Biomechanical Loading Modulates Proinflammatory and Bone Resorptive Mediators in Bacterial-Stimulated PDL Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oral squamous carcinoma cells secrete RANKL directly supporting osteolytic bone loss

Objective: Local invasion of bone is a frequent complication of oral squamous cell carcinoma (OSCC). Development of these osteolytic lesions is mediated by osteoclasts. Receptor activation of NF-kappa B ligand (RANKL) signaling, counteracted by osteoprotegerin (OPG), regulates osteoclastogenesis. Previous studies in rodent models have demonstrated that inhibition of RANKL decreases tumor growth and lesions within bone. However, the contributory role of OSCC cells to this disease process has yet to be defined.Methods: RANKL expression was assessed in a panel of OSCC cell lines by qPCR, flow cytometry, and ELISA. Induction of osteoclastogenesis was assessed by co-culture with macrophages or with OSCC-derived conditioned medium. In an animal model of bone invasion, nude mice were injected intratibially with UMSCC-11B cells expressing a RANKL luciferase promoter to detect tumor-derived RANKL activity. Osteolytic lesions were analyzed by X-ray, micro-CT, and histological methods. RANKL expression was assessed in human OSCC tissues by immunohistochemistry.Results: We demonstrated that OSCCs express varied levels of all RANKL isoforms, both membrane-bound and soluble RANKL. Both co-culture and treatment with OSCC-conditioned media induced osteoclastogenesis. In mice, we demonstrated human RANKL promoter activity during bone invasion. Over the course of the experiment, animals suffered osteolytic lesions as RANKL-driven luciferase expression increased with time. After 8 weeks, human-derived RANKL was detected in areas of bone resorption by immunohistochemistry. Similar epithelial RANKL expression was detected in human OSCC tissues.Conclusion: These data demonstrate the ability of OSCCs to produce RANKL, directly altering the tumor microenvironment to increase osteoclastogenesis and mediate local bone invasion. (C) 2012 Elsevier Ltd. All rights reserved.

Effect of the concentration of phenothiazine photosensitizers in antimicrobial photodynamic therapy on bone loss and the immune inflammatory response of induced periodontitis in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cimetidine Reduces Alveolar Bone Loss in Induced Periodontitis in Rat Molars

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

RANKL expression is differentially modulated by TLR2 and TLR4 signaling in fibroblasts and osteoblasts

Resident, non-immune cells express various pattern-recognition receptors and produce inflammatory cytokines in response to microbial antigens, during the innate immune response. Alveolar bone resorption is the hallmark of destructive periodontitis and it is caused by the host response to bacteria and their mediators present on the biofilm. The balance between the expression levels of receptor activator of nuclear factorkappa B ligand (RANKL) and osteoprotegerin (OPG) is pivotal for osteoclast differentiation and activity and has been implicated in the progression of bone loss in periodontitis. To assess the contribution of resident cells to the bone resorption mediated by innate immune signaling, we stimulated fibroblasts and osteoblastic cells with LPS from. Escherichia coli (TLR4 agonist), Porphyromonas gingivalis (TLR2 and -4 agonist), and interleukin-1 beta (as a control for cytokine signaling through Toll/IL-1receptor domain) in time-response experiments. Expression of RANKL and OPG mRNA was studied by RT-PCR, whereas the production of RANKL protein and the activation of p38 MAPK and NF-kB signaling pathways were analyzed by western blot. We used biochemical inhibitors to assess the relative contribution of p38 MAPK and NF-kB signaling to the expression of RANKL and OPG induced by TLR2, -4 and IL1β in these cells. Both p38 MAPK and NFkB pathways were activated by these stimuli in fibroblasts and osteoblasts, but the kinetics of this activation varied in each cell type and with the nature of the stimulation. E. coli LPS was a stronger inducer of RANKL mRNA in fibroblasts, whereas LPS from P. gingivalis downregulated RANKL mRNA in periodontal ligament cells but increased its expression in osteoblasts. IL-1β induced RANKL in both cell types and without a marked effect on OPG expression. p38 MAPK was more relevant than NF-kB for the expression of RANKL and OPG in these cell types.

Avaliação do reparo ósseo na interface osso/implante em ratas com osteoporose induzida tratadas com raloxifeno ou alendronato: análise histométrica, imunoistoquímica, por epifluorescência e biomecânica

Pós-graduação em Odontologia - FOA

Effects of electroacupuncture on experimental periodontitis in rats

Background: Acupuncture has shown the capability of modulating the immuno-inflammatory response of the host. This study aims to evaluate the effects of electroacupuncture (EA) on ligature-induced periodontitis in rats. Methods: Thirty-two animals were divided into four groups: 1) control; 2) experimental periodontitis (EP); 3) sham-treated (EP/EA-sham); and 4) treated with EA (EP/EA). For the EP groups, a ligature was placed around the right mandibular first molars at day 1. Sessions of EA or EA-sham were assigned every other day. For EA treatment, large intestine meridian points LI4 and LI11 and stomach meridian points ST36 and ST44 were used. EA-sham was performed in off-meridian points. Animals were euthanized at day 11. Histomorphometric and microtomographic analyses were performed. Immunolabeling patterns for the receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), and tartrate-resistant acid phosphatase (TRAP) were assessed. Expressions of interleukin (IL)-1 beta, matrix metalloproteinase (MMP)-8, IL-6, and cyclooxygenase (COX)-2 messenger RNAs (mRNAs) were evaluated by quantitative reverse transcription-polymerase chain reaction. Data were analyzed statistically (P < 0.05, analysis of variance). Results: Histomorphometric and microtomographic analyses demonstrated that group EP/EA presented reduced alveolar bone loss when compared to group EP (P < 0.05). Reduced RANKL immunolabeling and fewer TRAP-positive multinucleated cells were observed in the EA-treated group in relation to group EP. No differences were observed in OPG expression among groups. EA treatment decreased the genic expression of IL-1 beta and MMP-8 (P < 0.05), increased the mRNA expression of IL-6 (P < 0.05), and did not modify the genic expression of COX-2 in animals with EP (P > 0.05). Conclusion: It can be concluded that EA reduced periodontal tissue breakdown and the expression of some proinflammatory mediators and a proresorptive factor in EP in rats.

