Estudo histológico e imunohistoquímico da degeneração da região central da cartilagem de Meckel na mandíbula em desenvolvimento de ratos / Histological and immunohistochemical study of the degeneration of the central region of Meckel's cartilage in the developing mandible of rats

A cartilagem de Meckel é uma estrutura transitória embrionária presente durante os estágios iniciais da formação da mandíbula, localizada em toda sua extensão e dividida em três porções, anterior, intermediária e posterior. O enfoque deste trabalho foi direcionado à elucidação do destino final da porção intermediária por meio de um estudo temporal sequenciado. Por isso, foi investigado a presença de células de reabsorção e a presença de fibras colágenas, bem como da proteína óssea osteopontina (OPN) na cartilagem de Meckel na região do germe do 1º molar inferior e no seu entorno. Foram utilizados fetos de ratos Wistar em períodos gestacionais pré-estabelecidos, G18 a G21 (grupos de dias intrauterinos), bem como P0 e P1 (recém-nascidos) para remoção das cabeças. Em sequência, os espécimes foram fixados em solução de formaldeído 4% + glutaraldeído 0,1% com tampão fosfato 0,1M, descalcificados em EDTA 4,13%, desidratados em concentrações crescentes de etanol e incluídos em parafina. As amostras foram coradas em hematoxilina e eosina (HE) e tricrômico de Mallory para análise histológica. Adicionalmente, os grupos G19 a P0 foram submetidos à reação histoquímica de TRAP para determinação da presença de células clásticas. Além disso, os grupos G21 e P0 (dia do nascimento) passaram por reações de imunomarcação para análise da expressão de OPN. Foi observado a degeneração gradual da cartilagem com a observação de mudanças estruturais, a justaposição de células clásticas na superfície da cartilagem por reação histoquímica TRAP a partir do G21, o aparecimento de colágeno tipo I nas fases terminais da degeneração, assim como a marcação positiva para a osteopontina na superfície de G21 e em todo o remanescente da cartilagem de Meckel no grupo P0. O estudo apontou um processo de degeneração da cartilagem com evidências de formação de matriz mineralizada de natureza óssea, a qual foi reabsorvida por células clásticas, sugerindo a ossificação da porção intermediária da cartilagem de Meckel.

Curcumin alleviated oxidation stress injury by mediating osteopontin in nephrolithiasis rats

Purpose: To explore the role and mechanism of curcumin (Cur) in reducing oxidative stress damage in rats with nephrolithiasis induced by ethylene glycol (EG). Methods: Thirty male rats were divided into normal control, model, positive (10% potassium citrate), Cur-10 (10 mg/kg curcumin) and Cur-20 (20 mg/kg curcumin) groups. Results: The results of kidney tissue section stained by hematoxylin-eosin and von Kossa showed that curcumin treatment can inhibit the formation of kidney stones. The biochemical test results showed that the urea (Ur), creatinine (Cr), uric acid (UA), inorganic phosphorus and Ca2+ concentrations in urine decreased after being treated with curcumin. There were significant differences between different doses of curcumin (P < 0.05). Compared with the Cur-10 group, Cur-20 had a more significant inhibitory effect on malondialdehyde (MDA) (P < 0.05). In addition, reverse transcription polymerase chain reaction (PCR) detection and immunohistochemical results indicated that the osteopontin (OPN) in the kidney was significantly reduced after curcumin treatment. Conclusion: Curcumin could reduce the oxidative stress damage caused by EG-induced kidney stones.

Estudo histológico e imunohistoquímico da degeneração da região central da cartilagem de Meckel na mandíbula em desenvolvimento de ratos / Histological and immunohistochemical study of the degeneration of the central region of Meckel's cartilage in the developing mandible of rats

