Análise da influência de solução de biovidro no procedimento endodôntico regenerativo / Analysis of the influence of bioglass solution on the regenerative endodontic procedure

Este estudo avaliou os efeitos de uma solução experimental de vidro bioativo F18 dopado com cobalto (F18Co) no reparo tecidual após procedimento endodôntico regenerativo (REP, do inglês regenerative endodontic procedure) em molares de ratos. A solução de F18Co foi preparada misturando o pó de F18Co com água destilada na proporção de 1:5. Os primeiros molares superiores direito ou esquerdo de 12 ratos Wistar foram utilizados, nos quais as polpas foram expostas, removidas e os canais irrigados com hipoclorito de sódio (NaOCl) 2,5%, seguido de ácido etilenodiaminotetraacético (EDTA) 17% (5 min cada). Em seguida, os molares foram divididos em dois grupos (n = 6): REP-SS e REP-F18Co, nos quais receberam irrigação final (5 min) com solução salina (SS) ou solução de F18Co, respectivamente. Em seguida, foi induzido sangramento intracanal e o dente foi selado. Molares não tratados foram usados como controles (n = 3). Após 21 dias, os ratos foram eutanasiados e as peças processadas para análise da formação de tecido mineralizado e tecido mole dentro do canal radicular, pela técnica de hematoxilinaeosina. A presença e maturação do colágeno foram avaliadas por coloração de tricrômio de Masson e picrosírius red. Análises da imunomarcação do antígeno nuclear de proliferação celular (PCNA) e osteocalcina (OCN) foram realizadas. Os dados foram submetidos ao teste de Mann-Whitney U (p < 0,05). Houve formação semelhante de tecido mineralizado em espessura e comprimento nos grupos REP-SS e REP-F18Co (p > 0,05). Em relação à presença de tecido neoformado no interior dos canais radiculares, a maioria dos espécimes de REP-F18Co apresentou formação de tecido até o terço cervical do canal radicular, enquanto do grupo REP-SS, até o terço médio (p < 0,05), e houve maior maturação colágena em REP-F18Co (p < 0,05). O número de células positivas para PCNA encontradas no terço apical do canal radicular foi significativamente maior em REP-F18Co, assim como a imunomarcação de OCN, que foi severa na maior parte dos espécimes do grupo REP-F18Co, e leve em REPSS. Conclui-se que a irrigação final com solução de biovidro F18Co em REP não influenciou a formação de tecido mineralizado, mas induziu a formação de tecido conjuntivo no interior dos canais radiculares, com maior maturação colágena, aumento no número de células positivas para PCNA e na imunomarcação de OCN.

This study evaluated the influence of an experimental solution of cobalt-doped F18 bioactive glass (F18Co) on tissue repair following regenerative endodontic procedure (REP) in rat molars. The F18Co solution was prepared at a ratio of 1:5 F18Co powder to distilled water. The right or left upper first molars of 12 Wistar rats were used, where the pulps were exposed, removed, and irrigated with 2.5% sodium hypochlorite (NaOCl), followed by 17% ethylenediaminetetraacetic acid (EDTA) (5 min each). Subsequently, the molars were divided into two groups (n = 6): REP-SS and REP F18Co, where they received a final irrigation (5 min) with saline solution (SS) or F18Co solution, respectively. Then, intracanal bleeding was induced, and the tooth was sealed. Untreated molars were used as controls (n = 3). At 21 days, the rats were euthanized, and the specimens were processed for analysis of mineralized tissue and soft tissue formation inside the root canal using haematoxylin-eosin. The presence and maturation of collagen was evaluated by Masson's trichrome and picrosirius red staining. Immunolabeling analyses of proliferating cell nuclear antigen (PCNA) and osteocalcin (OCN) were performed. The data were submitted to the Mann-Whitney U test (P < 0.05). There was similar formation of mineralized tissue in thickness and length in REP-SS and REP-F18Co groups (P > 0.05). Regarding the presence of newly formed soft tissue, most specimens of the REP-F18Co had tissue formation up to the cervical third of the canal, while the REP-SS specimens showed formation up to the middle third (P < 0.05), and there was higher maturation collagen in REP-F18Co (P < 0.05). The number of PCNA-positive cells found in the apical third of the root canal was significantly higher in the F18Co group, as well as the OCN immunolabeling, which was severe in most specimens of REP-F18Co, and low in most specimens of REP SS. In conclusion, the final irrigation with F18Co bioactive glass solution in REP did not influence mineralized tissue formation but induced soft tissue formation inside the root canals, with higher collagen maturation, and an increase in PCNA-positive cells and OCN immunolabeling

Influência do LED Violeta associado ou não ao gel clareador na inflamação, mineralização e maturação colágena do tecido dentinário e pulpar / Influence of violet LED associated or not with peroxide gel on inflammation, mineralization, and collagen fiber maturation in dentin and pulp tissue

Objetivo: Avaliar a influência do LED Violeta, associado ou não ao gel clareador a base de peróxido de hidrogênio (PH) a 17,5% no complexo dentino-pulpar de ratos. Materiais e métodos: Molares superiores de 80 ratos foram distribuídos nos grupos (n = 10): CONT ­ sem tratamento, PH ­ 1 aplicação de 30 minutos de PH a 17,5%, LED ­ 1 aplicação de 20 minutos do LED Violeta, e PH+LED - aplicação do PH e LED Violeta. Imediatamente (T0), e aos 7 (T1), 15 (T2) e 30 dias (T3) após o tratamento, os ratos foram eutanasiados e as maxilas processadas para avaliação histológica, imunoistoquímica (IL-17, IL-23 e osteocalcina), e de picrosírius red, sendo realizados os testes de Wilcoxon e Mann-Whitney e Teste-T pareado e Teste-T, respectivamente. (α = 0,05). Resultados: Necrose e infiltrado inflamatório severo foram observados nos grupos PH e PH+LED. Apenas o grupo PH+LED manteve a imunomarcação severa para IL-17 e IL-23, diferindo do grupo LED e PH que apresentaram moderada imunomarcação em T0. Os grupos PH e PH+LED apresentaram severa imunomarcação de OCN em T2 e moderada imunomarcação em T3. O grupo LED apresentou menor quantidade de fibras imaturas em T2 e T3 que o grupo CONT. Conclusão: A terapia com LED violeta não induziu inflamação e fibrose no tecido pulpar, apesar de acelerar a maturação das fibras de colágeno da dentina e, quando associada ao peróxido de hidrogênio, pode tornar os dentes mais sensíveis(AU)

