMK II pathway

ABSTRACT Objective This study aimed to investigate the potential role of CAMK II pathway in the compression-regulated OPG expression in periodontal ligament cells (PDLCs). Material and Methods The PDL tissue model was developed by 3-D culturing human PDLCs in a thin sheet of poly lactic-co-glycolic acid (PLGA) scaffolds, which was subjected to static compression of 25 g/cm2 for 3, 6 and 12 h, with or without treatment of KN-93. After that, the expression of OPG, RANKL and NFATC2 was investigated through real-time PCR and western blot analysis. Results After static compression, the NFATC2 and RANKL expression was significantly up-regulated, while partially suppressed by KN-93 for 6 and 12 h respectively. The OPG expression was significantly down-regulated by compression in 3 h, started to elevate in 6 h, and significantly up-regulated in 12 h. The up-regulation after 12 h was significantly suppressed by KN-93. Conclusions Long-term static compression increases OPG expression in PDLCs, at least partially, via the CAMK II pathway.

Effect of sumac extract on serum oxidative status, RANKL/OPG system and alveolar bone loss in experimental periodontitis in rats

Objectives Sumac (Rhus coriaria L.) is widely used spice which has several properties such as antioxidant, anti-inflammatory and antimicrobial. The purpose of this animal study was to evaluate the effects of sumac extract on levels of receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG) expression, serum oxidative status, and alveolar bone loss in experimental periodontitis. Material and Methods Twenty-four Wistar rats were separated into three groups: non-ligated (NL, n=8), ligature only (LO, n=8), and ligature and treated with sumac extract (S, n=8) (20 mg/kg per day for 11 days). A 4/0 silk suture was placed around the mandibular right first molars subgingivally; after 11 days, the rats were sacrificed, and alveolar bone loss was histometrically measured. The detection of RANKL and OPG were immunohistochemically performed. Levels of serum total antioxidant status (TAS)/total oxidant status (TOS), and oxidative stress index (OSI) were also analyzed. Results Alveolar bone loss was significantly greater in the LO group compared to the S and NL groups (p<0.05). The number of inflammatory cell infiltrate (ICI) and osteoclasts in the LO group was significantly higher than that of the NL and S groups (p<0.05). The number of osteoblasts in the LO and S groups was significantly higher than that of the NL group (p<0.05). There were significantly more RANKL-positive cells in the LO group than in the S and NL groups (p<0.05). OPG-positive cells were higher in S group than in LO and NL groups (p<0.05). TOS and OSI levels were significantly reduced in S group compared to LO group (P<0.05) and TAS levels were similar in S and NL group (p>0.05). Conclusions The present study showed that systemic administration of sumac extract may reduce alveolar bone loss by affecting RANKL/OPG balance, TOS and OSI levels in periodontal disease in rats. .

Efeito da pioglitazona sobre o remodelamento ósseo em diabetes tipo 2 / Pioglitazone effect on bone remodeling in type 2 diabetes