A cartilagem de Meckel é uma estrutura transitória embrionária presente durante os estágios iniciais da formação da mandíbula, localizada em toda sua extensão e dividida em três porções, anterior, intermediária e posterior. O enfoque deste trabalho foi direcionado à elucidação do destino final da porção intermediária por meio de um estudo temporal sequenciado. Por isso, foi investigado a presença de células de reabsorção e a presença de fibras colágenas, bem como da proteína óssea osteopontina (OPN) na cartilagem de Meckel na região do germe do 1º molar inferior e no seu entorno. Foram utilizados fetos de ratos Wistar em períodos gestacionais pré-estabelecidos, G18 a G21 (grupos de dias intrauterinos), bem como P0 e P1 (recém-nascidos) para remoção das cabeças. Em sequência, os espécimes foram fixados em solução de formaldeído 4% + glutaraldeído 0,1% com tampão fosfato 0,1M, descalcificados em EDTA 4,13%, desidratados em concentrações crescentes de etanol e incluídos em parafina. As amostras foram coradas em hematoxilina e eosina (HE) e tricrômico de Mallory para análise histológica. Adicionalmente, os grupos G19 a P0 foram submetidos à reação histoquímica de TRAP para determinação da presença de células clásticas. Além disso, os grupos G21 e P0 (dia do nascimento) passaram por reações de imunomarcação para análise da expressão de OPN. Foi observado a degeneração gradual da cartilagem com a observação de mudanças estruturais, a justaposição de células clásticas na superfície da cartilagem por reação histoquímica TRAP a partir do G21, o aparecimento de colágeno tipo I nas fases terminais da degeneração, assim como a marcação positiva para a osteopontina na superfície de G21 e em todo o remanescente da cartilagem de Meckel no grupo P0. O estudo apontou um processo de degeneração da cartilagem com evidências de formação de matriz mineralizada de natureza óssea, a qual foi reabsorvida por células clásticas, sugerindo a ossificação da porção intermediária da cartilagem de Meckel.

Assessing the level and diagnostic accuracy of osteopontin and oral health status in periodontitis patients with/without Type-2 Diabetes Mellitus / Avaliação do nível e precisão do diagnóstico de Osteopontina e estado de saúde bucal em pacientes com periodontite com/sem Diabetes Mellitus tipo 2

Objective: Investigating osteopontin (OPN) level in gingival crevicular fluid (GCF) of patients affected by periodontitis with or without Type-2 diabetes mellitus (T2DM). The aim of this study is to explore the possibility of OPN to differentiate between periodontal health and disease. Material and Methods: A total number of 36 participants seeking periodontal treatment were recruited in this pilot study and divided into three study groups. Periodontitis [systemically healthy participants with periodontitis (probing pocket depth) PPD (probing pocket depth) ≥ 4mm], periodontitis and poorly controlled type 2 diabetes mellitus (), and control (systemically and periodontally healthy periodontium) groups. Plaque index (PI), gingival index (GI) and PPD were examined. OPN level was measured in the GCF and analysed, using Enzyme-Linked Immunosorbent assay. Results: PI and GI were significantly higher in T2DM with periodontitis compared to periodontitis and control groups. Both periodontitis and P-T2DM groups showed significant increase in the OPN levels compared to control group (p<0.001). PPD showed the only significant positive association with OPN (p<0.001) compared to other clinical parameters. The receiver operating characteristics curve analysis demonstrated that OPN had higher area under the curve value (AUC: 0.95) in periodontitis compared to P-T2DM patients (AUC: 0.86). Conclusion: In periodontitis groups, clinical parameters were equally deteriorated together with significant increase in the expression of OPN compared to control. Furthermore, GCF levels of OPN were sensitive and specific enough to discriminate between health and periodontitis even with T2DM. This could introduce OPN to be as a candidate diagnostic biomarker of periodontal disease. (AU)

Objetivo: Investigar o nível de osteopontina (OPN) no fluido gengival crevicular (GCF) de pacientes com periodontite com ou sem diabetes mellitus tipo 2 (T2DM). O objetivo deste estudo foi explorar a possibilidade da OPN diferenciar entre saúde e doença periodontal. Material e Métodos: No total, para este estudo piloto foram recrutados 36 participantes que estavam em busca de tratamento periodontal e divididos em três grupos de estudo: grupos periodontite [participantes sistemicamente saudáveis com periodontite (profundidade de sondagem) PPD ≥ 4 mm], grupo periodontite e Diabetes Mellitus tipo 2 mal controlada (P-T2DM) e grupo controle (saudáveis sistemicamente e periodontalmente). Índice de placa (PI), índice gengival (GI) e PPD foram examinados. O nível de OPN foi medido no GCF e analisado usando o ensaio ELISA (Enzyme-Linked Immuno Sorbent Assay). Resultados: PI e GI foram significativamente maiores no T2DM com periodontite em comparação aos grupos com periodontite e controle. Os grupos com periodontite e P-T2DM apresentaram aumento significativo nos níveis de OPN em comparação ao grupo controle (p <0,001). PPD mostrou a única associação positiva significativa com OPN (p <0,001) em comparação com outros parâmetros clínicos. A análise da curva de características operacionais do receptor demonstrou que OPN teve maior área sob o valor da curva (AUC: 0,95) na periodontite em comparação com pacientes com P-T2DM (AUC: 0,86). Conclusão: Nos grupos com periodontite, os parâmetros clínicos foram igualmente deteriorados juntamente com aumento significativo na expressão de OPN em comparação com o grupo controle. Além disso, os níveis de OPN no GCF foram sensíveis e específicos o suficiente para discernir entre saúde e periodontite, mesmo com T2DM. Isso poderia apresentar a OPN como um candidato a biomarcador diagnóstico de doença periodontal.(AU)