Objective: Was to evaluated the influence of the Violet LED, associated or not with a 17.5% hydrogen peroxide (HP) bleaching gel in the dentin-pulp complex of rats. Materials and methods: Upper molars of eighty Wistar rats were distributed in the groups (n = 10): CONT - without treatment, HP - 1 application of 30 minutes of 17.5% HP, LED - 1 application of 20 minutes of the Violet LED, and HP+LED - application of HP and LED Violet. Immediately (T0), and at 7 (T1), 15 (T2) and 30 days (T3) after treatment, the rats were euthanized and the jaws were processed for histological, immunohistochemical evaluation (IL-17, IL-23 and osteocalcin), and picrosirius red, with Wilcoxon and Mann-Whitney tests and paired T-test and T-test, respectively (α = 0.05). Results: Necrosis and severe inflammatory infiltrate were observed in the PH and PH+LED groups. Only the PH+LED group maintained severe immunostaining for IL-17 and IL-23, differing from the LED and PH group which presented moderate T0 immunostaining. The PH and PH+LED groups presented severe immunostaining of OCN in T2 and moderate immunostaining in T3. The LED group had a lower amount of immature fibers in T2 and T3 than the CONT group. Conclusion: Violet LED therapy induced no inflammation and fibrosis in the pulp tissue, however accelerating the maturation of dentin collagen fibers and, when associated with hydrogen peroxide, can make the teeth more sensitive(AU)

Experimental acute sepsis reduced number of osteocalcin immunolabeled cells in periodontal ligament

Abstract The aim of this study was to evaluate the effect of acute sepsis in the periodontal ligament, alveolar and furcation bone in absence of periodontitis induction through histological and immunohistochemical analyses. A septic rat model was established by cecal ligation and puncture (CLP). Twelve rats were randomly divided into CLP (n=6) and Sham (n=6) groups. The animals were euthanized at 24 h and hemimandibles were submitted to histomorfometric (bone matrix, collagenous fibers, fibroblasts, osteocytes, inflammatory cells, and blood vessels) and immunohistochemical (BMP-2/4, RANKL and osteocalcin) evaluation in alveolar bone, furcation bone and periodontal ligament. Our results demonstrated that histomorphometric parameters were similar in alveolar bone, furcation bone and periodontal ligament of Sham and CLP rats. Regarding to immunohistochemical analyses, the number of BMP-2/4 and RANKL immunolabeled cells was also similar in both groups. Furthermore, it was detected a reduction in the osteocalcin immunolabeled cells in periodontal ligaments of CLP compared to Sham rats (p=0.0014). In conclusion, the acute sepsis induction resulted in reduced number of osteocalcin labelled cells in periodontal ligament region. Moreover, no significant histological differences were observed in the periodontium of rats under acute sepsis. Considering the role of osteocalcin in bone remodeling, the study contributes to revealing the importance of careful periodontal evaluation in the presence of sepsis.

Resumo O objetivo deste estudo foi avaliar o efeito da sepse aguda no ligamento periodontal, osso alveolar e osso da furca por meio de análise histológica e imunohistoquímica. O modelo de sepse em ratos foi estabelecido pelo procedimento de ligação e perfuração do ceco (CLP). Doze ratos foram divididos de forma randomizada em ratos sépticos (n=6) e controle - grupo Sham (n=6). Os animais foram eutanasiados após 24 horas e suas hemimandíbulas foram submetidas aos procedimentos histotécnicos para análise histomorfométricos (matriz óssea, fibras colágenas, fibroblastos, osteócitos, células inflamatórias e vasos sanguíneos) e imunohistoquímicos (BMP-2/4, RANKL e osteocalcina) no osso alveolar, osso de furca e ligamento periodontal. Nossos resultados demonstraram que os parâmetros histomorfométricos foram similares no osso alveolar, osso de furca e ligamento periodontal dos animais do grupo sepse e do grupo Sham. Em relação à análise por imunohistoquímica, o número de células imunomarcadas para BMP-2/4 e RANKL também foi similar em ambos os grupos. Entretanto, houve redução (p=0.0014) no número de células imunomarcadas para osteocalcina no ligamento periodontal de ratos sépticos em relação ao grupo Sham. Como conclusão, o estabelecimento de sepse aguda resultou em um número reduzido de células imunomarcadas para osteocalcina na região do ligamento periodontal (p=0,0014). Além disso, não foram observadas diferenças histológicas significativas no periodonto de ratos na presença de sepse aguda. Considerando o papel da osteocalcina na remodelação óssea, este estudo contribui para revelar a importância da avaliação periodontal cuidadosa na presença de sepse.

A influência da ocitocina e do antagonista do receptor de ocitocina no metabolismo ósseo, estresse oxidativo, padrões da marcha e análise do tipo ansioso de ratas senescentes / The influence of oxytocin and the oxytocin receptor antagonist on bone metabolism, oxidative stress, gait and analysis of the anxious type of senescent rats

O objetivo deste estudo foi avaliar a ação da ocitocina (OT) endógena, bem como o efeito potencializador da OT exógena sobre o metabolismo ósseo, estresse oxidativo, marcha e análise do tipo ansioso de ratas na periestropausa. Ao completar 19 meses, os animais receberam injeções de solução salina (0,15M/ip), Atosiban (AT) (At; 300 µg/Kg/ip), OT (Ot; 134 µg/Kg/ip) ou At+Ot (injeções de OT 5 minutos após AT), sendo duas injeções de cada substância por dia, com intervalos de 12 horas entre elas, a cada 30 dias até a idade de 21 meses. Após trinta dias sem tratamentos, foi realizada a coleta de amostras biológicas. Aspartato aminotransferase (AST), marcador de dano hepático, foi menor em Ot e At+Ot. Substância ácida reativa ao ácido tiobarbitúrico (TBARs É¥mol/L), marcador do dano oxidativo lipídico, foi maior no grupo Ot comparado ao At (p = 0,0093), e menor no At+Ot em relação ao Ot (p = 0,0040). Houve maior defesa antioxidante enzimática avaliada por meio da superóxido dismutase (SOD) no grupo Ot em comparação ao Veh (p < 0,0312). Por sua vez, no grupo At houve maior atividade enzimática da fosfatase alcalina (FAL) em relação ao Veh e Ot (p < 0,0001; At+Ot: p = 0,0015). A espessura do tecido ósseo compacto foi menor no grupo At em relação ao Veh (p = 0,0228), no entanto, foi maior no grupo Ot em relação ao Veh e At (p = 0,0132, p < 0,0001); no grupo At+Ot foi menor quando comparado ao grupo Ot (p = 0,0003). O número de trabéculas ósseas foi menor no grupo At comparado ao Veh (p = 0,0240), e maior em Ot em relação ao At (p = 0,0084). Quanto a análise imunoistoquímica realizada no osso cortical do colo do fêmur, o grupo Ot apresentou maior expressão de osteocalcina (OCN) em comparação aos grupos Veh e At (p = 0,05 e 0,0033), e menor expressão no grupo At+Ot em relação ao grupo Ot (p = 0,05). A expressão de fosfatase ácida resistente ao tartarato (TRAP) foi menor no grupo Ot comparado aos grupos Veh e At (p = 0,05 e 0,0033), contudo foi maior no grupo At+Ot comparado ao Ot (p = 0,05). A densidade mineral óssea areal (DMO) foi significativamente maior nos grupos Ot e At+Ot em relação à Veh (p < 0,0001) e grupo At (p = 0,0231, p = 0,0418). Por sua vez, a relação mineral-matriz (vPO4/Proline) foi maior e a substituição de carbonato tipo B (CO3/vPO4) foi menor no grupo Veh. O teste de deambulação por comprimento (cm) usado para avaliar função musculoesquelética, aumentou em última análise no grupo Ot em relação ao grupo Veh - F (p = 0,0078), At - F (p = 0,0023), bem como aumentou sobre Ot - I (p = 0,0094). O teste do labirinto, usado para estudar o comportamento chamado "tipo ansioso", demonstrou que a OT inverte a redução nas entradas dos braços fechados, reduz o tempo gasto no centro causado pelo At. Os resultados obtidos neste estudo demonstram que a OT ajuda a modular o ciclo de remodelação óssea de ratas senescentes, melhorando os parâmetros de densitometria óssea e os parâmetros funcionais musculoesquelético(AU)