Evaluation of the progression and treatment of experimental periodontitis in rats subjected to chemotherapy with 5-fluorouracil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Adjuvant therapy with sodium alendronate for the treatment of experimental periodontitis in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Raloxifene modulates regulators of osteoclastogenesis and angiogenesis in an oestrogen deficiency periapical lesion model

Aim To analyse the local regulatory mechanisms of osteoclastogenesis and angiogenesis during the progression of periapical lesions in female rats with oestrogen deficiency and treatment with raloxifene (RLX). Methodology Female Wistar rats were distributed into groups: SHAM-veh, subjected to sham surgery and treated with a vehicle; OVX-veh, subjected to ovary removal and treated with a vehicle; and OVX-RLX, subjected to ovary removal and treated with RLX. Vehicle or RLX was administered orally for 90 days. During treatment, the dental pulp of mandibular first molars was exposed to the oral environment for induction of periapical lesions, which were analysed after 7 and 30 days. After the experimental periods, blood samples were collected for measurement of oestradiol, calcium, phosphorus and alkaline phosphatase. The rats were euthanized and the mandibles removed and processed for immunohistochemical detection of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), hypoxia-inducible factor-1 alpha (HIF-1α) and bone-specific alkaline phosphatase (BALP). Data were compared using Kruskal–Wallis followed by Dunn test (nonparametric values) and anova followed by the Tukey's test (parametric values). Results The plasma concentration of oestradiol showed hypo-oestrogenism in the rats subjected to ovary removal. On day 7, alkaline phosphatase activity, calcium and phosphorus were higher in the OVX-RLX group than in the OVX-veh group (P < 0.001), but immunolabelling for RANKL and HIF-1α was lower in OVX-RLX group (P < 0.001). On day 30, the OVX-veh group had higher immunolabelling for RANKL than the OVX-RLX group (P < 0.05). There were no significant differences in the immunoreactivity of OPG and BALP between any groups at either time-point (P > 0.05). Conclusion RLX therapy reversed the increased levels of the local regulators of both osteoclastogenesis and angiogenesis induced by oestrogen deficiency.

Immunohistochemical, tomographic, and histological study on onlay bone graft remodeling. Part III: allografts

Objective In the last decades aroused the interest for bone tissue bank as an alternative to autogenous grafting, avoiding donor sites morbidity, surgical time, and costs reduction. The purpose of the study was to compare allografts (ALg) with autografts (AUg) using histology, immunochemistry, and tomographic analysis. Material and methods Fifty-six New Zealand White rabbits were submitted to surgical procedures. Twenty animals were donors and 36 were actually submitted to onlay grafting with ALg (experimental group) and AUg (control group) randomly placed bilaterally in the mandible. Six animals of each group were sacrificed at 3, 5, 7, 10, 20, and 60 postoperative days. Immunolabeling was accomplished with osteoprotegerin (OPG); receptor activator of nuclear factor-k ligand (RANKL); alkaline phosphatase (ALP); osteopontin (OPN); vascular endothelial growth factor (VEGF); tartrate-resistant acid phosphatase (TRAP); collagen type I (COL I); and osteocalcin (OC). Density and volume of the grafts was evaluated on tomography obtained at the surgery and sacrifice. Results The ALg and AUg exhibited similar patterns of density and volume throughout the experiments. The intra-group data showed statistical differences at days 7 and 60 in comparison with other time points (P = 0.001), in both groups. A slight graft expansion from fixation until day 20 (P = 0.532) was observed in the AUg group and then resorbed significantly at the day 60 (P = 0.015). ALg volume remained stable until day 7 and decreased at day 10 (P = 0.045). The light microscopy analysis showed more efficient incorporation of AUg onto the recipient bed if compared with the ALg group. The immunohistochemical labeling picked: at days 10 and 20 with OPG in the AUg group and at day 7 with TRAP in the ALg group (P = 0.001 and P = 0.002, respectively). Conclusions ALg and AUg were not differing in patterns of volume and density during entire experiment. Histological data exhibit more efficient AUg incorporation into recipient bed compared with the ALg group. Immunohistochemistry outcomes demonstrated similar pattern for both ALg and AUg groups, except for an increasing resorption activity in the ALg group mediated by TRAP and in the AUg group by higher OPG labeling. However, this latter observation does not seem to influence clinical outcomes.

Efeitos do chá mate (llex paraguariensis) na estrutura óssea de ratas na perimenopausa

Pós-graduação em Ciências Fisiológicas - FOA

Influência da neuromodulação parassimpática durante a indução da periodontite em camundongos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeitos da perda de peso sobre o metabolismo ósseo de pacientes submetidos à cirurgia bariátrica de Bypass Gástrico em Y de Roux: efeitos da cirurgia bariátrica sobre o metabolismo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeitos da perda de peso sobre o metabolismo ósseo de pacientes submetidos à cirurgia bariátrica de Bypass Gástrico em Y de Roux: efeitos da cirurgia bariátrica sobre o metabolismo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)