Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion

ABSTRACT Objective: This study aimed to evaluate the effects of systemic teriparatide on sutural bone formation after premaxillary suture expansion in rats. Material and Methods: Twenty Wistar male rats (8-10 weeks old) were randomly divided into two groups, namely, control (C, n=10) and teriparatide (T, n=10). An expansion force was applied to the maxillary incisors using helical spring for a seven-day expansion period, for both groups. On the eighth day, the rats were kept for a seven-day consolidation period, and then 60 µg/kg teriparatide (once a day) was administered to group T subcutaneously for seven days. Then, all the rats were sacrificed, and histological sections were stained with hemotoxylin-eosin for examination. Anti-osteonectin, anti-osteocalcin, anti-Vascular endothelial growth factor (VEGF) and anti-transforming growth factor beta (TGF-β) were evaluated by immunohistochemical analysis in the midpalatal suture area. Results: Histologically, the newly formed bone tissue was observed to be larger in group T than in group C. The number of immunoreactive osteoblasts for osteonectin, osteocalcin and VEGF antibodies was significantly higher in group T than in group C (p = 0.0001). The TGF-β antibody showed a mild reaction in group T, but did not reach significance in comparison with group C (p ˃ 0.05). Conclusion: Systemic teriparatide application following the premaxillary expansion of the suture area may stimulate bone formation and add to the consolidation of the expansion in rats by regulating osteonectin, osteocalcin and VEGF.

RESUMO Objetivo: O presente estudo teve como objetivo avaliar os efeitos do uso sistêmico da teriparatida na formação óssea sutural após a expansão da pré-maxila em ratos. Material e Métodos: Vinte ratos machos da raça Wistar (com oito a dez semanas de vida) foram divididos aleatoriamente em dois grupos: controle (C, n=10) e teriparatida (T, n=10). Uma força de expansão foi aplicada aos incisivos superiores, usando uma mola helicoidal, por um período de expansão de sete dias em ambos os grupos. No oitavo dia, os ratos iniciaram um período de sete dias de consolidação, nos quais 60 µg/kg de teriparatida foram administrados (uma vez ao dia), por via subcutânea, para o grupo T. Posteriormente, todos os ratos foram sacrificados e cortes histológicos corados com hemotolixina-eosina foram examinados. Por meio de análise imuno-histoquímica da região da sutura palatina mediana, avaliou-se a presença de anti-ostenectina, anti-osteocalcina, anti-fator de crescimento endotelial vascular (VEGF) e anti- fator transformador de crescimento (TGF-β). Resultados: Histologicamente, observou-se que o tecido ósseo recém-formado foi maior no grupo T do que no grupo C. O número de osteoblastos imunorreativos para anticorpos de osteonectina, osteocalcina e VEGF foi significativamente maior no grupo T do que no grupo C (p = 0,0001). O anticorpo TGF-β mostrou uma pequena reação no grupo T; porém, sem diferença significativa para o grupo C (p ˃ 0,05). Conclusão: O uso sistêmico de teriparatida após a expansão da sutura na região da pré-maxila pode estimular a formação óssea e melhorar a consolidação da expansão em ratos, por meio da regulação de osteonectina, osteocalcina e VEGF.

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.

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.

Effects of melatonin on tibia bone defects in rats / Efectos de la melatonina sobre defectos óseos en la tibia de ratas

The aim of this study was to evaluate the effects of melatonin healing in a tibial bone defect model in rats by means of histopathological and immunohistochemistry analysis. Twenty one male Wistar albino rats were used in this study. In each animal, bone defects (6 mm length ) were created in the tibias. The animals were divided into three groups. In group 1 control group (rats which tibial defects). Group 2 melatonin (10 mg/kg) + 14 days in the tibial defect group) was administered intraperitoneally to rats. Group 3 melatonin (10 mg/kg) + 28 days in the tibial defect group) was administered intraperitoneally to rats. Histopathological analysis of samples was performed to evaluate the process of osteoblastic activity, matrix formation, trabecular bone formation and myeloid tissue in bone defects. Immunohistochemical and immunoblot analysis demonstrated non-collagenous proteins (osteopontin and osteonectin) differences in tibial bone defects. The expression of osteopontin on tibia was increased by 14 days melatonin treatment. The expression of osteonectin on tibia was dramatically increased by 14 days melatonin treatment.

El objetivo fue evaluar por medio de análisis histopatológico e inmunohistoquímico los efectos cicatrizantes de la melatonina en un modelo de defecto óseo tibial en ratas. Se utilizaron 21 ratas albinas Wistar macho. En cada animal, se crearon defectos óseos en las tibias de 6 mm de longitud. Los animales se dividieron en tres grupos. El Grupo 1 correspondió al grupo control (defectos tibiales sin tratamiento). Al Grupo 2 se administró melatonina por vía intraperitoneal (10 mg/kg) 14 días posteriores al defecto tibial. Al Grupo 3 se administró melatonina por vía intraperitoneal (10 mg/kg) 28 días posteriores al defecto tibial. Se realizó un análisis histopatológico para evaluar los procesos de actividad osteoblástica, formación de matriz, formación de hueso trabecular y tejido mieloide en los defectos óseos. Los análisis inmunohistoquímicos y de inmunotransferencia mostraron diferencias de proteínas no colágenas (osteopontina y osteonectina). La expresión de osteopontina en defectos óseos tibiales se incrementó en el Grupo 2. La expresión de osteonectina en la tibia se incrementó fuertemente bajo el tratamiento con melatonina por 14 días.