The objective of this study was to evaluate the endogenous oxytocin (OT) action, as well as the potentiating effect of exogenous OT on the bone metabolism, oxidative stress, gait and analysis of the anxious type of rats in periestropause. Upon completing 19 months, the animals received injections of saline solution (0.15M/ip), Atosiban (AT) (At; 300 µg/Kg/ip), OT (Ot; 134 µg/Kg/ip) or At+Ot (OT injections 5 minutes after AT), being two injections of each substance per day, with intervals of 12 hours between them, every 30 days until the age of 21 months. After thirty days without treatment, biological samples were collected. Aspartate aminotransferase (AST), a marker of liver damage, was lower after Ot and At+Ot. Acid reactive substance to thiobarbituric acid (TBARs µmol/L), marker of lipid oxidative damage, was higher in the Ot group compared to At (p = 0.0093), and lower in At+Ot compared to Ot (p = 0.0040). There was a higher antioxidant enzymatic defense evaluated by means of superoxide dismutase (SOD) in the Ot group compared to Veh (p < 0.0312). In turn, in the At group there was greater alkaline phosphatase (FAL) enzymatic activity in relation to Veh and Ot (p < 0.0001; At+Ot: p = 0.0015). The thickness of the compact bone tissue was smaller in the At group in relation to Veh (p = 0.0228), however, it was greater in the Ot group in relation to Veh and At (p = 0.0132, p < 0.0001); in the At+Ot group it was smaller when compared to Ot (p = 0.0003). The number of bone trabecules was smaller in the At group compared to the Veh (p = 0.0240), and greater in Ot in relation to the At (p = 0.0084). As for the immunohistochemical analysis performed on the cortical bone of the femoral neck, the Ot group presented a higher expression of osteocalcin (OCN) compared to the Veh and At groups (p = 0.05 and 0.0033), and lower expression in the At+Ot group compared to the Ot group (p = 0.05). The tartrate-resistant acid phosphatase (TRAP) expression was lower in the Ot group compared to the Veh and At groups (p = 0.05 and 0.0033), however it was higher in the At+Ot group compared to Ot (p = 0.05). The sandal mineral density (BMD) was significantly higher in the Ot and At+Ot groups compared to Veh (p < 0.0001) and At group (p = 0.0231, p = 0.0418). In turn, the parent mineral ratio (vPO4/Proline) was higher and the replacement of carbonate type B (CO3/vPO4) was lower in the Veh group. The walking test per length (cm) used to evaluate musculoskeletal function was ultimately increased in group Ot in relation to group Veh - F (p = 0.0078), At - F (p = 0.0023), as well as increased over Ot - I (p = 0.0094). The labyrinth test, used to study the so-called "anxious type" behavior, demonstrated that the OT reverses the reduction in the entries of the closed arms, reducing the time spent in the center caused by At. The results obtained in this study show that the OT helps to modulate the cycle of bone remodeling of senescent rats, improving the parameters of bone densitometry and the musculoskeletal functional parameters(AU)

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.

Avaliação da regeneração óssea guiada por meio histológico, histométrico, imunoistoquímico e perfil inflamatório de membranas reabsorvíveis de colágeno porcino em defeitos críticos em calotas de ratos / Evaluation histological, histometric, immunohistochemistry analysis and inflammatory profile of bone-guided regeneration with membranes of reabsorbable porcine collagen in critical defects in rodent skullcaps

A regeneração óssea guiada (RGO) tornou-se uma prática comum e importante na odontologia, sendo necessário o uso de membranas para sua realização, uma vez que são barreiras que evitam o crescimento de tecido mole nas áreas de defeitos ósseos. Entre as características mais relevantes das membranas absorvíveis estão: o suporte sanguíneo (diretamente relacionado com a porosidade do material) e suporte mecânico ósseo que depende do tempo de reabsorção da membrana. O objetivo desse estudo foi avaliar e comparar duas membranas de colágeno por meio de estudo histológico, histomorfométrico, imunoistoquímico e por contagem de células inflamatórias o processo de regeneração óssea guiada utilizando a membrana de colágeno derivada de pericárdio porcino (Jason®-Instituto Straumann AG, Suíça) em defeitos críticos de 7 mm de diâmetro criados em setenta e duas calvárias de ratos (Rattus Albinus, variedade Wistar). Esses animais foram divididos em 3 grupos: grupo membrana de colágeno porcino (BioGide® - Geistlich Wohlhusen, Suíça), grupo membrana de colágeno de pericárdio porcino (Jason®-Instituto Straumann AG, Suíça) e grupo coágulo, sendo este preenchidos somente com coágulo sem membrana. Esses 3 grupos forma subdivididos em quatro subgrupos de acordo com os tempos avaliados: 7, 15, 30 e 60 dias. Como resultado tivemos na análise histológica e histométrica maior neoformação óssea com o grupo de membrana pericárdio porcino nos períodos de 7 dias (199 pontos) e não foi significante, com 15 dias (494 pontos) estatisticamente significativo, com 30 dias (979 pontos) não significante. Esses valores se modificam 60 dias, mostrando maior superioridade a membrana de colágeno porcino (BioGide®- Geistlich Wohlhusen, Suíça) (1151 pontos) estatisticamente significativo. No analises global a membrana de colágeno porcino (Jason®-Instituto Straumann AG, Suíça) foi superior que a membrana de pericárdio porcino (BioGide®- Geistlich Wohlhusen, Suíça) (p= 0,021) estatisticamente significativo. A análise imunoistoquímica confirmou os achados histométricos, demostrando maior presença da osteocalcina no grupo de pericárdio porcino aos 7 e 15 dias e presença da osteopontina pouco evidente. Já com a membrana de colágeno porcino a osteopontina foi mais imunomarcada aos períodos de 7 e 15 dias, e a presencia de osteocalcina mais evidente aos 30 e 60 dias, corroborando com os resultados iniciais. Na contagem de células inflamatórias para o tempo de 7 dias não houve diferenças estatísticas, já na contagem de vasos sanguíneos houve diferença significativa no período de 15 dias com maior quantidade de vasos para membrana de colágeno porcino, com estes achados concluímos que tanto a membrana de colágeno de pericárdio porcino (Jason® -Instituto Straumann AG, Suíça) quanto a membrana de colágeno porcino (BioGide®-Geistlich Wohlhusen, Suíça) podem ser consideradas como material de escolha apropriada para regeneração óssea guiada, com maior proporção de osso neoformado com a membrana de colágeno porcino(AU)