Alterações morfológicas no tecido ósseo têm sido descritas nos usuários de hipoglicemiantes orais da classe das tiazolidinedionas (TZDs). Hipotetiza-se que alguns genes relacionados com a osteogênese e osteoclastogênese podem ser influenciados pelo tratamento farmacológico, entretanto, o exato mecanismo ainda não está bem esclarecido. O objetivo do estudo foi avaliar o efeito da pioglitazona no remodelamento ósseo através de genes envolvidos na osteoclastogênese em indivíduos recentemente diagnosticados com DM2 e modelos animais, com a finalidade de identificar marcadores genéticos sensíveis de alterações ósseas. Foram convidados para participar do estudo 199 indivíduos (100 diabéticos e 99 normoglicêmicos), no ambulatório de dislipidemias do Instituto Dante Pazzanese de Cardiologia. Os indivíduos diabéticos foram tratados com pioglitazona (15, 30, 45, 45 mg/ dia/ via oral) por 16 semanas. Foram colhidas amostras de sangue, antes e após o tratamento para avaliações laboratoriais, extração de DNA genômico e de RNA total. Os polimorfismos e a expressão do mRNA nas células sanguíneas foram determinados pela PCR em tempo real através do sistema TaqMan®. Para o estudo em modelo animal após a indução da dieta hiperlipídica por 32 semanas, foram utilizados 12 camundongos machos da linhagem C57BL/J6, os quais foram divididos em três grupos: controle (n=4); diabéticos induzidos pela dieta hiperlipídica (DH, n=4) e diabéticos induzidos pela dieta hiperlipídica e tratados com pioglitazona 35mg/Kg/dia por 16 semanas (DHP, n=4). Para os grupos experimentais foram colhidos: amostras de sangue, para exames laboratoriais; fêmures, para a extração do RNA total; e tíbias, para determinação dos parâmetros histomorfométricos. Os pacientes DM2 apresentaram diminuição nas concentrações séricas de osteocalcina e na expressão de OPG e aumento na expressão de VDR em comparação ao grupo NG (p<0,05). A expressão de RANKL e IL6 foi maior entre as mulheres, enquanto que a expressão de PPARG foi maior entre os homens com DM2 em comparação ao grupo NG (p=0,032). Pacientes DM2 antes do tratamento apresentaram glicemia e expressão do mRNA de IL6 negativamente associados ao cálcio ionizado, enquanto que as transcrições de TNFA e VDR foram associadas positivamente e negativamente com bALP respectivamente (p<0,05). O tratamento com pioglitazona reduziu a glicemia de jejum, glicemia pós-prandial, insulina, HOMA-IR, triglicerídeos, VLDL-C, tALP e bALP e aumentou a HDL, tACP, TNF-α e a transcrição de OPG (p<0,05). A glicemia basal associou-se positivamente com o cálcio ionizado. A expressão basal de OPG foi associado negativamente com tALP, enquanto que a expressão basal de TNFA foi associada positivamente com tALP e negativamente com tACP. A expressão basal IL6 foi associada positivamente com tALP, enquanto que a expressão basal de VDR foi associada negativamente com osteocalcina e positivamente com bALP em resposta ao tratamento (p<0,05). O polimorfismo RANK rs1805034 foi associado com redução na transcrição do gene RANK nos indivíduos DM2 e com o remodelamento ósseo após o tratamento com pioglitazona (p<0,05). O polimorfismo RANKL rs9525641 foi associado com aumento da transcrição gênica de RANKL nos indivíduos NG e DM2 e melhora da resposta farmacológica nos indivíduos DM2 tratados com pioglitazona (p<0,05). O polimorfismo rs3102735 do gene OPG foi associado com aumento da formação óssea nos indivíduos DM2 antes e após o tratamento (p<0,05). O genótipo CG do polimorfismo OPG rs2073618 foi associado com alteração da transcrição de OPG no grupo DM2 pré e pós-tratamento (p<0,05). O polimorfismo PPARG rs1801282 foi associado com menor risco para o desenvolvimento de diabetes (p<0,05). O polimorfismo PPARG rs2972162 foi associado com melhora da resistência insulínica nos indivíduos DM2 tratados com pioglitazona (p=0,017). O polimorfismo ESRI rs9340799 foi associado com redução da formação óssea nos indivíduos DM2 (p=0,038). Nos camundongos, após a indução da dieta hiperlipídica por 32 semanas, observou-se aumento do peso, da glicemia, do colesterol total, da expressão do mRNA de RANK, RANKL, IL6 e TNFA em fêmures e aumento de Tb.Sp e diminuição de BV/TV em comparação ao grupo controle (p<0,05). O tratamento com pioglitazona diminuiu a expressão de TNFA (p=0,028). As medidas histomorfométricas não alteraram-se após o tratamento (p>0,05). Os resultados sugerem que o estado hiperglicêmico e o tratamento influenciam os marcadores bioquímicos e moleculares. Os polimorfismos dos genes RANK, RANKL, OPG e ESRI parecem estar envolvidos no remodelamento ósseo independentemente da hiperglicemia e do tratamento e os polimorfismos do gene PPARG parecem estar envolvidos com menor risco para desenvolver diabetes e com a melhora da resistência insulínica em resposta ao tratamento com pioglitazona