Graft application and dexamethasone treatment influences new bone formation in rat tibial bone defects / Aplicación de injerto y tratamiento con dexametasona influyen en la formación de hueso nuevo en los defectos en la tibia de rata

SUMMARY: The objective of this study were bone defect complications that occur due to traumas or infections. Bone grafts are required to provide support, fill gaps and improve biological repair in skeletal damage. Dexamethasone plays role in calcium signaling modulation and used in diseases. Aim of this study was to evaluate osteonectin and osteopontin expressions in new bone development after dexamethasone application on tibial bone defects. Rats were divided into defect, defect+graft and defect+graft+dexamethasone treated groups. Tibial bone defect created, and rats were kept immobile for 28 days. Alloplastic material was placed in defect area in second and group third groups. 2.5 mg/kg Dex and normal saline were injected to dexamethasone and defect groups twice a week for 56 days. Inflammation and congestion were increased in defect and defect+graft groups. Defect+graft+dexamethasone group; increased number of osteoblast and osteocyte cells, dense bone matrix, formation of new bone trabeculae was observed. Defect+graft group; osteonectin expression in graft regions, osteoblast cells, some connective tissue cells and fibers were seen whereas in defect+graft+dexamethasone group; osteopontin expression in osteoblast and osteocyte cells of new bone trabeculae were observed. Dexamethasone may lead to formation of new bone trabeculae into the graft material resulting in increased osteoconduction and osteoinductive effect for differentiation of osteon.

RESUMEN: Los defectos óseos son complicaciones que ocurren debido a traumas o infecciones. Se requieren injertos óseos para proporcionar apoyo, llenar los espacios y mejorar la reparación biológica en el hueso dañado. La dexametasona desempeña un papel importante en la modulación de la señalización del calcio y se usa en enfermedades. El objetivo de este estudio fue evaluar las expresiones de osteonectina y osteopontina en el desarrollo óseo después de la aplicación de dexametasona en defectos óseos tibiales. Las ratas se dividieron en grupos: defecto, defecto + injerto y defecto + injerto + grupos tratados con dexametasona. Se creó un defecto óseo tibial, y las ratas se mantuvieron inmóviles durante 28 días. El material aloplástico se colocó en el área del defecto en el segundo y tercer grupo. Se inyectaron 2,5 mg / kg de dexametasona y solución salina normal a grupos de defectos dos veces por semana durante 56 días. La inflamación y la congestión aumentaron en los grupos de defectos y defectos + injerto; En el grupo defecto + injerto + grupo tratado con dexametasona se observó un aumento en el número de osteoblastos y osteocitos, de matriz ósea densa y en la formación de nuevas trabéculas óseas. En el grupo defecto + grupo de injerto se observó la expresión de osteonectina en las áreas de injerto, osteoblastos, algunas células y fibras de tejido conectivo, mientras que en el grupo defecto + injerto + dexametasona se observó la expresión de osteopontina en osteoblastos y osteocitos y formación de nuevas trabéculas óseas . En conclusión la dexametasona puede conducir a la formación de nuevas trabéculas óseas en el material de injerto, lo que resulta en un aumento de la osteoconducción y un efecto osteoinductivo para la diferenciación del osteón.