Guided bone regeneration (RGO) has become a common and important practice in dentistry, requiring the use of membranes to perform it, since they are barriers that prevent the growth of soft tissue in areas of bone defects. Among the most relevant characteristics of absorbable membranes are: blood support (directly related to the porosity of the material) and mechanical bone support that depends on the time of membrane resorption. The goal of this study was to evaluate and compare two collagen membranes by means of histological, histomorphometric, immunohistochemical study and by inflammatory cell counting the guided bone regeneration process using the collagen membrane derived from porcine pericardium (Jason®-Instituto Straumann AG, Switzerland) in critical defects of 7 mm in diameter created in seventy-two calvaria of rats (Rattus Albinus, Wistar variety). These animals were divided into 3 groups: porcine collagen membrane group (BioGide® - Geistlich Wohlhusen, Switzerland), porcine pericardium collagen group (Jason®-Instituto Straumann AG, Switzerland) and clot group, which were filled with only a clot without membrane. These 3 groups were subdivided into four subgroups according to the evaluated times: 7, 15, 30 and 60 days. As a result, we had a greater bone neoformation in the histological and histometric analysis with the porcine pericardial membrane group in the periods of 7 days (199 points) and it was not statistically significant with 15 days (494 points), with 30 days (979 points) not significant. These values change 60 days, showing greater superiority to the porcine collagen membrane (BioGide®- Geistlich Wohlhusen, Switzerland) (1151 points) statistically significant. In the global analysis, the porcine collagen membrane (Jason®Instituto Straumann AG, Switzerland) was superior than the porcine pericardium membrane (BioGide®- Geistlich Wohlhusen, Switzerland) (p = 0.021) statistically significant. The immunohistochemical analysis confirmed the histometric findings, showing a greater presence of osteocalcin in the porcine pericardium group at 7 and 15 days and the presence of osteopontin little evident. As for the porcine collagen membrane, osteopontin was more immunostained at 7 and 15 days, and the presence of osteocalcin was more evident at 30 and 60 days, corroborating the initial results. In the inflammatory cell count for the 7-day period, there were no statistical differences, whereas in the blood vessel count, there was a significant difference in the 15-day period with a greater number of vessels for porcine collagen membrane, with these findings we conclude that both the porcine pericardial collagen (Jason® - Straumann AG Institute, Switzerland) and porcine collagen membrane (BioGide®-Geistlich Wohlhusen, Switzerland) can be considered as the material of choice for guided bone regeneration, with a higher proportion of neoformed bone according to porcine collagen membrane(AU)

Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells

Abstract Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. Results RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.

A SERM increasing the expression of the osteoblastogenesis and mineralization-related proteins and improving quality of bone tissue in an experimental model of osteoporosis

Abstract Raloxifene is an antiresorptive drug, selective estrogen receptor modulator (SERM) used in the treatment of osteoporosis. Objective To evaluate proteins related to bone repair at the peri-implant bone in a rat model of osteoporosis treated with raloxifene. Material and Methods 72 rats were divided into three groups: SHAM (healthy animals), OVX (ovariectomized animals), and RLX (ovariectomized animals treated with raloxifene). Raloxifene was administered by gavage (1 mg/kg/day). Tibial implantation was performed 30 days after ovariectomy, and animals were euthanized at 14, 42, and 60 days postoperatively. Samples were collected and analyzed by immunohistochemical reactions, molecular analysis, and microtomographic parameters. Results RLX showed intense staining of all investigated proteins at both time points except for RUNX2. These results were similar to SHAM and opposite to OVX, showing mild staining. The PCR gene expression of OC and ALP values for RLX (P<0.05) followed by SHAM and OVX groups. For BSP data, the highest expression was observed in the RLX groups and the lowest expression was observed in the OVX groups (P<0.05). For RUNX2 data, RLX and SHAM groups showed greater values compared to OVX (P<0.05). At 60 days postoperatively, microtomography parameters, related to closed porosity, showed higher values for (Po.N), (Po.V), and (Po) in RLX and SHAM groups, whereas OVX groups showed lower results (P<0.05); (BV) values (P=0.009); regarding total porosity (Po.tot), RLX group had statistically significant lower values than OVX and SHAM groups (P=0.009). Regarding the open porosity (Po.V and Po), the SHAM group presented the highest values, followed by OVX and RLX groups (P<0.05). The Structural Model Index (SMI), RLX group showed a value closer to zero than SHAM group (P<0.05). Conclusions Raloxifene had a positive effect on the expression of osteoblastogenesis/mineralization-related proteins and on micro-CT parameters related to peri-implant bone healing.

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.

In-office tooth bleaching with 38% hydrogen peroxide promotes moderate/severe pulp inflammation and production of ll-1ß, TNF-ß, GPX, FGF-2 and osteocalcin in rats

Abstract Objectives: To study the intensity of inflammatory infiltrate and production of interleukin-1β (ll-1β), tumor necrosis factor-β (TNF-β), fibroblast growth factor-2 (FGF-2), glutathione peroxidase (GPX), and osteocalcin in response to in-office tooth bleaching in rats. Material and Methods: Twenty male Wistar rats were randomized into four groups (n=5) according to the received treatment (tooth bleaching or no treatment - control) and the period of euthanasia after treatment (24 h or 10 days). We performed tooth bleaching using a 38% hydrogen peroxide gel on maxillary and mandibular incisors. After euthanasia, incisors (20 per group) were processed for histological analysis, immunohistochemistry staining of ll-1β, TNF-β, FGF-2 and GPX and osteocalcin by immunofluorescence. We analyzed data using the Mann-Whitney and Kruskal-Wallis/Dunn tests (p<0.05). Results: The bleached groups presented statistically significant differences regarding the pulp inflammation stage compared with the control groups. Bleached teeth showed moderate/severe inflammatory infiltrate and control groups presented absent inflammatory cells or a negligible number of mononuclear cells (p<0.001) at two times (24 h and 10 days). There was strong staining for ll-1β, TNF-β, and GPX in bleached groups at 24 h and strong staining for ll-1β, TNF-β, GPX and FGF-2 at 10 days. After 10 days of tooth bleaching, the bleached group showed a statistically superior amount of osteocalcin than the other groups (p<0.01). Conclusions: Tooth bleaching with 38% hydrogen peroxide causes severe pulp inflammation, but characteristics of tissue repair after 10 days.

