Green tea extract rich in epigallocatechin gallate impairs alveolar bone loss in ovariectomized rats with experimental periodontal disease.

Periodontal disease and osteoporosis are characterized by bone resorption, and researchers have shown an association between these two diseases through increasing loss of systemic bone mass and triggering alveolar bone loss. Green tea is a common and easily accessible beverage, and evidences show that flavonoid epigallocatechin gallate (EGCG) could decrease bone loss in pathologies such as osteoporosis and periodontal disease. In order to verify its possible effects and apply them in the treatment and prevention of these diseases, this investigation aimed to evaluate the influence of green tea extract (GTE) on bone metabolism of ovariectomized rats after experimental periodontal disease (EPD) by histological, morphological and microtomographic parameters. Wistar female rats were divided into Sham, Sham + EPD, Sham + EPD + GTE, OVX, OVX + EPD and OVX + EPD + GTE groups. Immediately after surgery, gavage administration of 50 mg/kg of green tea extract (GTE) was performed for 60 days, with subsequent induction of periodontal disease by ligature 15 days before euthanasia. Mandible and femur samples were collected for histological, morphometric and microtomographic analysis. The results were analysed by means of statistical software with significance set at 5%. Histological and morphometric analysis showed a significant decrease in alveolar and femoral trabecular bone loss in groups that received GTE. Microtomographic results showed that trabecular thickness and bone surface density values in alveolar bone interradicular septum of the OVX + EPD + GTE groups were similar to the Sham group. The results obtained suggest that green tea extract may improve bone metabolism in osteoporotic rats with periodontal disease.

Challenges of the minimum postmortem interval (PMImin) estimation in forensic scenarios: Is dental histology an alternative?

Postmortem interval (PMI) estimates the time since death. Teeth are perennial elements capable of remaining intact in taphonomic environmental circumstances. The objective was to evaluate the feasibility of estimating the minimum postmortem interval (PMImin) through histological analyses of dental tissues exposed to burial and drowning conditions, simulating common scenarios in forensic practice. A total of n = 99 teeth were analyzed and divided into four groups: control (T0), one month (T1), three months (T2), and six months (T3). The control sample comprised 10 teeth, while T1, T2 e T3 were divided into three different subgroups: controlled environment, buried, and drowned. For each subgroup, ten samples were used. Following exposure to taphonomic conditions, the specimens were processed, and histological sections were obtained. The two-way ANOVA test and the Tukey's post-hoc test were employed for the quantitative analysis of dentin collagen fibrils, revealing statistically significant differences (α = 5 %). This allowed for the estimation of the PMImin at three months by observing pixel counts exceeding 13e+05 in drowned teeth and greater than 8e+05 in buried teeth. Qualitative analysis revealed that the PMImin of drowned teeth was estimated at one month due to the absence of the periodontal ligament (PDL) and at six months due to the absence of predentin and partial degradation of the cementum. For buried teeth, the three-month PMImin was indicated by the absence of PDL and partial cementum degradation. The absence of pulp and remnants of predentin characterized the six-month PMImin. Qualitative and quantitative histological characteristics and parameters are potential to estimate PMImin in forensic scenarios spanning up to six months.

Histological and histochemical effects after occlusion alteration in suprahyoid muscles.

This study verified the effect of unilateral teeth extraction on the suprahyoid muscles in gerbils (Meriones unguiculatus). Ten adult male gerbils weighing about 50g had induced occlusal alterations by upper molar teeth extraction on the left side while the other ten animals were only subjected to surgical stress, control group. After 60 days, animals of both groups, experimental and control had the suprahyoid muscles removed and processed for histological and histochemical (adenosine triphosphatase (ATPase), nicotine adenine dinucleotide tetrazolium reductase (NADH-TR) and succinate dehydrogenase (SDH)) purposes. The fiber type area was estimated in % according to Weibel method (point-counting method) using a test-system. The myosinic ATPase pH 4.7 activity in the control group of the digastric, milohyoid and geniohyoid muscles presented a small area of type I fiber and a larger area of type IIa fibers; in the experimental group, significant contractile capacity alteration was not observed. Samples of the digastric, milohyoid and geniohyoid muscles, after SDH activity, showed a small area with high metabolic activity fibers, and a large area with intermediary and low metabolic activity fibers in the control group. The milohyoid muscle of the experimental group presented low metabolic fibers in a reduced area, in both sides, however without significant difference. In the experimental group, high metabolic fibers were observed on the left side in a reduced area in the geniohyoid muscle, but without statistical significance. Thus, the geniohyoid muscle did not change the metabolic activity after occlusal alteration. In conclusion, 60 days of unilateral malocclusion induced was able to alter the fibers oxidative activity of the suprahyoid muscles, however, it does not affect the contractile property of the fibers. The digastric muscle has adequate fibers to produce fast contraction and able to resist to fatigue in intermediate degrees, but became more fatigable after unilateral exodontia.