Melatonin has a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/secretion / Melatonina tem efeito estimulador em osteoblastos, pela regulação positiva da expressão/secreção de COL-I e OPN

ABSTRACT Melatonin (MLT) is a potential signaling molecule in the homeostasis of bone metabolism and may be an important mediator of bone formation and stimulation. The aim of this in vitro study was to evaluate the effect of MLT on the viability, mRNA/protein expression and mineralization of pre-osteoblastic cells. The concentrations 5, 2.5, 1, 0.1 and 0.01 mM MLT were tested on pre-osteoblastic cells (MC3T3) compared to control (no MLT), evaluating proliferation and cell viability (C50), gene expression (RT-PCR) and secretion (ELISA) of COL-I and OPN at 24h, 48h and 72h, and the formation of mineral nodules (alizarin red and fast red) after 10 days of treatment. MLT at 5 and 2.5 mM proved to be cytotoxic (C50), so only 0.01, 0.1 and 1 mM were used for the subsequent analyses. OPN mRNA expression increased with MLT at 0.1 mM - 1 mM, which was followed by increased secretion of OPN both at 24h and 72h compared to the remaining groups (p <0.05). COL-I mRNA and COL-1 secretion followed the same pattern as OPN at 0.1 mM MLT at 72h of treatment (p <0.05). Regarding mineralization, all MLT doses (except 1mM) caused an increase (p <0.05) in the formation of mineral nodules compared to the control. Melatonin at 0.01mM - 1mM had a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/ secretion and mineralization, thereby fostering osteogenesis.

RESUMO A melatonina (MLT) é uma molécula potencial de sinalização na homeostase do metabolismo ósseo e pode ser um importante mediador da formação e estimulação óssea. O objetivo deste estudo in vitro foi avaliar o efeito da MLT na viabilidade, na expressão do mRNA da proteína e mineralização de células préosteoblásticas. As concentrações de MLT 5, 2,5, 1, 0,1 e 0,01 mM foram testadas em células pré-osteoblásticas da linhagem MC3T3 em comparação ao controle (sem MLT), avaliando a proliferação e a viabilidade celular (C50), expressão gênica (rtPCR) e secreção (Elisa) de Colágeno tipo 1 (COL-I) e osteopontina (OPN) às 24, 48 e 72 horas, além da formação de nódulos minerais por meio do teste vermelho de Alizarina fast red após 10 dias de tratamento. MLT a 5 e 2,5 mM provou ser tóxico (C50). Portanto, as concentrações de 0,01, 0,1 e 1 mM foram utilizadas para as análises subsequentes. A expressão do mRNA da OPN aumentou com MLT a 0,1 mM-1mM, seguida pela secreção aumentada de OPN às 24 e 72 horas em comparação aos demais grupos (p<0,05). O mRNA de COL-I e a secreção de COL-I seguiram o mesmo padrão do OPN a 0,1 mM de MLT em 72 horas de tratamento (p<0,05). Em relação à mineralização, todas as doses de MLT (exceto 1mM) causaram aumento (p<0,05) na formação de nódulos minerais em comparação ao controle. A MLT na concentração entre 0,01mM a 1 mM teve um efeito estimulador sobre os osteoblastos, ao regular positivamente a expressão e secreção de COL-I e OPN, além da mineralização, favorecendo a osteogênese.

Osteopontin expression and its relationship with prognostic biomarkers in canine mammary carcinomas / Expressão de osteopontina e sua relação com biomarcadores prognóstico nos carcinomas mamários caninos

Osteopontin is a glycophosphoprotein implicated in different physiologic and pathologic processes and is known to be involved in progression and metastasis of various cancers in humans, but this relation is still little explored in the veterinary. The aim was to evaluate the expression of osteopontin in canine mammary carcinomas and its relation with well-established canine mammary tumor biomarkers. For that, expression of OPN, EGFR, HER2, and c-Kit were evaluated along with Ki67 rate in 43 mammary carcinomas. Osteopontin was demonstrated to be expressed by neoplastic epithelial cells in all carcinomas as well as in stromal cells from the tumor microenvironment. Relation between high osteopontin expression and EGFR positivity (P<0.001) and HER2 overexpression (P=0.012) was demonstrated. In conclusion, high OPN expression seems to be related to poor prognosis and MAPK pathway activation, given the association with EGFR and HER2, members of the MAPK signaling pathway.(AU)

A osteopontina é uma glicofosfoproteina implicada em diferentes processos fisiológicos e patológicos, sendo conhecida por estar envolvida na progressão e metástase de vários cânceres nos humanos, no entanto, essa relação é ainda pouco explorada na veterinária. O objetivo deste trabalho foi avaliar a expressão da osteopontina nos carcinomas mamários caninos e sua relação com biomarcadores bem estabelecidos para esta neoplasia. Para isto, foi avaliada a expressão de OPN, EGRH, HER2 e c-Kit juntamente com a taxa de Ki67 em 43 carcinomas mamários. A osteopontina foi expressa pelas células epiteliais neoplásicas em todos os carcinomas, assim como, nas células estromais do microambiente tumoral. Foi demonstrada uma relação entre uma alta expressão de osteopontina e positividade para EGFR (P<0.001) e superexpressão de HER2 (P=0.012). Em conclusão, alta expressão de OPN parece estar relacionada com mau prognóstico e ativação da via MAPK, devido a sua associação com EGRF e HER2, os quais são membros desta via de sinalização.(AU)