Daily melatonin administration improves osseointegration in pinealectomized rats

Abstract The hypothesis of this study was that the peri-implant bone healing of the group of pinealectomized rats would differ from the control group. The samples were subjected to immunohistochemical, microtomographic (total porosity and connectivity density), and fluorochrome (mineralized surface) analyses. Objectives The goal of this study was to investigate the cellular changes and bone remodeling dynamics along the bone/implant interface in pinealectomized rats. Material and Methods The total of 18 adult male rats (Rattus norvegicus albinus, Wistar) was divided into three groups (n=6): control (CO), pinealectomized without melatonin (PNX) and pinealectomized with melatonin (PNXm). All animals were submitted to the first surgery (pinealectomy), except the CO group. Thirty days after the pinealectomy without melatonin, the second surgery was conducted, in which all animals received an implant in each tibia (36 titanium implants with surface treatment were installed - Implalife® São Paulo, SP, Brazil). By gavage, the rats of the PNX group received the vehicle solution, and the procedure. Results Immunohistochemical analysis for runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC) showed that the bone repair process in the PNXm group was similar to that of the CO group, whereas the PNX group showed a delay. The microtomographic parameters of total porosity [Po(tot)] and bone surface (BS) showed no statistically significant differences, whereas for the connective density (Conn.Dn) a statistical difference was found between the CO and PNXm groups. Fluorochrome analysis of the active mineralized surface showed statistically significant difference between the CO and PNX and between the CO and PNXm groups. Conclusion The absence of the pineal gland impaired the bone repair process during osseointegration, however the daily melatonin replacement was able to restore this response.

Bone repair access of BoneCeramic™ in 5-mm defects: study on rat calvaria

Abstract Objective: The aim of this study was to evaluate the osteoconductive potential of BoneCeramic™ on bone healing in rat calvaria 5-mm defects. Material and Methods: A 5-mm calvaria bone defect was induced in three groups and the defect was not filled with biomaterial [Clot Group (CG)], autogenous bone (AG), or Bone Ceramic Group (BCG). Animals were euthanized after 14 or 28 days and the bone tissue within the central area of the bone defect was evaluated. Results were compared using ANOVA and Tukey test (p<0.05). Immunohistochemistry was performed using primary antibodies against osteocalcin, RUNX-2, TRAP, VEGF proteins, and 3-dimensional images of the defects in μCT were obtained to calculate bone mineral density (BMD). Results: In BCG, the defect was completely filled with biomaterial and new bone formation, which was statistically superior to that in the GC group, at both time-points (p<0.001 for 14 days; p=0.002 for 28 days). TRAP protein showed weak, RUNX-2 showed a greater immunolabeling when compared with other groups, VEGF showed moderate immunostaining, while osteocalcin was present at all time-points analyzed. The μCT images showed filling defect by BCG (BMD= 1337 HU at 28 days). Conclusion: Therefore, the biomaterial tested was found to be favorable to fill bone defects for the reporting period analyzed.

Effects of Syzygium aromaticum, Cinnamomum zeylanicum, and Salvia triloba extracts on proliferation and differentiation of dental pulp stem cells

Abstract Hypersensitivity, local irritative and cytotoxic effects are known for the chemical components of Syzygium aromaticum and Cinnamomum zeylanicum contained in dental materials. However, there is no intimate data in dentistry using the whole extracts of these plants and introducing new ones. Salvia triloba is a well-known anti-inflammatory plant that correspondingly could be used in several dental traumas. Objectives: We aimed to show and compare the effect of S. aromaticum, C. zeylanicum, and S. triloba extracts on dental pulp stem cells (DPSCs) proliferation, differentiation, and immune responses. Material and Methods: Using xCELLigence, a real time monitoring system, we obtained a growth curve of DPSCs with different concentrations of the Extracts. A dose of 10 μg/mL was the most efficient concentration for vitality. Osteogenic differentiation and anti-inflammatory activities were determined by using an ELISA Kit to detect early and late markers of differentiation. Results: The level of osteonectin (ON, early osteogenic marker) decreased, which indicated that the osteogenic differentiation may be accelerated with addition of extracts. However, the level of osteocalcin (OCN, late osteogenic marker and sign of calcium granulation) differed among the extracts, in which S. aromaticum presented the highest value, followed by S. triloba and C. zeylanicum. Surprisingly, the determined calcium granules were reduced in S. aromaticum and S. triloba. In response to tumor necrosis factor alpha (TNF-α), S. triloba-treated DPSCs showed the most reduced level of IL-6 cytokine level. We suggest C. zeylanicum as a promising osteogenic inducer and S. triloba as a potent anti-inflammatory agent, which could be used safely in biocomposite or scaffold fabrications for dentistry. Conclusions: Because calcium granule formation and cell viability play a critical role in hard tissue formation, S. aromaticum in dentistry should be strictly controlled, and the mechanism leading to reduced calcium granule formation should be identified.

Short term sodium alendronate administration improves the peri-implant bone quality in osteoporotic animals

Abstract Sodium alendronate is a bisphosphonate drug that exerts antiresorptive action and is used to treat osteoporosis. Objective The aim of this study was to evaluate the bone repair process at the bone/implant interface of osteoporotic rats treated with sodium alendronate through the analysis of microtomography, real time polymerase chain reactions and immunohistochemistry (RUNX2 protein, bone sialoprotein (BSP), alkaline phosphatase, osteopontin and osteocalcin). Material and Methods A total of 42 rats were used and divided in to the following experimental groups: CTL: control group (rats submitted to fictitious surgery and fed with a balanced diet), OST: osteoporosis group (rats submitted to a bilateral ovariectomy and fed with a low calcium diet) and ALE: alendronate group (rats submitted to a bilateral ovariectomy, fed with a low calcium diet and treated with sodium alendronate). A surface treated implant was installed in both tibial metaphyses of each rat. Euthanasia of the animals was conducted at 14 (immunhostochemistry) and 42 days (immunohistochemistry, micro CT and PCR). Data were subjected to statistical analysis with a 5% significance level. Results Bone volume (BV) and total pore volume were higher for ALE group (P<0.05). Molecular data for RUNX2 and BSP proteins were significantly expressed in the ALE group (P<0.05), in comparison with the other groups. ALP expression was higher in the CTL group (P<0.05). The immunostaining for RUNX2 and osteopontin was positive in the osteoblastic lineage cells of neoformed bone for the CTL and ALE groups in both periods (14 and 42 days). Alkaline phosphatase presented a lower staining area in the OST group compared to the CTL in both periods and the ALE at 42 days. Conclusion There was a decrease of osteocalcin precipitation at 42 days for the ALE and OST groups. Therefore, treatment with short-term sodium alendronate improved bone repair around the implants installed in the tibia of osteoporotic rats.