Bone repair using mineral trioxide aggregate combined to a material carrier, associated or not with calcium hydroxide in bone defects.

Mineral trioxide aggregate (MTA) is a powder aggregate containing mineral oxides with a good biological action and may facilitate the regeneration of the periodontal ligament and formation of bone. Calcium hydroxide demonstrates antibacterial properties, enhances tissue dissolution, and induces bone formation. The objective of this study was to evaluate the MTA in the bone healing process and verify if the calcium hydroxide P.A. can improve and accelerate this process. It was used forty male Wistar rats, which were divided into two groups, considering or not the use of calcium hydroxide P.A. solution before treatment. Thus, each one of these groups was divided in four groups with five animals each, according to the treatment and the defect filled by: animal's coagulum, monoolein gel, MTA in aqueous solution, and MTA combined with monoolein gel. After 10 days, the animals were perfused and the right hemimandibles removed for histological analysis. Statistical analysis of the data showed significant difference between all analyzed groups when it was made comparisons using or not calcium hydroxide P.A. (p<0.0001). There was found statistical difference between the groups that was inserted or not MTA, independently the calcium hydroxide application (p<0.05). Results showed that the MTA used was able to induce bone regeneration and had its action optimized when combined to calcium hydroxide P.A.

Lycopene enhances bone neoformation in calvaria bone defects of ovariectomized rats

Abstract Osteoporosis can affect a significant part of the population and fractures are the most common complications associated with this disease, leading to high public health costs. Thus, the prevention of fractures is relevant to individuals with signs and symptoms as well as to the health system. Postmenopausal osteoporosis has been associated with oxidative stress, emphasizing the importance of an efficient defense system to maintain bone health. Lycopene is a carotenoid with antioxidant properties that may stimulate osteoblastogenesis and inhibit osteoclastogenesis. The purpose of this investigation was to analyze the influence of lycopene in the bone neoformation of calvaria defects in ovariectomized rats utilizing the concentration of 45 mg/kg. Wistar Hannover female rats were divided into ovariectomized and sham groups. The ovariectomized animals received 45 mg/kg lycopene (OvxL) or water (Ovx) by daily gavage the day after ovariectomy/sham surgery for 16 weeks. Twelve weeks after ovariectomy, there were performed 5-mm calvaria defects followed by euthanasia after 4 weeks. Samples of bone tissue were collected to perform morphological and morphometrical analysis of the neoformed bone area, and percentage with Software Image J. Morphological evaluation showed mature bone with more osteocytes in the group OVxL when compared to the other groups. The morphometrical analysis demonstrated a significant increase of bone neoformation in the group OvxL (p<0.05). The data obtained suggest that lycopene benefits bone repair in the absence of estrogenic hormones.