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)

A comparative study of osteopontin and MMP-2 protein expression in peripheral and central giant cell granuloma of the jaw / Estudo comparativo da expressão das proteínas osteopontina e MMP-2 no granuloma central e periférico de células gigantes de mandíbula

Abstract Introduction: Oral peripheral and central giant cell granulomas are lesions with little-known etiology and pathogenesis. Objective: The aim of this study was to compare matrix metalloproteinases-2 and osteopontin protein expression in the multinucleated giant cells and mononuclear cells of the peripheral and central giant cell granuloma lesions. Methods: In this retrospective study, the presence of matrix metalloproteinases-2 and osteopontin in 37 cases of central giant cell granuloma and 37 cases of peripheral giant cell granuloma paraffin blocks were assessed by streptavidin-biotin immunohistochemistry. Independent sample t-test, Chi-square, Mann-Whitney tests and Spearman's rank correlation coefficient were used. Results: The osteopontin was expressed in both multinucleated giant cells and mononuclear cells in all cases of peripheral and central giant cells granulomas. However, the matrix metalloproteinases-2 expression was positive in 86.5% of giant cells and it was positive in all of mononuclear cells in peripheral giant cells granuloma. In central giant cells granulomas, 91.8% of giant cells and all mononuclear cells were positive for matrix metalloproteinases-2 marker. Percentage and Intensity of staining were significantly higher in central than peripheral giant cells lesions, for both markers (p ˂ 0.05). Conclusion: This study showed that the expression of osteopontin in giant cells supports the theory of osteolcastic nature of these cells. Also, the presence of osteopontin and matrix metalloproteinases-2 in mononuclear cells may indicate the monocyte-macrophage origin of these cells, as the differentiation of the precursors of the mononuclear stromal monocyte/macrophage to osteoclasts is possibly affected by the expression of osteolytic factors. Also, may be differences in biological behaviors of these lesions are associated with the level of osteopontin and matrix metalloproteinases-2 expression.

Resumo Introdução: Os granulomas periféricos e centrais de células gigantes são lesões com etiologia e patogênese pouco conhecidas. Objetivo: Comparar a expressão das proteínas metaloproteinases da matriz-2 e osteopontina nas células gigantes multinucleadas e células mononucleares no granuloma periférico e central de células gigantes. Método: Neste estudo retrospectivo, a presença de metaloproteinases da matriz-2 e osteopontina em 37 casos de granuloma central de células gigantes e 37 casos de granuloma periférico de células gigantes em blocos de parafina foi avaliada por imuno-histoquímica pela estreptavidina-biotina. Foram usados teste t para amostra independente, teste de qui-quadrado, Mann-Whitney e coeficiente de correlação de Spearman. Resultados: A osteopontina foi expressa em células gigantes multinucleadas e células mononucleares em todos os casos de granuloma periférico de células gigantes e granuloma central de células gigantes. No entanto, a expressão de metaloproteinases da matriz-2 foi positiva em 86,5% de células gigantes e foi positiva em todas as células mononucleares em granuloma periférico de células gigantes. Em granuloma central de células gigantes, 91,8% das células gigantes e todas as células mononucleares foram positivas para o marcador metaloproteinases da matriz-2. A porcentagem e intensidade de coloração em granuloma central de células gigantes foram significantemente maiores do que em granuloma periférico de células gigantes para ambos os marcadores (p ˂ 0,05). Conclusão: Este estudo mostrou que a expressão de osteopontina em células gigantes apoia a teoria da natureza osteoclástica dessas células. Além disso, a presença de osteopontina e metaloproteinases da matriz-2 em células mononucleares pode indicar a origem dos monócitos-macrófagos dessas células, uma vez que a diferenciação dos precursores do monócito/macrófago estromal mononuclear em osteoclastos é possivelmente afetada pela expressão de fatores osteolíticos. Além disso, as diferenças nos comportamentos biológicos dessas lesões estão associadas ao nível de expressão de osteopontina e metaloproteinases da matriz-2.

