Testosterone replacement relieves ligature-induced periodontitis by mitigating inflammation, increasing pro-resolving markers and promoting angiogenesis in rats: A preclinical study.

OBJECTIVES: This study aimed to evaluate the inflammatory profile as well as the resolution of inflammation in a ligature-induced periodontal inflammation in rats with depletion and/or supraphysiological testosterone replacement. DESIGN: Sixty male rats (Holtzman) were used in the present study. Study groups were created as following: (1) Sham (no testicle removal); (2) Orchiectomy (OCX), 3) OCX + Testosterone (OCX + T); (4) Sham + Ligature (SH + L); (5) OCX+L; and 6) OCX + T + L. The surgeries were performed on day 1, and testosterone was administered weekly since day 1. On day 15, a cotton ligature was placed around the lower first molars and maintained for 15 days. Morphological changes in periodontal tissues were determined by histopathological analysis. Immunohistochemistry (factor VIII) and immunoenzymatic assay were performed to evaluate angiogenesis process and (pro- and anti-) inflammatory markers, respectively. RESULTS: Ligature promoted a marked inflammatory gingival infiltrate and bone loss (P < 0.05). Supraphysiological testosterone treatment increased the percentage of blood vessels, extracellular matrix and fibroblasts in the presence and absence of periodontal inflammation (P < 0.05). A high dose of testosterone increased factor VIII+ blood vessels and IL-10 expression in inflamed gingival tissue, while PGE2, LXA4 and MPO were reduced as a result of supraphysiological testosterone administration (P < 0.05). CONCLUSIONS: These results, in our experimental model, suggest that supraphysiological testosterone treatment stimulated gingival tissue repair during ligature-induced periodontitis, and it seems to be related to an anti-inflammatory and pro-resolutive mechanism resulting by the modulatory effect on PGE2 and IL-10 related to an enhanced angiogenesis.

Effects of red and infrared laser on post extraction socket repair in rats subjected to alendronate therapy

Abstract This study evaluated the effect of photobiomodulation therapy (PBMT) with a red or infrared laser on the repair of post extraction sockets in rats administered alendronate (ALN). Forty male rats were randomly allocated into four groups: Control Group (CTR): subcutaneous administration of saline solution throughout the experimental period; Alendronate Group (ALN): subcutaneous administration of alendronate during the entire experimental period; Alendronate/Red Laser Group (ALN/RL): administration of ALN and irradiation with a GaAlAs laser (λ 660 nm); and Alendronate/Infrared Laser Group (ALN/IRL): administration of ALN and irradiation with a GaAlAs laser (λ 830 nm). The first lower molars were extracted 60 days after the beginning of the administration of the drugs. The PBMT was applied after tooth extraction (7 sessions with intervals of 48 hours between sessions). Thirty days after tooth extraction, the animals were euthanized. Micro-CT and histometric analysis were performed to assess the bone healing and soft tissue repair of the tooth socket. The ALN group presented with more bone than the CTR; however, most of this bone was necrotic. ALN does not affect the bone microarchitecture. On the other hand, PBMT with IRL enhances the bone density due to the increase in the number and reduction in the spacing of the trabeculae. The amount of vital bone and connective tissue matrix was higher in the ALN/RL and ALN/IRL groups than in the ALN and CTR groups. PBMT enhanced the healing of the post extraction sockets in rats subjected to ALN administration. Furthermore, IRL improved the new bone microarchitecture.

Systemic Dietary Hesperidin Modulation of Osteoclastogenesis, Bone Homeostasis and Periodontal Disease in Mice.