Resumo A osteoporose afeta grande parte da população e as fraturas são as complicações mais importantes relacionadas a essa doença, gerando altos gastos para o poder público. Dessa forma, a prevenção de fraturas decorrentes da osteoporose torna-se relevante tendo em vista que gera benefícios tanto para o indivíduo acometido pela doença quanto para o sistema de saúde. A osteoporose pós menoupasa tem sido associada ao estresse oxidativo, portanto, um eficiente sistema de defesa antioxidante é primordial para a manutenção da saúde óssea. O licopeno é um carotenoide antioxidante que aparentemente estimula a osteoblastogênese e inibe a osteoclastogênese. O objetivo deste estudo foi analisar a influência do licopeno na neoformação óssea em defeitos de calvária em ratas ovariectomizadas utilizando a concentração de 45 mg/kg. Foram utilizados 15 ratas Wistar Hannover pesando aproximadamente 200g, sendo que 10 animais foram submetidos à ovariectomia bilateral e 5 (Grupo Sham) foram submetidos à simulação da cirurgia de ovariectomia bilateral. Os animais ovariectomizados foram divididos aleatoriamente em 2 grupos: Ovariectomizado (Ovx) e Ovariectomizado Licopeno (OvxL) que receberam água e licopeno respectivamente, por sonda gástrica, diariamente. As administrações iniciaram-se no dia seguinte à cirurgia de ovariectomia e/ou da exposição dos ovários e foram mantidas por 120 dias, data de realização da eutanásia. O grupo Sham recebeu água diariamente. Noventa dias após a ovariectomia bilateral foram confecionados defeitos ósseos nas calvárias de todos os animais e após trinta dias as ratas foram eutanasiadas. As amostras de tecido ósseo foram coletadas e foi realizado o processamento para a obtenção das lâminas histológicas. Foram realizadas as análises morfológicas e morfométrica, onde foi estimada a área (mm2) e porcentagem (%) relativa de osso neoformado utilizando o Software Image J. A avaliação morfológica evidenciou a ação benéfica do licopeno pois os animais que receberam esse antioxidante apresentaram um tecido ósseo mais maduro, com maior presença de osteócitos quando comparados aos demais grupos. Por meio das análises morfométricas verificou-se maior neoformação óssea para os animais que receberam o licopeno (p<0,05). Diante dos resultados obtidos, concluiu-se que o licopeno na concentração de 45 mg/Kg teve efeito benéfico no processo de reparação, promovendo significante formação óssea frente à ausência de hormônios estrogênicos.

Osteoinductivity potential of rhBMP-2 associated with two carriers in different dosages.

The objective of this study was to evaluate bone formation after application of different doses of recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with monoolein or poloxamer gels, in critical bone defects of rats. Forty-five Wistar rats were divided into nine treatment groups with five animals each: I: application of 1 µg rhBMP-2 + monoolein; II: 3 µg rhBMP-2 + monoolein; III: 7 µg rhBMP-2 + monoolein; IV: 1 µg rhBMP-2 + poloxamer; V: 3 µg rhBMP-2 + poloxamer; VI: 7 µg rhBMP-2 + poloxamer; VII: monoolein only; VIII: poloxamer only; and IX: critical bone defect only. A critical-sized defect of 6 mm diameter was produced in the left parietal bone and it was filled with gels of the above mentioned treatments. After 2 weeks, the calvarial bones were removed for histological processing. Bone formation in the groups that received poloxamer gel and rhBMP-2 was not significantly different from the control group (IX). Groups receiving monoolein and rhBMP-2 (1 and 3 µg) and those that received only the carriers (VII and VIII) had less bone formation in relation to the control. The association of rhBMP-2 to both poloxamer and monoolein did not exhibit any significant differentiation in bone formation in comparison with the control group.

Collagen fibers evaluation after rhBMP-2 insertion in critical-sized defects.

The objective of this investigation was to assess the quantity of collagen fibers with different dosages of recombinant human bone morphogenetic protein, type 2 (rhBMP-2) associated with two different carriers, monoolein and poloxamer gels, in critical bone defects created in the calvaria of Wistar rats. Forty male adult Wistar rats were divided into eight groups of 5 animals each-group I: critical bone defect with application of 1 microg of rhBMP-2 combined with monoolein gel; group II: 3 microg of rhBMP-2 combined with monoolein gel; group III: 7 microg of rhBMP-2 combined with monoolein gel; group IV: 1 microg of rhBMP-2 combined with poloxamer gel; group V: 3 microg of rhBMP-2 combined with poloxamer gel; group VI: 7 microg of rhBMP-2 combined with poloxamer gel; group VII: monoolein gel only and group VIII: poloxamer gel only. A critical-sized defect of 6mm diameter was produced in the left parietal bone using a surgical round bur and a high-speed micromotor. The bone defects were filled according to the group that animals belonged and after two weeks the rats were perfused and their calvarial bones were removed for histological processing, and collagen fibers quantification. Differences among the eight groups were statistically analyzed by Anova and Bonferroni test (p<0.05). The results did not show statistical difference between the groups, in exception, between the comparisons II and III. According to the experimental methodology used in this research, it was observed, in a general way, a qualitative inverse relationship between collagen fibers presence and rhBMP-2 quantity inserted in the critical bone defect, associated or not to a material carrier.

Microwave-induced fast decalcification of rat bone for electron microscopic analysis: an ultrastructural and cytochemical study.