Expression of Osteopontin mRNA During Periodontal Healing Following Scaling and Root Planing

Objective: To analyze osteopontin mRNA expression levels in subjects with periodontitis prior to (baseline) and 7, 14, and 28 days following scaling and root planing (SRP). Material and Methods: Gingival crevicular fluid was collected as clinical samples from four subjects with periodontitis (pocket depth, 4-5 mm) aged 35-54 years old as well as from three healthy subjects (controls). The osteopontin mRNA expression levels were measured by quantitative real-time polymerase chain reaction. Spearman's rank correlation between osteopontin levels in gingival crevicular fluid and the modified gingival index (MGI) was also performed. Results: The Wilcoxon signed-rank test showed no significant difference in osteopontin mRNA expression levels between baseline and 28 days following SRP (p=0.068). The Friedman test showed no significant difference in osteopontin mRNA expression levels between baseline and following SRP (7, 14, or 28 days) (p>0.05). Spearman's rank correlation showed no significant correlation between osteopontin mRNA expression levels and MGI (r=0.087; p=0.749). Conclusion: Following SRP of periodontal tissue, there was a decreasing trend in osteopontin mRNA expression; however, this finding was not statistically significant. Nevertheless, osteopontin can be used as a biomarker to monitor the healing process; however, further studies are required to clarify our results.

The influence of Aloe vera with mesenchymal stem cells from dental pulp on bone regeneration: characterization and treatment of non-critical defects of the tibia in rats

Abstract Objective This study aimed to evaluate the inflammatory effect and bone formation in sterile surgical failures after implantation of a collagen sponge with mesenchymal stem cells from human dental pulp (hDPSCs) and Aloe vera. Material and Methods Rattus norvegicus (n=75) were divided into five experimental groups according to treatment: G1) control (blood clot); G2) Hemospon®; G3) Hemospon® in a culture medium enriched with 8% Aloe vera; G4) Hemospon® in a culture medium containing hDPSCs and G5) Hemospon® in a culture medium enriched with 8% Aloe vera and hDPSCs. On days 7, 15 and 30, the animals were euthanized, and the tibia was dissected for histological, immunohistochemistry and immunofluorescence analyses. The results were analyzed using nonparametric Kruskal-Wallis test and Dunn's post-test. Results On days 7 and 15, the groups with Aloe vera had less average acute inflammatory infiltrate compared to the control group and the group with Hemospon® (p<0.05). No statistically significant difference was found between the groups regarding bone formation at the three experimental points in time. Osteopontin expression corroborated the intensity of bone formation. Fluorescence microscopy revealed positive labeling with Q-Tracker® in hDPSCs before transplantation and tissue repair. Conclusion The results suggest that the combination of Hemospon®, Aloe vera and hDPSCs is a form of clinical treatment for the repair of non-critical bone defects that reduces the inflammatory cascade's effects.

Short-term use of resveratrol in alloplastic graft material applied with calvarial bone defects in rats

Abstract Purpose: The effects of resveratrol administration on calvarial bone defects with alloplastic graft material was investigated for osteoinductive reaction and bone development in rats. Methods: Healthy male rats were randomly divided into 3 groups consisting of 10 rats. Groups were as follows: control (defect) group, defect + graft group, and defect + graft + resveratrol group. A calvarial bone defect was created in all groups, alloplastic bone grafts were applied to the defect in the 2nd and 3rd group, resveratrol (5 mg/kg/day) was added to the drinking water of the animals following graft application for 28 days in the 3rd group. Results: Increase in osteoclasts and necrotic changes were observed histopathologically in the control group. In the 2nd group, reduction of inflammation, congestion of blood vessels, increased osteblastic activity, osteoinductive effect, progression of osteocyte development and increased collagen fibers in connective tissue were observed. In the 3rd group, osteoblasts seemed to secrete bone matrix and accelerate osteoinductive effect with increased osteopregenitor activity and positive osteopontin and osteonectin expressions. Conclusion: Resveratrol treatment was thought to be an alternative and supportive drug for implant application by inducing new bone formation in the calvaral defect region as a result of short-term treatment.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro

Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Effects on alveolar bone of diabetes mellitus induced by streptozotocin in rats: histopathologic and immunohistochemical study / Efectos de la diabetes mellitus inducida por streptozotocina en el hueso alveolar de ratas: estudio histopatológico e inmunohistoquímico