This study aimed to evaluate the effects of hesperidin (HE) on in vitro osteoclastogenesis and dietary supplementation on mouse periodontal disease and femoral bone phenotype. RAW 264.7 cells were stimulated with RANKL in the presence or absence of HE (1, 100 or 500 µM) for 5 days, and evaluated by TRAP, TUNEL and Western Blot (WB) analyses. In vivo, C57BL/6 mice were given HE via oral gavage (125, 250 and 500 mg/kg) for 4 weeks. A sterile silk ligature was placed between the first and second right maxillary molars for 10 days and microcomputed tomography (µCT), histopathological and immunohistochemical evaluation were performed. Femoral bones subjected or not to dietary HE (500 mg/kg) for 6 and 12 weeks were evaluated using µCT. In vitro, HE 500 µM reduced formation of RANKL-stimulated TRAP-positive(+) multinucleated cells (500 µM) as well as c-Fos and NFATc1 protein expression (p < 0.05), markers of osteoclasts. In vivo, dietary HE 500 mg/kg increased the alveolar bone resorption in ligated teeth (p < 0.05) and resulted in a significant increase in TRAP+ cells (p < 0.05). Gingival inflammatory infiltrate was greater in the HE 500 mg/kg group even in the absence of ligature. In femurs, HE 500 mg/kg protected trabecular and cortical bone mass at 6 weeks of treatment. In conclusion, HE impaired in vitro osteoclastogenesis, but on the contrary, oral administration of a high concentration of dietary HE increased osteoclast numbers and promoted inflammation-induced alveolar bone loss. However, HE at 500 mg/kg can promote a bone-sparing effect on skeletal bone under physiological conditions.

Physiological testosterone replacement effects on male aged rats with orchiectomy-induced osteoporosis in advanced stage: a tomographic and biomechanical pilot study.

Aim: This study aimed to evaluate the effect of physiological testosterone replacement on male aged rats with orchiectomy-induced osteoporosis in advanced stage.Methods: Thirty male rats (Rattus norvegicus albinus, Holtzman lineage) were randomly distributed into 3 groups (n = 10): 1-sham, 2-orchiectomy (OCX), 3-OCX + testosterone replacement (OCX + T). On day 0, a sham or orchiectomy surgery was performed according to the groups. Thirty and sixty days after surgeries, the animals from OCX + T group received testosterone intramuscularly, and the rats in all groups were euthanized on day 77. The femurs were removed for micro-CT scanning and biomechanical test.Results: Orchiectomy resulted in a marked trabecular bone damage (p < 0.05), which was not reversed with testosterone treatment (OCX + T group). The femoral strength was lower in orchiectomized animals (p < 0.05), while the bone strength in OCX + T group was similar to that observed in the sham animals (p > 0.05) and correlated to this parameter the deformation of rupture was smaller in OCX + T group.Conclusion: In conclusion, testosterone depletion induced by orchiectomy established an osteoporotic environment, mainly affecting the trabecular bone. Moreover, even though testosterone treatment did not enhance these variables, the hormonal replacement improved the femoral fracture strength and promoted beneficial effects on the biomechanical parameters compromised by castration in femoral bone.

Leukotriene receptor antagonist reduces inflammation and alveolar bone loss in a rat model of experimental periodontitis.

BACKGROUND: Leukotrienes (LTs) participate in the process of tissue damage in periodontal disease by leukocyte chemotaxis and osteoclastic activation. The activation of Cysteinyl-LT receptor is associated with increased expression of proinflammatory molecules and osteoclastogenesis. However, its implications on periodontal disease progression have not been studied. The present study evaluated the effect of the cysteinyl-LT receptor antagonist (montelukast [MT]) on ligature-induced experimental periodontitis (EP) in rats. METHODS: Adult male Wistar rats were subjected to bilateral ligature-induced periodontitis and orally treated with MT (at doses of 10 or 30 mg/kg/d, MT10, and MT30, respectively). Sham animals had the ligatures immediately removed and received placebo treatment. Sets of animals were euthanized 7, 14, or 21 days after ligature placement and the mandibles were removed for macroscopic evaluation of alveolar bone loss (ABL). In addition, histological analysis of periodontal tissues, myeloperoxidase (MPO) activity of gingival tissues, and periodontal tissue expression of collagen type I, RUNX2, RANK, RANKL, OPG, BLT1, Cys-LTR1, LTA4H, and LTC4S were also analyzed. RESULTS: MT significantly reduced ABL at 14 (MT10 and MT30) and 21 days (MT10) (P < 0.05), gingival MPO at 7 (MT10) and 14 days (MT30) (P < 0.05), LTA4H, BLT1 and LTC4S gene expression on day 14 day (MT30, P < 0.05) and increased RUNX2 expression on day 14 (MT30, P < 0.05). CONCLUSION: Systemic therapy with MT decreases periodontal inflammation and ABL in ligature-induced periodontitis in rats.

Silencing matrix metalloproteinase-13 (Mmp-13) reduces inflammatory bone resorption associated with LPS-induced periodontal disease in vivo.