Morphological changes in bone tissue have been reported in users of oral hypoglycemic class of thiazolidinediones (TZDs). It is hypothesized that some genes related to osteogenesis and osteoclastogenesis may be influenced by pharmacological treatment, however, was not aware exact mechanism. The study aims was to evaluate pioglitazone effect on bone remodeling through genes involved in osteoclastogenesis in individuals newly diagnosed with DM2 and animal models, in order to identify sensibles genetics markers of bone alterations. Were invited to participate in study 199 patients (100 diabetics and 99 normoglycemic), in dyslipidemia ambulatory of Institute Dante Pazzanese of Cardiology. Diabetic subjects were treated with pioglitazone (15, 30, 45 or 45 mg /day/oral) for 16 weeks. Blood samples were collected before and after treatment for laboratory evaluations, extraction of genomic DNA and total RNA. Polymorphisms and mRNA expression in blood cells was determined by real time PCR using TaqMan® system. For study in animal model after 32 weeks of fat diet induction, was used 12 male mice C57BL/J6, which were divided into three groups: control (n=4); induced diabetic fat diet (DH, n=4) and induced diabetic fat diet and treated with pioglitazone 35mg/Kg/day for 16 weeks (DHP, n=4). For experimental groups were collected: blood samples for laboratory tests; femurs, for extraction of total RNA; and tibias, to determine histomorphometric parameters. DM2 patients showed decrease in serum osteocalcin and OPG expression and increased VDR expression compared to NG group (p<0.05). RANKL and IL6 expression were higher among women, whereas PPARG expression was higher among men with DM2 compared to NG group (p=0,032). DM2 patients before treatment showed blood glucose and IL6 mRNA expression negatively associated with ionized calcium, whereas TNFA and VDR transcription are positively and negatively associated with bALP respectively (p<0.05). Pioglitazone treatment reduced fasting glucose, postprandial glucose, insulin, HOMA-IR, triglycerides, VLDL-C, tALP and bALP and increased HDL, tACP, TNF-α and OPG transcription (p<0.05). Basal blood glucose was positively associated with ionized calcium. Basal OPG expression was negatively associated with tALP, whereas basal TNFA expression was positively associated with tALP and negatively with tACP. Basal IL6 expression was positively associated with tALP, whereas basal VDR expression was negatively associated with osteocalcin and positively with bALP in response to treatment (p<0.05). RANK rs1805034 polymorphism was associated with RANK gene transcription reduction in subjects with DM2 and bone remodeling after treatment with pioglitazone (p<0.05). RANKL rs9525641 polymorphism was associated with increased RANKL gene transcription in NG and DM2 subjects and pharmacological response improvement in DM2 subjects treated with pioglitazone (p<0.05). OPG rs3102735polymorphism was associated with increased bone formation in DM2 subjects before and after treatment (p<0.05). CG genotype of OPG rs2073618 polymorphism was associated with OPG transcription change in DM2 group before and after treatment (p<0.05). PPARG rs1801282 polymorphism was associated with lower risk for diabetes development (p<0.05). PPARG rs2972162 polymorphism was associated with insulin resistance improvement in DM2 subjects treated with pioglitazone (p=0,017). ESRI rs9340799 polymorphism was associated with reduced bone formation in DM2 subjects (p=0,038). In mice, after 32 weeks of fat diet induction, was observed increase weight, blood glucose, total cholesterol and RANK, RANKL, IL6 and TNFA mRNA expression in femurs and Tb.Sp increase and BV/TV decrease compared to control group (p<0.05). Treatment with pioglitazone decrease TNFA (p=0,028). Histomorphometrics measurements not change after treatment (p>0.05). Results suggest that hyperglycemic state and treatment influence biochemical and molecular markers. RANK, RANKL, OPG and ESRI polymorphisms seens to be involved in bone remodeling regardless of hyperglycemia and treatment and PPARG gene polymorphisms seens to be associated with lower risk for diabetes development and with insulin resistance improvement in response to treatment with pioglitazone