SUMMARY: The purpose of this study was to investigate effects of diabetes mellitus (DM) on the alveolar bone with histopathological and immunohistochemical methods. Wistar rats were divided into two groups, control and diabetes group. Control group was fed standard rat chow and drinking water for 8 weeks. Single dose (Streptozotocin) STZ (55 mg/kg), was dissolved in sodium citrate buffer and introduced intraperitoneal injection. Diabetes group and control group were compared in terms of glucose values. The blood glucose concentration in diabetic rats was significantly high (p <0.05). In diabetes group; periodontal membrane and the dilation of blood vessels, hemorrhage has also been a significant increase in inflammatory cells. In the diabetes group, osteonectin showed positive expression in periodontal membrane and showed negative expression in osteocytes of alveolar bone. Osteopontin expression in fibroblast cells and periodontal membrane collagen fibrils was positive, alveolar cells, osteocytes and bone matrix bone was found positive. Diabetes results showed that there formed periodontitis; due to the increase in inflammation inhibiting bone formation delaying the development of early bone cells.

RESUMEN: El objetivo de este estudio fue investigar los efectos de la diabetes mellitus (DM) sobre el hueso alveolar con métodos histopatológicos e inmunohistoquímicos. Las ratas Wistar se dividieron en dos grupos, grupo control y grupo de diabetes. El grupo control fue alimentado con comida estándar y agua potable durante 8 semanas. La dosis única Streptozotocina (STZ) (55 mg/ kg), se disolvió en tampón de citrato de sodio y se introdujo mediante inyección intraperitoneal. El grupo diabetes y el grupo control se compararon en términos de valores de glucosa. La concentración de glucosa en sangre en ratas diabéticas fue significativamente alta (p <0,05). En el grupo diabetes hubo un aumento significativo de la membrana periodontal y dilatación de los vasos sanguíneos y hemorragia, con un aumento significativo de células inflamatorias. En el grupo diabetes, la osteonectina mostró una expresión positiva en la membrana periodontal además se observó expresión negativa en los osteocitos del hueso alveolar. La expresión de osteopontina en fibroblastos y fibrillas de colágeno en membrana periodontal fue positiva, las células alveolares, osteocitos y hueso de la matriz ósea dio positivo. Los resultados de la diabetes mostraron que existía periodontitis, debido al aumento de la inflamación que inhibió la formación ósea retardando el desarrollo de células óseas tempranas.

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.

Evaluation of apoptosis and osteopontin expression in osteocytes exposed to orthodontic forces of different magnitudes

The in vivo response of osteocytes to different force magnitudes soon after they are applied remains to be elucidated. The aim of this study was to examine the early effects of applying a very light (LF: 0,16 N) and a very strong (SF: 2,26 N) orthodontic force during one hour on apoptosis and osteopontin (OPN) expression on alveolar bone osteocytes, in rats. Results: LF: compared to the control group, they showed a significant increase in OPN expression, and a significant decrease in the number of TUNELpositive osteocytes. SF: compared to the control group, they showed a significant increase in OPN expression and a significant decrease in the number of TUNELpositive osteocytes. Our results show that osteocytes respond very early to the application of tension and pressure forces of different magnitudes, and application of forces decreases the number of apoptotic osteocytes and increases OPN expression. These results allow concluding that osteocytes activate rapidly when subjected to locally applied forces, whether these forces be pressure or tension, light or strong forces. Grants: UBACyT 200201301002270 BA and School of Dentistry, University of Buenos Aires (AU)

Hasta el momento no se ha dilucidado la respuesta temprana in vivo de los osteocitos a la aplicación de fuerzas de diferentes magnitudes sobre el hueso. El objetivo de este estudio fue examinar la respuesta temprana de la aplicación de una fuerza ortodóncica muy liviana (FL: 0,16 N) y muy fuerte (FF: 2,26 N) durante una hora sobre la expresión de apoptosis y osteopontina (OPN) en los osteocitos del hueso alveolar, en ratas. Resultados: FL: en comparación con el grupo control, mostraron un aumento significativo en la expresión de OPN y una disminución significativa en el número de osteocitos TUNELpositivos. FF: en comparación con el grupo control, mostraron un aumento significativo en la expresión de OPN y una disminución signi ficativa en el número de osteocitos TUNELpositivos. Nuestros resultados muestran que los osteocitos responden muy temprano a la aplicación de fuerzas de tensión y presión de diferentes magnitudes, y la aplicación de fuerzas disminuye el número de osteocitos apoptóticos y aumenta la expresión de OPN. Estos resultados permiten concluir que los osteocitos se activan rápidamente cuando se los somete a fuerzas aplicadas localmente, ya sean estas fuerzas de presión o tensión, livianas o fuertes (AU)