OBJECTIVES: The aim of this study was to evaluate the effect of specific inhibition of MMP-13 on inflammation and inflammatory bone resorption in a murine model of lipopolysaccharide (LPS)-induced periodontitis. MATERIALS AND METHODS: Periodontitis was induced in mice by micro-injections of LPS into the gingival tissues adjacent to the palatal surfaces of maxillary molars twice a week for 15 days. Matrix metalloproteinase-13 (Mmp-13) shRNA or a specific biochemical inhibitor were also injected into the same sites in alternating days with the LPS injections. Efficacy of shRNA-mediated silencing of Mmp-13 was verified by quantitative real-time polymerase chain reaction (qPCR) and immunoblot. Bone resorption was assessed by microcomputed tomography (uCT). Histological sections stained with hematoxylin/eosin (H/E) were used in the stereometric analysis of the inflammatory infiltrate. Gingival tissues were used to evaluate expression of Mmp-13, Il-6, Tnf-α, Ptgs2, and Rankl (qPCR). Protein levels of TGF-ß and IL-10 in the tissues were determined by enzyme-linked immunosorbent assays (ELISA) or by MMP-13 and p38 immunoblot. RESULTS: Silencing Mmp-13 expression reduced bone resorption significantly. Expression of Mmp-13, Il-6, and Tnf-α, as well as the protein levels of IL-6 and TNF-α, was reduced in the animals treated with adenovirus-delivered shRNA; however, these effects were not associated with modulation of p38 MAPK signaling. Interestingly, inhibition Mmp-13 did not affect the severity of inflammatory infiltrate. CONCLUSIONS: Site-specific inhibition of MMP-13 reduced bone resorption and production of inflammatory mediators associated with periodontal disease. CLINICAL RELEVANCE: The results suggest that site-specific inhibition of MMP-13 may be an interesting strategy to modulate inflammation and reduce bone resorption in osteolytic inflammatory diseases.

Testosterone Increases Fibroblast Proliferation in vitro Through Androgen and Estrogen Receptor Activation.

BACKGROUND: Skin-related disorders and periodontitis are distinct diseases that have been associated with altered levels of testosterone. Understanding the mechanisms through which testosterone mediates gingival enlargement in animals and humans is crucial for preventing or treating this condition. In this study, we investigated the impact of different doses of androgens, the role of aromatase inhibition, and the effects of testosterone association with sex hormone receptor antagonists or aromatase inhibitors on human gingival fibroblast proliferation and migration in vitro. METHODS: Fibroblasts were cultivated in Dulbecco's Modified Eagle's Medium in a humidified atmosphere and treated with different doses of testosterone or dihydrotestosterone, and testosterone in association with: aromatase inhibitor - anastrozole; antagonist of androgen receptors - flutamide; and antagonist of estrogen receptors - fulvestrant. RESULTS: Low (1nM) and high (1µM) doses of testosterone significantly increased cell migration, but the higher dose did not alter cell proliferation. Those effects were related to both androgen and estrogen receptors activation, as evidenced by the dihydrotestosterone and drug interaction groups. CONCLUSIONS: Testosterone association with sex hormone receptor antagonists flutamide and fulvestrant suggests that not only androgen receptors, but also estrogen receptors, may take part in fibroblast cell proliferation and migration in vitro.

Topical application of lectin Artin M improves wound healing in defects created in the palatal mucosa: an in vivo study in dogs.

Previous studies have shown that topical application of lectin Artin-M accelerates wound healing in the rat oral mucosa. The aim of this study was to evaluate, by means of histology and immunohistochemistry (IHC) the effects of Artin-M on wound healing in the palatal mucosa in dogs. Three full thickness wounds of 6 mm diameter were surgically created in the palatal mucosa of twenty dogs and randomly divided into three groups according to one of the treatment assigned: Group C-Control (coagulum); Group A-Artin-M gel; Group V-Vehicle (carboxymethylcellulose 3%). Each animal received all the three experimental treatments. Afterwards, four animals were killed at 2, 4, 7, 14 and 21 days post-surgery. Wounded areas were photographed and scored for macroscopic evaluation. Biopsies were harvested and used for descriptive histological analysis, proliferating cell nuclear antigen IHC and measurement of myeloperoxidase activity. The results demonstrated faster wound closure in group A in comparison to the other groups in all the periods evaluated. Histological analyses exhibited improved re-epithelialization and collagen fiber formation resulting in faster maturation of granulation tissue in group A compared to the other groups by day 14. Treatment with Artin-M gel significantly induced cell proliferation and increased volumetric density of fibroblasts at day 2 and 4 (p < 0.05). Neutrophil infiltration in group A was significantly higher than the other groups (p < 0.05) at the same time points. Collectively, our findings demonstrated that Artin-M may potentially favor wound healing on palatal mucosa lesions via recruitment of neutrophils and promotion of cell proliferation.