Immunolocalization of bmp2/4, tgfβ-1 and osteonectin in fibro-osseous lesions of the jaws / Imunolocalização de bmp2/4, tgfß-1 e osteonectina em lesões-fibro-óssea benignas

Objetivo: as lesões fibro-ósseas benignas (LFOB) correspondem a um grupo diverso de patologias caracterizadas pela substituição do tecido ósseo por tecido conjuntivo e matriz extracelular mineralizada. Pouco se conhece a respeito da etiologia desse grupo de lesões. Propomo-nos a analisar por meio da técnica imunohistoquímica a expressão de 3 moléculas (osteonectina, TGFβ-1 e BMP 2/4) envolvidas no metabolismo ósseo. Métodos: Trinta e dois casos diagnosticados como osso normal (ON,8), displasia fibrosa (DF,8), displasia cemento-óssea (DCO,8) e fibroma cemento-ossificante (FCO,8) foram selecionados. Resultados: A osteonectina e a BMP2/4 foram positivas em todos os casos. O TGFβ-1 revelou positividade em 1 caso de DCO e FCO. Conclusão: Os achados imunohistoquímicos sugerem que as LFOB tem processos diferentes de produção de tecido ósseo.

Background: Benign fibro-osseous lesions (BFOL) comprise a diverse group of pathologies characterized by the replacement of normal bone by fibrous tissue and a mineralized product. Little is known about the biology of this group of lesions. We have analyzed the immunohistochemical expression of three molecules involved in bone metabolism, namely osteonectin, TGF-b1, and BMP2/4. Methods: Thirty-two cases diagnosed as normal jaw bone (NJB, 8 cases), fibrous dysplasia (FD, 8 cases), cemento-osseous dysplasia (COD, 8 cases), and cemento-ossifying fibroma (COF, 8 cases) were selected. Results: Osteonectin and BMP2/4 antibodies were positive in all cases. TGFb-1 labeling was seen in one case of COD and COF. Conclusion: The immunohistochemistry findings suggest that BFOL have different processes of osseous tissue production.

Avaliação imunohistoquímica de proteínas da matriz extracelular na reparação de defeitos agudos de furca classe II em cães / Immunohistochemical evaluation of extracellular matrix proteins in the repair of acute class II furcation defects in dogs

O estudo da reparação periodontal deve se basear em modelos que forneçam os dados necessários para a análise deste fenômeno. O modelo proposto, de defeitos agudos de furca classe II, parece ser o mais indicado, pois possui um grau previsível de regeneração espontânea, fornecendo um ambiente em que é possível o estudo dos fatores que favorecem a regeneração dos tecidos periodontais. O objetivo deste trabalho foi avaliar a qualidade da reparação de defeitos agudos de furca classe II por meio da imunoexpressão de proteínas da matriz extracelular (MEC). Após a elevação de retalhos de espessura total, em quatro cães, foram criados defeitos agudos de furca classe II nos segundos, terceiros e quartos pré-molares de um dos lados da mandíbula. Os retalhos foram posicionados coronariamente e suturados. Após 45 dias, os espécimes foram coletados, descalcificados e analisados, em um plano vestíbulo- lingual, por meio de técnica imunohistoquímica para osteopontina (OPN), sialoproteína óssea (BSP) e osteonectina (BON). Nos tecidos periodontais originais, a marcação para os anticorpos testados foi fracamente positiva na MEC, caracterizando a presença de tecidos maduros. Já no interior dos defeitos, houve diferença na marcação, com marcação mais pronunciada para BSP, OPN e BON. Conclui-se que os resultados evidenciam maior atividade metabólica nos tecidos reparativos.

The study of periodontal repair should be based on models that provide the data required for analysis of thisphenomenon. The proposed model of acute class II furcation defects seems to be the most appropriate, as it provides apredictable degree of spontaneous regeneration, creating an environment where the factors that promote the regeneration of periodontal tissues can be studied. The objective of this study was to evaluate the quality of repair ofacute class II furcation defects, by means of immunoexpression of extracellular matrix (ECM) proteins.After lifting full thickness flaps in 4 dogs, acute class II furcation defects were created in the second, third and fourth premolar in one side of the mandible. The flaps were coronallypositioned and sutured. After 45 days, the specimens were collected, decalcified and analyzed in a buccal-lingual plane, by immunohistochemistry technique for osteopontin (OPN), bone sialoprotein (BSP) and osteonectin (BON). In original periodontal tissues, the marking for the tested antibodieswas weakly positive in the ECM, characterizing the presence of mature tissues. Inside the defects, there was a difference in the marking, with markings more pronounced for BSP, OPN and BON. It was concluded that the results showed greater metabolic activity in the reparative tissues.