Efeito do tratamento periodontal nos níveis de osteocalcina sérica em pacientes com diabetes tipo 2 e periodontite crônica severa / Effect of periodontal therapy on serum osteocalcin levels in patients with type 2 diabetes and severe chronic periodontitis

O tecido ósseo é metabolicamente ativo e sofre um processo contínuo de renovação e remodelação. A osteocalcina é um peptídeo secretado pelos osteoblastos. Apesar de ser primariamente depositada na matriz óssea recém-formada, uma pequena fração entra em circulação na corrente sanguínea, sendo considerada como um marcador de formação óssea e interferir na homeostasia da glicose. O objetivo deste estudo foi investigar se o tratamento periodontal afeta os níveis séricos de osteocalcina em pacientes com DM2 e periodontite crônica severa. 26 pacientes foram divididos em grupo teste (n=13, idade média de 55,8 ± 8,4 anos, 9 homens e 4 mulheres) e controle. (n=13, idade média de 59,4± 8.4, 5 homens e 8 mulheres). O grupo teste recebeu tratamento periodontal e grupo controle nãorecebeu tratamento periodontal imediato. O exame periodontal incluiu medidas de IP (Índice de placa), SS (Sangramento à sondagem), PBS (Profundidade de bolsa à sondagem), NIC (Nível de inserção clínica). Exames laboratoriais incluíram HBA1c, glicemia estimada, glicose, triglicerídeo, colesterol total e frações. A concentração de osteocalcina sérica foi analisada pelo ELISA. O exame periodontal, exames laboratoriais e concentração de osteocalcina foram reavaliados 90 dias após. Os resultados mostraram que não houve alterações significativas nos parâmetros periodontais, exames laboratoriais, e níveis de osteocalcina 90 dias após o tratamento periodontal. Conclusão: o tratamento periodotnal não influenciou nos níveis séricos da osteocalcina em pacientes com diabetes melllitus tipo 2 e periodontite crônica severa.

Bone tissue is metabolically active and undergoes a continuous process of resorption and formation. Osteocalcin is a peptide secreted by osteoblasts. Although primarily deposited in newly formed bone matrix, a small fraction enters into circulation in the bloodstream and is considered as a marker of bone formation and interferes with glucose homeostasis. The aim of this study was to investigate whether periodontal treatment affects serum osteocalcin levels in patients with type 2 diabetes and severe chronic periodontitis. 26 patients were divided into test group (n = 13, mean age 55.8 ± 8.4 years, 9 men and 4 women) and control. (n = 13, mean age 59.4 ± 8.4, 5 men and 8 women). The test group received periodontal treatment and untreated control group. Periodontal examination included PI measures (plaque index), BOP (bleeding on probing), PPD (probing pocket depth), CAL (clinical attachment level). Laboratory tests included HbA1c, glucose, triglyceride, total cholesterol and fractions. The concentration of serum osteocalcin was analyzed by ELISA. Periodontal examination, laboratory tests and concentration of osteocalcin were reassessed after 90 days. The results showed no significant changes in periodontal parameters, laboratory tests, and osteocalcin levels 90 days after periodontal treatment. In conclusion, periodontal treatment did not influence the serum levels of osteocalcin in patients with type 2 diabetes and severe chronic periodontitis.

Osteoclacin expression during autogenous onlay bone grafts with or without resorbable collagen membrane in diabetic rats / Expressão da osteocalcina no reparo de enxertos ósseos autógenos onlay recobertos ou não por membrana colágena reabsorvível em ratos diabéticos

Objetivo: O objetivo deste estudo foi quantificar, através da técnica imunoistoquímica, a expressão do marcador de formação óssea Osteocalcina no processo de reparo do enxerto ósseo autógeno onlay, associado ou não à membrana colágena reabsorvível e comparar esses achados com a presença da Diabetes Mellitus. Material e Métodos: Foram utilizados 60 ratos (Rattus norvegicus, variação albinus, Wistar) com 90 dias de idade, divididos em dois grupos , cada um contendo 30 animais: Grupo Teste com Diabéticos , composto por ratos com diabetes induzida e Grupo Controle composto por ratos normoglicêmicos. Todos receberam enxertos na hemi mandíbula esquerda com recobrimento de membrana colágena e na hemi mandíbula direita sem recobrimento. Os animais foram eutanasiados nos períodos 0h, 7, 14, 21, 45 e 60 dias. A análise imunoistoquímica foi realizada com o marcador Osteocalcina na interface leito-enxerto. Para analise da expressão imunoistoquímica da Osteocalcina, realizou-se uma fotografia com visão panorâmica do enxerto e duas fotografias em maior aumento. Resultados: Os resultados mostraram que houve diferença estatisticamente significante ao nível de 5% intragrupo para Diabético e Controle com e sem membrana e também quando comparamos o grupo Diabético com o Controle com a presença da membrana. Conclusão: Dentro dos limites do presente estudo, pode-se concluir que a marcação da osteocalcina pode sofrer alguma influência do diabetes, sendo expressa de forma mais tardia na presença dessa condição. Porém, a associação da membrana ao enxerto pode melhorar esse atraso e deixar a expressão semelhante ao grupo controle.

Objective: This study aimed to quantify through immunohistochemistry the expression of bone formation marker osteocalcin during the repair process of autogenous onlay bone graft, associated or not to the resorbable collagen membrane and to compare these findings with the presence of Diabetes Mellitus. Material and Methods: Sixty rats (Rattus norvegicus, albinus variation, Wistar) aged 90 days, were divided into two groups with 30 animals: test group – rats with induced diabetes; control group - normoglycemic rats. All rats received grafts on the left and right hemi-mandible with or without collagen membrane coverage, respectively. The animals were euthanized at the following periods: 0h, 7, 14, 21, 45, and 60 days. Immunohistochemistry analysis was performed by the marker osteocalcin at receptor site-graft interface. To analyze osteocalcin immunohistochemical expression, a panoramic view photograph of the graft was taken followed by two photographs at larger magnification. Results: No statistically significances at 5% level were observed between diabetic and control group with and without membrane; and diabetic and control groups with membrane coverage. Conclusion: Within the limits of this present study, it can be concluded that the osteocalcin marker might be influenced by diabetes so that it was late expressed during this condition. However, the association of the graft with the membrane could improve this delay by reaching expression values similar to those of control group.