Role of testosterone and androgen receptor in periodontal disease progression in female rats.

BACKGROUND: Sex hormone therapy has strict recommendations in the treatment of postmenopausal symptoms, in which testosterone (TES) replacement may play a potential role. However, it remains unclear whether TES affects the course of chronic inflammation and alveolar bone loss in females. Herein, we investigated the role of androgen receptor and TES on the inflammatory response and alveolar bone resorption associated with ligature-induced periodontal disease in female rats. METHODS: Fifty female Holtzman rats were divided in five groups (n = 10/group): androgen receptor antagonist (flutamide); estrogen receptor antagonist (fulvestrant); TES supplementation; aromatase inhibitor (anastrozole); and TES plus anastrozole. Periodontitis was induced by ligatures around the lower first molars for 2 weeks. Twenty animals (n = 10/group) were used as untreated ligated or non-ligated controls. Bone loss and the number of osteoclasts were measured through radiographic and immunohistochemical analysis, respectively. Inflammatory cytokines, chemokines and bone markers were measured by multiplex immunoassay and ELISA in serum samples and periodontal tissues. RESULTS: The blockage of androgen receptor significantly increased radiographic bone loss and tissue levels of IL-1α (P <0.05), IL-1ß (P <0.001) and IL-10 (P <0.01) compared with the periodontitis group. Testosterone supplementation significantly increased EGF levels in tissue samples, whereas when combined with aromatase inhibitor anastrozole significantly increased both EGF and VEGF (P <0.05). None of the treatment conditions significantly impacted the number of osteoclasts compared with the periodontitis group. CONCLUSIONS: Androgen receptor activation is an important factor in the regulation of several inflammatory markers, and its blockage significantly increases bone loss.

Evaluation of bone turnover after bisphosphonate withdrawal and its influence on implant osseointegration: an in vivo study in rats.

OBJECTIVES: The aim of this study was to investigate bone turnover alterations after alendronate (ALD) withdrawal and its influence on dental implants osseointegration. MATERIALS AND METHODS: Seventy female Wistar rats were randomly divided in 2 groups that received on day 0 either placebo (control group-CTL; n = 10) or 1 mg/kg sodium alendronate (ALD; n = 60) once a week for 4 months. At day 120, ALD treatment was suspended for 50 animals. Then, a titanium implant was placed in the left tibia of each rat that were randomly allocated in five subgroups of ten animals each, according to the period of evaluation: day 0 (INT-0), day 7 (INT-7), day 14 (INT-14), day 28 (INT-28), and day 45 (INT-45) after ALD withdrawal. CTL group and a group that received ALD until the end of the experimental period (non-interrupted group-non-INT; n = 10) underwent implant placement on day 120. Animals were euthanized 28 days after implant surgery. Bone mineral density (BMD) of femur and lumbar vertebrae were evaluated by DXA, biochemical markers of bone turnover were analyzed by ELISA, and bone histomorphometry was performed to measure bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO). RESULTS: All groups receiving ALD showed higher BMD values when compared to CTL group, which were maintained after its withdrawal. Decreased concentrations in all bone turnover markers were observed in the non-INT group, and in the groups in which ALD was discontinued compared to the CTL group. The non-INT group showed lower %BIC and notably changes in bone quality, which was persistent after drug withdrawal. CONCLUSION: Collectively, the findings of this study demonstrated that ALD therapy decreased bone turnover and impaired bone quality and quantity around dental implants, and that its discontinuation did not reverse these findings. CLINICAL RELEVANCE: The severe suppression of bone turnover caused by the prolonged use of ALD may alter the capacity of bone tissue to integrate with the implant threads impairing the osseointegration process.

Dose-response assessment of chemically modified curcumin in experimental periodontitis.