Effects of the photobiomodulation using different energy densities on the periodontal tissues under orthodontic force in rats with type 2 diabetes mellitus

Abstract To evaluate the impact of the GaAlAs diode laser with energy densities of 160 J/cm2, 320 J/cm2, and 640 J/cm2 on the periodontal tissues under continuous orthodontic force application and on the rate of orthodontic tooth movement in rats with type-2 diabetes mellitus. The intensity of primary alveolar bone formation was also investigated through the immune-positive osteocytes for OPN antibody. Forty adult male Wistar rats were divided into eight groups of 5 rats: normoglycemic (N), 160 J-laser-normoglycemic (160 J-LN), 320 J-laser-normoglycemic (320 J-LN), 640 J-laser-normoglycemic (640 J-LN), diabetic (D), 160 J-laser-diabetic (160 J-LD), 320 J-laser-diabetic (320 J-LD), and 640 J-laser-diabetic (640 J-LD) rats. Diabetes mellitus was induced by a single intravenous injection of 40 mg/kg monohydrated-alloxan. An orthodontic force magnitude of 20cN was applied. The laser parameters were continuous emission of 780-nm wavelength, output power of 20mW, and fiber probe with a spot size of 0.04 cm in diameter. Radiographic, histomorphological, and immunohistochemical analysis were performed after a period of 21 days. The photobiomodulation using the energy density of 640 J/cm2 strongly stimulated the alveolar bone formation and contributed the reorganization of the soft periodontal tissues, followed by the 320 J/cm2. Extensive alveolar bone loss, intense infiltration of inflammatory cells, and degradation of the PDJ tissue were mainly found in the D and 160 J-LD groups. The rate of orthodontic tooth movement was represented by the interdental distance between the cementoenamel junctions of the right mandibular first and second molars . This distance was larger in the diabetic groups (D: 39.98±1.97, 160 J-LD: 34.84±6.01, 320 J-LD: 29.82±1.73, and 640 J-LD: 35.47±4.56) than in the normoglycemic groups (N: 21.13±1.19; 160 J-LN: 22.69±0.72, 320 J-LN: 22.28±0.78, and 640 J-LN: 24.56±2.11). The number of osteopontin-positive osteocytes was significantly greater in the 640 J-LD (14.72 ± 0.82; p < 0.01) and 640 J-LN (13.62 ± 1.33; p < 0.05) groups than with D (9.82 ± 1.17) and 160 J-LD (9.77 ± 1.10) groups. Therefore, the energy density of 640 J/cm2 provided the best maintenance and integrity of the periodontal tissue microarchitecture under continuous orthodontic force when compared with the other dosages, mainly in the uncontrolled diabetic rats. The interdental distance was greater in the D and 160 J-LD groups due to presence of severe periodontitis caused by diabetes plus the mechanical stress generated by continuous orthodontic forces, implying, thus, an insufficient biostimulatory effect for the dosage of 160 J/cm2.

Systemic melatonin application increases bone formation in mandibular distraction osteogenesis

Abstract This study aimed to investigate the effects of different doses of systemic melatonin application on new bone formation during mandibular distraction osteogenesis (DO) in rats. Mandibular DO was performed on 30 adult female Sprague-Dawley rats, which were randomly divided into three groups: control group (CNT), melatonin dose 1 (MLT-D1), and melatonin dose 2 (MLT-D2). A five-day latent waiting period and a ten-day distraction phase followed the surgery. After the surgery, rats from the MLT-D1 and MLT-D2 groups received 25 and 50 mg/kg melatonin, respectively, at 7, 14, 21, 28, and 35 days. The animals were euthanised 28 days after distraction, i.e. at 43 days after surgery. Histological and histomorphometric analyses revealed that the distracted bone area was completely filled with new bone formation in all three groups. The MLT-D2 group exhibited the most new bone formation, followed by MLT-D1 and CNT. The melatonin groups had more osteoclasts than the CNT (p < 0.05). The number of osteoblasts was higher in the melatonin groups than in the CNT group, and the MLT-D2 had more osteoclasts than the MLT-D1 group (p < 0.05). Finally, the osteopontin (OPN) and vascular endothelial growth factor (VEGF) levels were higher in the melatonin groups than in the CNT group, and the MLT-D2 had higher OPN and VEGF levels than the MLT-D1 (p < 0.05). This study suggests that systemic melatonin application could increase new bone formation in DO.

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.