Immunolocalization of markers for bone formation during guided bone regeneration in osteopenic rats

Objective The aim of this paper was to evaluate the repair of onlay autogenous bone grafts covered or not covered by an expanded polytetrafluoroethylene (e-PTFE) membrane using immunohistochemistry in rats with induced estrogen deficiency. Material and Methods Eighty female rats were randomly divided into two groups: ovariectomized (OVX) and with a simulation of the surgical procedure (SHAM). Each of these groups was again divided into groups with either placement of an autogenous bone graft alone (BG) or an autogenous bone graft associated with an e-PTFE membrane (BGM). Animals were euthanized on days 0, 7, 21, 45, and 60. The specimens were subjected to immunohistochemistry for bone sialoprotein (BSP), osteonectin (ONC), and osteocalcin (OCC). Results All groups (OVX+BG, OVX+BMG, SHAM+BG, and SHAM+BMG) showed greater bone formation, observed between 7 and 21 days, when BSP and ONC staining were more intense. At the 45-day, the bone graft showed direct bonding to the recipient bed in all specimens. The ONC and OCC showed more expressed in granulation tissue, in the membrane groups, independently of estrogen deficiency. Conclusions The expression of bone forming markers was not negatively influenced by estrogen deficiency. However, the markers could be influenced by the presence of the e-PTFE membrane. .

Reparação óssea em alvéolos de ratas ovarectomizadas após implante com biovidro: análise histomorfométrica e imunoistoquímica / Alveolar bone repair in ovarectomized rats after implantantion with bioglass: histomorphometric and immunohistochemistry analysis

Objetivos: Avaliar o processo de reparo alveolar em ratas ovarectomizadas após implante com Biosilicato cristalino. Materiais e Métodos: Sessenta ratas foram divididas em quatro grupos (n = 15) de acordo com o tratamento: Grupo 1- ratas submetidas à cirurgia sham com alvéolos preenchidos com coágulo; Grupo 2-ratas submetidas à cirurgia sham com alvéolos preenchidos com Biosilicato cristalino; Grupo 3-ratas ovarectomizadas com alvéolos preenchidos com coágulo; e Grupo 4- ratas ovarectomizadas com alvéolos preenchidos com Biosilicato cristalino. Após 7, 14 e 28 dias os animais foram sacrificados e análises histomorfométricas (histologia e histometria) e imunoistoquímicas (Osteopontina e Osteocalcina) foram realizadas. A comparação dos resultados foi realizada utilizando o programa estatístico Assistat 2013 para Windows através da Análise de Variância e teste de Mann-Whitney, com resultados significantes sendo encontrados quando p≤0.05. Resultados: No período de 7 dias, os Grupos 1 e 3 apresentaram resultados superiores e estatisticamente maiores do que os Grupos 2 e 4 (p<0.05) quanto à formação óssea. Após o período de 14 dias as diferenças estatísticas mantiveram-se, com os Grupos 1 e 3 apresentando diferença estatisticamente significante maiores aos Grupos 2 e 4 (p<0.05). Aos 28 dias, a diferença estatística significativa permaneceu entre os mesmo Grupos estudados, sendo que nos grupos implantados com o Biosilicato cerca de 60% do alvéolo já havia sido preenchido por osso novo. A imunoistoquímica revelou expressão das proteínas Osteocalcina e Osteopontina, principalmente nos animais submetidos à cirurgia de ovarectomia. Conclusões: Os Grupos 1 e 3 foram os que apresentaram os melhores resultados durante todo o estudo, com o Biosilicato cristalino apresentando resultado favorável na reparação óssea sem contudo ser observada a absorção do biovidro. As análises imunoistoquímicas demonstraram que os animais submetidos a ovarectomia apresentaram uma maior...

Objectives: To evaluate the process of alveolar repair in ovariectomized rats after implantation with Biosilicate. Materials and Methods: Sixty rats were divided into four groups (n = 15) according to treatment: Group 1 - rats underwent sham surgery with alveoli filled with blood clot; Group 2 - rats underwent sham surgery with alveoli filled with crystalline Biosilicate; Group 3 - ovariectomized rats with alveoli filled with blood clot; Group 4 - ovariectomized rats with alveoli filled with crystalline Biosilicate. After 7, 14 and 28 days the animals were sacrificed and histomorphometric analysis (histology and histometric) and immunohistochemical (Osteopontin and Osteocalcin) were measured . The comparison of the results was performed using the statistical program Assistat 2013 for Windows by Analysis of Variance and Mann-Whitney tests, with significant results being found when p ≤ 0:05 . Results: During the 7- day period, Groups 1 and 3 showed better and significantly higher results in bone formation than Groups 2 and 4 (p<0.05). After 14 days the statistical differences remained between Groups 1 and 3 and Groups 2 and 4 (p<0,05). At 28 days, the statically difference remained significant between the same Groups, while the groups implanted with Biosilicate presented about 60% of the socket filled by new bone. Immunohistochemistry revealed expression of proteins osteocalcin and osteopontin, especially in animals subjected to ovariectomy surgery. Conclusions: Groups 1 and 3 were those with the best results throughout the study, with the crystalline Biosilicate presenting favorable result in bone repair without bioglass resorption. The immunohistochemical analysis showed that animals subjected to ovariectomy had a higher signaling in Ostecalcina and Osteopontin proteins

Análise histomorfométrica e imunoistoquímica do processo de reparo alveolar de ratas ovariectomizadas com dieta pobre em cálcio e submetidas à terapia com raloxifeno ou com alendronato / Histomorphometric and immunohistochemistry analysis of the alveolar healing process of ovariectomized rats with a low calcium diet submitted to raloxifene or alendronate therapy

Objetivos: Avaliar a influência do alendronato e raloxifeno no processo de reparo alveolar em ratas com osteoporose induzida (ovariectomizadas e submetidas a uma dieta pobre em cálcio). Materiais e Métodos: Sessenta e quatro ratas foram divididas em quatro grupos (n = 16) de acordo com o tratamento em ratas sham com dieta normal, ratas ovariectomizadas com uma dieta pobre em cálcio sem tratamento medicamentoso, ratas ovariectomizadas com uma dieta pobre em cálcio tratadas com alendronato e ratas ovariectomizadas com uma dieta pobre em cálcio tratadas com raloxifeno. Assim, a análise histomorfométrica foi realizada, bem como expressão da proteína TRAP, osteocalcina, osteoprotegerina (OPG) e RANKL, pela técnica de imunoistoquímica. Para comparar os valores médios obtidos nos diferentes grupos e períodos experimentais, os dados foram analisados pelos testes de Kruskal-Wallis e Nemenyi-Damico-Wolfe-Dunn como post-hoc técnicas (α =. 05). Resultados: No longo prazo, os grupos SHAM e raloxifeno mostraram a melhor taxa de formação óssea (P <0,05). A pior taxa de formação óssea foi observado no grupo OVX ST. Os grupos raloxifeno e alendronato melhoram a taxa de formação óssea, quando administrados em animais com osteoporose. O grupo raloxifeno apresentou uma melhor resposta no longo prazo. O grupo alendronato mostrou uma resposta favorável em 14 dias comparado ao grupo raloxifeno, mas uma resposta reduzida aos 42 dias pós-extração. A imunoistoquímica revelou a expressão da proteína TRAP, osteocalcina, osteoprotegerina (OPG) e fator de RANKL. Foi possível notar um osso maduro no grupo SHAM aos 14 dias, um osso imaturo no grupo OVX ST e uma qualidade óssea intermediária nos grupos OVX ALE e OVX RAL. Conclusões: A ovariectomia associada a uma dieta pobre em cálcio atrasou o processo de reparo alveolar. O tratamento com alendronato e raloxifeno melhorou o reparo alveolar em ratas osteoporóticas, mas não o suficiente para atingir os valores histométricos e de expressão...