BACKGROUND: CMC2.24, a novel tri-ketonic chemically modified compound based on natural di-ketonic curcumin, has been shown to reduce bone loss and inflammatory mediators in experimental periodontitis, however, a potential dose-response relationship was not determined. The purpose of this study was to assess the effects of different doses of CMC2.24 on inflammation and bone resorption in vivo and also to describe on the effects of CMC2.24 on macrophage response. METHODS: CMC2.24 was administered daily to animals for 28 days by oral gavage, at the following doses: 0 (control), 1, 3, 10, and 30 mg/kg of body weight. Experimental periodontitis was induced by injections of lipopolysaccharide (LPS) into the gingival tissues. Outcomes assessed were bone resorption, detection of tartrate-resistant acid phosphatase, and determination of gene expression. In vitro, macrophages (RAW264.7) were treated with different concentrations of CMC2.24: 1, 3, 10, and 30 µM and then subjected to different activation stimuli. Gene expression, phagocytic activity, production of reactive oxygen species (ROS) and cytokine production were evaluated. RESULTS: CMC2.24 inhibited bone resorption, osteoclastogenesis, and tumor necrosis factor (TNF)-α expression in vivo. These beneficial responses reached maximum levels at a dose of 1 mg/kg, i.e. no dose-dependent effect. In vitro, CMC2.24 reduced the production of TNF-α and interleukin-10, inhibited phagocytic activity and stimulated production of ROS. A dose-dependent effect was observed only for ROS production. CONCLUSION: Low doses of CMC2.24 (1 mg/kg/day) administered orally were sufficient to significantly inhibit alveolar bone resorption associated with the experimental periodontal disease; whereas in vitro macrophage inflammatory gene expression and phagocytosis were reduced, whereas production of ROS was stimulated.

Avaliação da combinação de poli (ácido láctico-co-glicólico) e poli-isopreno (Cellprene®): estudo histológico em ratos / Evaluation of the combination of poly (Lactic-Co-Glycolic Acid) and polyisoprene (Cellprene®) materials: histological study in rats

Resumo Introdução A quantidade e qualidade óssea na implantodontia é um fator de alta relevância quando se tem por objetivo instalar implantes e reabilitar pacientes. No entanto, essa disponibilidade é comprometida na maioria dos casos, havendo a necessidade da busca de novos biomateriais, membranas e substâncias para uma regeneração mais favorável. Objetivo: O objetivo deste estudo foi avaliar a resposta da neoformação óssea em defeitos críticos em calvárias de ratos utilizando scaffolds de fibras de blenda polimérica a partir de poli (ácido láctico-co-glicólico) e poli-isopreno (Cellprene®). O projeto foi aprovado pelo Comitê de Ética em Experimentação Animal. Material e método Neste estudo, foram utilizados 36 ratos (Rattus Norvegicus), variação albinus, Holtzman, adultos. Os animais foram submetidos à tricotomia na região da calota craniana e à confecção de defeitos ósseos circulares bilaterais com 5 mm de diâmetro. Os animais foram divididos em três grupos: GC - defeito sem colocação de biomaterial; GCol - scaffolds de colágeno (Bio-Gide, da empresa Geistlich Pharma Ag - Biomaterials); GPoli - scaffolds de fibras de blenda polimérica a partir de poli (ácido láctico-co-glicólico - Cellprene®). Cada grupo foi avaliado em quatro períodos experimentais (7, 15, 30 e 60 dias). Após esses períodos, os animais foram sacrificados, e as peças passaram por tramitação laboratorial de rotina e inclusão em parafina. Foram obtidos cortes semisseriados e corados pela técnica de hematoxilina e eosina para análise histométrica e histológica. Foi executada análise histométrica para avaliar a composição do tecido ósseo reparado (% osso). Os dados obtidos foram analisados estatisticamente com nível de significância de 95%. Resultado Foi verificado que o GCol apresentou maior preenchimento do defeito nos períodos de 30 e 60 dias em comparação aos GC e GPoli. Conclusão Os scaffolds de fibras de blenda polimérica a partir de poli (ácido láctico-co-glicólico) e poli-isopreno (Cellprene®) não apresentaram vantagens quando utilizados em defeitos críticos.