Bone decalcification is a time-consuming process. It takes weeks and preservation of the tissue structure depends on the quality and velocity of the demineralization process. In the present study, a decalcification methodology was adapted using microwaving to accelerate the decalcification of rat bone for electron microscopic analysis. The ultrastructure of the bone decalcified by microwave energy was observed. Wistar rats were perfused with paraformaldehyde and maxillary segments were removed and fixed in glutaraldehyde. Half of specimens were decalcified by conventional treatment with immersion in Warshawsky solution at 4 degrees C during 45 days, and the other half of specimens were placed into the beaker with 20 mL of the Warshawsky solution in ice bath and thereafter submitted to irradiation in a domestic microwave oven (700 maximum power) during 20 s/350 W/+/-37 degrees C. In the first day, the specimens were irradiated 9 times and stored at 40 degrees C overnight. In the second day, the specimens were irradiated 20 times changing the solution and the ice after each bath. After decalcification, some specimens were postfixed in osmium tetroxide and others in osmium tetroxide and potassium pyroantimonate. The specimens were observed under transmission electron microscopy. The results showed an increase in the decalcification rate in the specimens activated by microwaving and a reduction of total experiment time from 45 days in the conventional method to 48 hours in the microwave-aided method.

Microwave-induced fast decalcification of rat bone for electron microscopic analysis: an ultrastructural and cytochemical study

Bone decalcification is a time-consuming process. It takes weeks and preservation of the tissue structure depends on the quality and velocity of the demineralization process. In the present study, a decalcification methodology was adapted using microwaving to accelerate the decalcification of rat bone for electron microscopic analysis. The ultrastructure of the bone decalcified by microwave energy was observed. Wistar rats were perfused with paraformaldehyde and maxillary segments were removed and fixed in glutaraldehyde. Half of specimens were decalcified by conventional treatment with immersion in Warshawsky solution at 4ºC during 45 days, and the other half of specimens were placed into the beaker with 20 mL of the Warshawsky solution in ice bath and thereafter submitted to irradiation in a domestic microwave oven (700 maximum power) during 20 s/350 W/±37ºC. In the first day, the specimens were irradiated 9 times and stored at 40ºC overnight. In the second day, the specimens were irradiated 20 times changing the solution and the ice after each bath. After decalcification, some specimens were postfixed in osmium tetroxide and others in osmium tetroxide and potassium pyroantimonate. The specimens were observed under transmission electron microscopy. The results showed an increase in the decalcification rate in the specimens activated by microwaving and a reduction of total experiment time from 45 days in the conventional method to 48 hours in the microwave-aided method.

A preservação da estrutura de ossos é dependente da qualidade e da velocidade em que ocorre o processo de desmineralização. Neste estudo foi observada a ultraestrutura de maxila de rato descalcificada utilizando microondas. Ratos Wistar sofreram perfusão com paraformaldeído e o segmento de maxila retirado e fixado em glutaraldeído. Após esta etapa algumas amostras foram descalcificadas por imersão em solução de Warshawsky durante 45 dias a 4(0)C. Outras amostras foram submetidas a irradiação por microondas (forno de microondas doméstico 700 Watts de potência), durante 20 s/350 W/ ± 37ºC. No primeiro dia foram realizadas um total de 9 irradiações e os espécimes foram deixadas posteriormente a 4ºC por 12 h na solução descalcificadora sem agitação. No segundo dia, os fragmentos foram submetidos à nova irradiação totalizando 20 banhos, trocando-se a solução e o gelo a cada banho. A seguir algumas amostras foram pós-fixadas com tetróxido de ósmio e outras com tetróxido de ósmio e piroantimonato de potássio. As amostras foram observadas em microscópio eletrônico de transmissão. Os resultados mostraram que o processo de descalcificação ativado por microondas reduziu para 48 h o período de descalcificação, o qual pelo método tradicional ocorre em 45 dias.

Bone morphogenetic proteins: its application in the process of repairing the dentin pulp complex

Evolution of knowledge related to molecular Biology has been applied in different areas of clinical Biology, Dentistry among them, allowing the use of these new advances in the treatment of offenses to the pulpal organ. The use of bone morphogenetic proteins (BMPs) represents another possibility in the treatment of exposed pulps with no contact with aggressive agents and free from undesirable effects to maintain pulp vitality.