Objectives: To evaluate the influence of alendronate and raloxifene in the alveolar healing process of rats with induced osteoporosis (ovariectomized and low calcium diet). Materials and Methods: The sixty-four rats were divided into four groups (n = 16) according to the treatment in ovariectomized rats with a low calcium diet treated with alendronate, ovariectomized rats with a low calcium diet raloxifene-treated, ovariectomized rats with a low calcium diet without pharmacological treatment and sham rats with a balanced diet. Thus, histomorphometric analysis was performed, as well as expression of TRAP protein, osteocalcin, osteoprotegerin (OPG) and RANKL, by the immunohistochemistry technique. To compare the mean values obtained in different groups and experimental periods, the data were analyzed by Kruskal-Wallis, Nemenyi-Damico-Wolfe-Dunn tests were further used as post-hoc techniques (α=.05). Results: In the long term, the SHAM and raloxifene groups showed the best bone formation rate (P <.05). The worst bone formation rate was observed in the untreated OVX group. Raloxifene and Alendronate groups improved bone formation rate when administered in osteoporotic animals. Raloxifene group had a better response in the long term. Alendronate group showed a favorable response at 14 days compared to Raloxifene group, but a reduced response at 42 days post-extraction. Immunohistochemistry revealed the expression of TRAP protein, osteocalcin, osteoprotegerin (OPG) and RANKL, it was possible to note a mature bone at 14 days in the SHAM group balanced diet, and an immature bone in OVX group low calcium diet and an intermediate quality in the Alendronate and Raloxifene groups. Conclusion: Ovariectomy delays the alveolar wound healing process and interferes with the bone turnover. The ALE replacement and the RLX treatment improved the healing but not enough to reach histomorphometric and immunocolocalization values of the sham group.

Análise histomorfométrica e imunoistoquímica do processo de reparo alveolar de ratas ovariectomizadas com dieta pobre em cálcio e submetidas à terapia com raloxifeno ou com alendronato / Histomorphometric and immunohistochemistry analysis of the alveolar healing process of ovariectomized rats with a low calcium diet submitted to raloxifene or alendronate therapy

Objetivos: Avaliar a influência do alendronato e raloxifeno no processo de reparo alveolar em ratas com osteoporose induzida (ovariectomizadas e submetidas a uma dieta pobre em cálcio). Materiais e Métodos: Sessenta e quatro ratas foram divididas em quatro grupos (n = 16) de acordo com o tratamento em ratas sham com dieta normal, ratas ovariectomizadas com uma dieta pobre em cálcio sem tratamento medicamentoso, ratas ovariectomizadas com uma dieta pobre em cálcio tratadas com alendronato e ratas ovariectomizadas com uma dieta pobre em cálcio tratadas com raloxifeno. Assim, a análise histomorfométrica foi realizada, bem como expressão da proteína TRAP, osteocalcina, osteoprotegerina (OPG) e RANKL, pela técnica de imunoistoquímica. Para comparar os valores médios obtidos nos diferentes grupos e períodos experimentais, os dados foram analisados pelos testes de Kruskal-Wallis e Nemenyi-Damico-Wolfe-Dunn como post-hoc técnicas (α =. 05). Resultados: No longo prazo, os grupos SHAM e raloxifeno mostraram a melhor taxa de formação óssea (P <0,05). A pior taxa de formação óssea foi observado no grupo OVX ST. Os grupos raloxifeno e alendronato melhoram a taxa de formação óssea, quando administrados em animais com osteoporose. O grupo raloxifeno apresentou uma melhor resposta no longo prazo. O grupo alendronato mostrou uma resposta favorável em 14 dias comparado ao grupo raloxifeno, mas uma resposta reduzida aos 42 dias pós-extração. A imunoistoquímica revelou a expressão da proteína TRAP, osteocalcina, osteoprotegerina (OPG) e fator de RANKL. Foi possível notar um osso maduro no grupo SHAM aos 14 dias, um osso imaturo no grupo OVX ST e uma qualidade óssea intermediária nos grupos OVX ALE e OVX RAL. Conclusões: A ovariectomia associada a uma dieta pobre em cálcio atrasou o processo de reparo alveolar. O tratamento com alendronato e raloxifeno melhorou o reparo alveolar em ratas osteoporóticas, mas não o suficiente para atingir os valores histométricos e de expressão das...

Objectives: To evaluate the influence of alendronate and raloxifene in the alveolar healing process of rats with induced osteoporosis (ovariectomized and low calcium diet). Materials and Methods: The sixty-four rats were divided into four groups (n = 16) according to the treatment in ovariectomized rats with a low calcium diet treated with alendronate, ovariectomized rats with a low calcium diet raloxifene-treated, ovariectomized rats with a low calcium diet without pharmacological treatment and sham rats with a balanced diet. Thus, histomorphometric analysis was performed, as well as expression of TRAP protein, osteocalcin, osteoprotegerin (OPG) and RANKL, by the immunohistochemistry technique. To compare the mean values obtained in different groups and experimental periods, the data were analyzed by Kruskal-Wallis, Nemenyi-Damico-Wolfe-Dunn tests were further used as post-hoc techniques (α=.05). Results: In the long term, the SHAM and raloxifene groups showed the best bone formation rate (P <.05). The worst bone formation rate was observed in the untreated OVX group. Raloxifene and Alendronate groups improved bone formation rate when administered in osteoporotic animals. Raloxifene group had a better response in the long term. Alendronate group showed a favorable response at 14 days compared to Raloxifene group, but a reduced response at 42 days post-extraction. Immunohistochemistry revealed the expression of TRAP protein, osteocalcin, osteoprotegerin (OPG) and RANKL, it was possible to note a mature bone at 14 days in the SHAM group balanced diet, and an immature bone in OVX group low calcium diet and an intermediate quality in the Alendronate and Raloxifene groups. Conclusion: Ovariectomy delays the alveolar wound healing process and interferes with the bone turnover. The ALE replacement and the RLX treatment improved the healing but not enough to reach histomorphometric and immunocolocalization values of the sham group