Abstract Introduction The bone quantity and quality in implant dentistry is a highly relevant factor when it aims the use of implants and rehabilitation in patients. However, this availability is compromised in most cases, with the need to research new biomaterials, membranes and substances for more favorable regeneration. Objective: The aim of this study was to evaluate the response of bone neoformation in critical defects in rat calvaries using polymeric blend fiber scaffolds from Poly (Lactic-Co-Glycolic Acid) and Polyisoprene (Cellprene®). The project was approved by the Animal Experimentation Ethics Committee. Material and method In this study 36 rats (Rattus Norvegicus), variation albinus, Holtzman, adults were used. The animals had trichotomy in the region of the skull and the confection of bilateral circular bone defects with a diameter of 5 mm. The animals were divided into 3 groups: Group GC - defect without biomaterial placement, Group GCol - collagen scaffolds (Bio-Gide, from Geistlich Pharma Ag - Biomaterials), Group GPoli - polymeric blend fiber scaffolds from Poly (Lactic-Co-Glycolic Acid)-Polyisoprene. Each group was evaluated in 4 experimental periods (7, 15, 30 and 60 days). After these periods the animals were sacrificed and the pieces underwent routine laboratory procedures and paraffin embedding. Semi-serial sections were obtained and stained by hematoxylin and eosin technique for histometric and histological analysis. Histometric analysis was performed to evaluate the composition of repaired bone tissue (% Bone). The data obtained were statistically analyzed with a significance level of 95%. Result It was found that the GCol group presented greater defect filling in the 30 and 60 days periods compared to the GC and GPoli groups. Conclusion Polymer blend fiber scaffolds from Poly (Lactic-Co-Glycolic Acid) and Polyisoprene (Cellprene®) did not have advantages when used in critical defects.

Intestinal host defense outcome is dictated by PGE2 production during efferocytosis of infected cells.

Inflammatory responses are terminated by the clearance of dead cells, a process termed efferocytosis. A consequence of efferocytosis is the synthesis of the antiinflammatory mediators TGF-ß, PGE2, and IL-10; however, the efferocytosis of infected cells favors Th17 responses by eliciting the synthesis of TGF-ß, IL-6, and IL-23. Recently, we showed that the efferocytosis of apoptotic Escherichia coli-infected macrophages by dendritic cells triggers PGE2 production in addition to pro-Th17 cytokine expression. We therefore examined the role of PGE2 during Th17 differentiation and intestinal pathology. The efferocytosis of apoptotic E. coli-infected cells by dendritic cells promoted high levels of PGE2, which impaired IL-1R expression via the EP4-PKA pathway in T cells and consequently inhibited Th17 differentiation. The outcome of murine intestinal Citrobacter rodentium infection was dependent on the EP4 receptor. Infected mice treated with EP4 antagonist showed enhanced intestinal defense against C. rodentium compared with infected mice treated with vehicle control. Those results suggest that EP4 signaling during infectious colitis could be targeted as a way to enhance Th17 immunity and host defense.

The role of androgens on periodontal repair in female rats.

BACKGROUND: Testosterone replacement enhances cognitive function and musculoskeletal health in postmenopausal women. However, the biological role of testosterone on inflammation and bone metabolism in females is not well understood. Our objective was to measure the impact of androgens and their receptors on periodontal tissues during periodontal repair in female rats. METHODS: Seventy female Holtzman rats were divided into seven groups (n = 10/group): negative control; repair control; androgen receptor antagonist (flutamide, 50 mg/kg, every other day); estrogen receptor antagonist (fulvestrant, 1.5 mg/kg/day); testosterone supplementation (durateston, 250 mg/kg, weekly); aromatase inhibitor (anastrozole, 0.2 mg /kg/day); testosterone plus anastrozole. Cotton ligatures were kept for 13 days, when pharmacological treatment was initiated. On day 14, the ligatures were removed. The rats were euthanized on the 17th or the 28th day (n = 5/group/period) for the evaluation of markers related to inflammation and bone. The tissue and serum samples were evaluated using a multiplexed immunoassay for the inflammatory targets. Radiographic and histologic analyses were performed to assess changes in tissues. RESULTS: Blockage of androgen receptors had little effect on inflammatory cell count, although it tended to increase interleukin (IL)-4, vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) as well as decrease IL-1ß, tumor necrosis factor (TNF)-α, and IL-6. Flutamide also significantly impaired bone repair (P < 0.05) and had greater osteoclast count, although this last difference was not statistically significant. Testosterone supplementation significantly increased the inflammatory cell count, decreased the levels of IL-4, IL-10, IL-1ß, IL-6, and TNF-α; and increased VEGF and EGF. CONCLUSION: The blockage of androgen receptors significantly impair bone repair in females through mechanisms that are different from those related to estrogen receptors.

Influence of obesity on experimental periodontitis in rats: histopathological, histometric and immunohistochemical study.