La evolución del conocimiento relacionado a la Biología Molecular ha sido aplicado en diferentes áreas de la Biología Clínica, entre ellas la Odontología, permitiendo el uso de esos nuevos adelantos en el tratamiento de agresiones al órgano pulpar. El uso de proteínas óseas morfogenéticas (BMPs), representa otra posibilidad en el tratamiento de pulas expuestas sin la presencia de agentes agresores, libre principalmente de los efectos indeseables a la mantención de la vitalidad pulpar.

Dinámica de la descalcificación de tejido mineralizado de perros a través de microondas / Decalcification dynamic of dog mineralized tissue by microwaves

The aim of this article is to present the decalcification process dynamic of mineralized tissue in dogs, teeth and jaw, comparing the traditional decalcification method, immersion, and microwave, immersion followed by irradiation using a domestic microwave oven, accompanying the liberation of calcium through spectrophotometer of atomic absorption. It was used as decalcified agent, EDTA solution or nitric acid. The results showed that with the use of nitric acid (5 percent), after 15 days, the irradiated fragments could be processed for histological analysis, otherwise the tooth not irradiated need to be submerged for 65 days. The EDTA decalcified action was slower than the nitric acid. The histological observations of the irradiated samples showed an excellent preservation of the morphological characteristics, independently of the decalcified agent used.

El objetivo de este artículo fue presentar, un método de descalcificación dinámico de tejido mineralizado de perros, dientes y mandíbula, comparando el método de descalcificación tradicional e irradiación en horno de microondas, analizando la liberación de calcio en espectro fotómetro de absorción atómica. Usamos como agente descalcificante, solución de EDTA y acido nítrico. Los resultados mostraron que los fragmentos descalcificados con ácido nítrico después de 15 días, ya podían ser preparados para análisis histológico, el diente al ser irradiado tardó 65 días para ser descalcificado. El EDTA descalcificó lentamente, en relación al ácido nítrico. Las observaciones histológicas de las muestras irradiadas mostraron una excelente preservación de las características morfológicas independiente del agente descacificador usado.

Histological Evaluation of the Bone Repair Using Mineral Trioxide Aggregate Combined to a Material Carrier / Evaluación Histológica del Proceso de Reparación Ósea Utilizando Agregado Trióxido Mineral Combinado a un Material Cargador

The aim of this study was to present a possible carrier for MTA, monoolein gel, with the objective to maintain this material in the place that was inserted and verify if this procedure is able to optimize its action. The data were evaluated by histomorphometric method and submitted to statistical analysis. The histological responses observed in this study indicate that the MTA is a reliable material and should be considered effective in bone periapical defects and the monoolein gel was capable to maintain the MTA in situ.

El objetivo de este estudio fue presentar el gel de monoleína como un posible cargador para el MTA y verificar si es capaz de optimizar su acción. Los datos obtenidos fueron evaluados por métodos histomorfométricos y sometidos a análisis estadístico. El resultado histológico reveló que el MTA es un material efectivo para utilizarlo en defectos óseos periapicales y que el gel de monoleína es capaz de mantener el MTA in situ.

Clinical and Histochemical Alterations of the Periodontal Ligament in Gerbils after Malocclusion Induced / Alteraciones Clínicas e Histoquímicas del Ligamento Periodontal en Gerbiles Después de Maloclusión Inducida

The aim of this article is to show the clinical and histochemical alterations of the first periodontal ligament, on the right side, after upper molars teeth extraction on the left side in gerbils. After two months, the periodontal ligaments were removed and processed for histochemical analysis. The data showed that TRAP reaction was able to evidence the osteoclastic activity in the hyperfunction hemimandible, right side, explaining the functional changes in the periodontal ligament after teeth extraction, and a little gingival recession and radicular exposure of teeth without function was observed at inferior molars of the left side.

El objetivo de este artículo es mostrar las alteraciones clínicas e histoquímicas del primer ligamento periodontal del lado derecho, después de la extracción del molar superior izquierdo en gerbiles {Meriones unguiculatus). Luego de dos meses, los ligamentos periodontales fueron retirados y procesados para el análisis histoquímico. Los resultados mostraron que la reacción de TRAP es capaz de evidenciar la actividad osteoclástica en la hiperfunción de la semimandíbula derecha, explicando los cambios funcionales del ligamento periodontal después de la extracción dental, siendo observada una pequeña recesión gingival y exposición radicular de los dientes sin función, en los molares inferiores izquierdos.