OBJECTIVES: This study assessed the influence of obesity on the progression of ligature-induced periodontitis in rats. MATERIALS AND METHODS: Forty-eight adult Wistar rats were randomly divided into two groups: the HL group (n = 24) was fed high-fat animal food to induce obesity, and the NL group (n = 24) was fed normolipidic animal food. Obesity was induced within a period of 120 days, and the induction of experimental periodontitis (EP) was subsequently performed for 30 days. The animals were euthanized after 7, 15, and 30 days, and the jaws were removed for histopathological, histometric, and immunohistochemical analyses. Tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor kappa beta ligand (RANKL), and osteoprotegerin (OPG) were analyzed via immunolabeling. RESULTS: Histological findings indicated that the inflammation was more extensive and lasted longer in the HL/EP; however, advanced destruction also occurred in the NL/EP. Greater bone loss was verified in the HL/EP group (2.28 ± 0.35) in the period of 7 days than in the NL/EP group (1.2 ± 0.29). High immunolabeling was identified in the HL/EP group in the initial periods for RANKL and TRAP, whereas the NL/EP group presented with moderate immunolabeling for both factors. The HL/EP and NL/EP groups showed low immunolabeling for OPG. CONCLUSIONS: Obesity induced by a high-fat diet influenced alveolar bone metabolism when associated with experimental periodontitis and caused a more severe local inflammatory response and alveolar bone loss. CLINICAL RELEVANCE: Obesity is related to greater alveolar bone loss and an accentuated local inflammatory response, which may be reflected in the clinical severity of periodontitis and dental loss.

Comparison of two different methods for detecting periodontal pathogenic bacteria

Aim: To perform a comparative analysis between two methods for detecting Porphyromonas gingivalis, Tannerella forsythia and Porphyromonas endodontalis in periodontal plaque samples. Methods: The study sample consisted of twenty systemically healthy patients showing generalized chronic periodontitis. The subgingival samples for microbiological analysis were collected before (baseline) and 60 days after a basic periodontal therapy from 30 non-adjacent affected sites (Probing Depth (PD): 5-7 mm, Clinical Attachment Loss (CAL) ≥ 5 mm, positive for Bleeding on Probing (BOP)). Microbiological analysis was performed by PCR and qPCR. To allow a comparative analysis between both methods, qPCR was divided in three different scores (score 2: presence of more than 100 bacteria; score 1: presence of 10-100 bacteria, and score 0: absence of bacteria), in accordance to DNA quantity, while for PCR two scores were assigned: presence or absence of bacteria. Results: qPCR demonstrated higher sensitivity in the detection of these pathogens compared with PCR when scores 1 and 2 were considered positive. However, when only score 2 was considered positive, PCR and qPCR showed better agreement. Conclusions: qPCR demonstrated higher sensitivity than conventional PCR for detection of low numbers of microorganisms and can be useful for the quantification of periodontopathogens. (AU)

Experimental osteonecrosis: development of a model in rodents administered alendronate.

The main objective of this study was to cause bisphosphonate-related osteonecrosis of the jaws to develop in a rodent model. Adult male Holtzman rats were assigned to one of two experimental groups to receive alendronate (AL; 1 mg/kg/week; n = 6) or saline solution (CTL; n = 6). After 60 days of drug therapy, all animals were subjected to first lower molar extraction, and 28 days later, animals were euthanized. All rats treated with alendronate developed osteonecrosis, presenting as ulcers and necrotic bone, associated with a significant infection process, especially at the inter-alveolar septum area and crestal regions. The degree of vascularization, the levels of C-telopeptide cross-linked collagen type I and bone-specific alkaline phosphatase, as well as the bone volume were significantly reduced in these animals. Furthermore, on radiographic analysis, animals treated with alendronate presented evident sclerosis of the lamina dura of the lower first molar alveolar socket associated with decreased radiographic density in this area. These findings indicate that the protocol developed in the present study opens new perspectives and could be a good starting model for future property design.

NOD1 in the modulation of host-microbe interactions and inflammatory bone resorption in the periodontal disease model.

Periodontitis is a chronic inflammatory condition characterized by destruction of non-mineralized and mineralized connective tissues. It is initiated and maintained by a dysbiosis of the bacterial biofilm adjacent to teeth with increased prevalence of Gram-negative microorganisms. Nucleotide-binding oligomerization domain containing 1 (NOD1) is a member of the Nod-like receptors (NLRs) family of proteins that participate in the activation of the innate immune system, in response to invading bacteria or to bacterial antigens present in the cytoplasm. The specific activating ligand for NOD1 is a bacterial peptidoglycan derived primarily from Gram-negative bacteria. This study assessed the role of NOD1 in inflammation-mediated tissue destruction in the context of host-microbe interactions. We used mice with whole-genome deletion of the NOD1 gene in a microbe-induced periodontitis model using direct injections of heat-killed Gram-negative or Gram-negative/Gram-positive bacteria on the gingival tissues. In vitro experiments using primary bone-marrow-derived macrophages from wild-type and NOD1 knockout mice provide insight into the role of NOD1 on the macrophage response to Gram-negative and Gram-negative/Gram-positive bacteria. Microcomputed tomography analysis indicated that deletion of NOD1 significantly aggravated bone resorption induced by Gram-negative bacteria, accompanied by an increase in the numbers of osteoclasts. This effect was significantly attenuated by the association with Gram-positive bacteria. In vitro, quantitative PCR arrays indicated that stimulation of macrophages with heat-killed Gram-negative bacteria induced the same biological processes in wild-type and NOD1-deficient cells; however, expression of pro-inflammatory mediators was increased in NOD1-deficient cells. These results suggest a bone-sparing role for NOD1 in this model.

Overexpression of Bcl-2, SOCS 1, 3 and Cdh 1, 2 are associated with the early neoplasic changes in modified 4-nitroquinoline 1-oxide-induced murine oral cancer model.

BACKGROUND: The objective was to assess histopathological changes and the expression of proliferating cell nuclear antigen (PCNA), Bcl-2, suppressor of cytokine signaling (SOCS) 1 and 3, Vimentin, TWIST1, and Cdh 1 and 2 in early stages of experimental oral carcinogenesis process using a shorter period of exposure to 4-nitroquinoline oxide (4-NQO) model. METHODS: In this study, 20 rats were divided into control group (n = 10), sacrificed on the first day of the experiment, and experimental group (n = 10) treated with 50 ppm of 4-NQO solution dissolved in drinking water for 8 and 12 weeks. The histological sections were stained with H&E or subjected to immunohistochemistry for detecting PCNA, Bcl-2, SOCS 1 and 3, and STAT 3. Some specimens were used for verification of Vimentin expression, Cdh 1, Cdh 2, and TWIST1 by RT-qPCR. RESULTS: At both 8 and 12 weeks, morphological changes occurred mainly in the posterior portion of the tongue and were limited to the epithelial tissue, including moderate to severe dysplasia at 8 weeks, and severe dysplasia with exacerbation of atypical cells at 12 weeks. Expression of SOCS 1 and 3 increased from 8 to 12 weeks (P < 0.05), whereas STAT 3 expression was reduced mainly at 12 weeks (P < 0.05) in comparison with the control group. The expression of all epithelial-mesenchymal transition markers (EMT) was increased after 12 weeks, reaching statistical significance (P < 0.05) for Cdh 1 and 2. CONCLUSIONS: Together, the results suggested that overexpression of Bcl-2, SOCS 1 and 3, and Cdh 1 and 2 is associated with the early neoplasic changes in modified 4-nitroquinoline 1-oxide-induced murine oral cancer model.

Experimental osteonecrosis: development of a model in rodents administered alendronate

Abstract The main objective of this study was to cause bisphosphonate-related osteonecrosis of the jaws to develop in a rodent model. Adult male Holtzman rats were assigned to one of two experimental groups to receive alendronate (AL; 1 mg/kg/week; n = 6) or saline solution (CTL; n = 6). After 60 days of drug therapy, all animals were subjected to first lower molar extraction, and 28 days later, animals were euthanized. All rats treated with alendronate developed osteonecrosis, presenting as ulcers and necrotic bone, associated with a significant infection process, especially at the inter-alveolar septum area and crestal regions. The degree of vascularization, the levels of C-telopeptide cross-linked collagen type I and bone-specific alkaline phosphatase, as well as the bone volume were significantly reduced in these animals. Furthermore, on radiographic analysis, animals treated with alendronate presented evident sclerosis of the lamina dura of the lower first molar alveolar socket associated with decreased radiographic density in this area. These findings indicate that the protocol developed in the present study opens new perspectives and could be a good starting model for future property design.