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.

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 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.

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. .

Estudo do efeito do ozônio diluído em água na reparação óssea por meio de avaliação imunoistoquímica / Study of the effect of ozone dissolved in water in the bone repair by immunohistochemical

A aplicação de ozônio pode ser usada como uma alternativa no tratamento de inúmeras patologias. O objetivo é interferir positivamente na reparação tecidual e agir como anti-séptico, uma vez que o ozônio é um potente agente antimicrobiano e possui a capacidade de estimular a circulação sanguínea e a resposta imunomodulatória. Tais características justificam o interesse da sua aplicação na Medicina e na Odontologia. Quando em contato com fluidos orgânicos, o ozônio age como um oxidante gerando a formação de moléculas reativas do oxigênio e produtos de lipídeos oxidantes que influenciam em eventos bioquímicos do metabolismo celular. A proposição deste estudo foi avaliar a aplicação do ozônio diluído em água no processo de reparação óssea por meio de análise morfológica e imunoistoquímica em modelo animal. Os resultados mostraram que a irrigação da ferida cirúrgica com ozônio diluído em água ocasionou atraso na reparação e que esse era mais exuberante no início do processo

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.

Obtençäo e caracterizaçäo de linhagem de células osteoblásticas / Development and characterization of osteoblast-like cell line

Apresentamos metodologia para obtençäo de linhagem de células osteoblástica propagada a partir de tecido ósseo normal. Por meio de técnica de dispersäo enzimática células isoladas do osso parietal de ratos recém-nascidos foram cultivadas em meio de Eagle modificado por Dullbecco supplementado com soro fetal bovino (10 por cento). Após a primeira passagem as culturas foram induzidas pela suplementaçäo do meio com ß-glicerofosfato de sódio (10mM), ácido ascórbico (50ug/ml) e dexametasona (10 elevado a -8 M) para obtençäo de diferenciçäo fenotípica e funcional. A morfologia, presença de atividade de fosfatase alcalina, produçäo de nódulos calcificados, expressäo imunocitoquímica de proteínas do tecido ósseo colagênicas (colágeno tipo I) e näo colagênicas (osteonectina e sialoproteína óssea-II) comprovaram a natureza osteoblástica da linhagem obtida. O sistema desenvolvido confirmou que a técnica de cultivo celular primário a partir de tecido ósseo normal é viável e origina células com as características básicas dos osteoblastos. Essa metodologia de obtençäo de células osteoblásticas pode ser utilizada amplamente em Odontologia, em especial, nos estudos da fisio-patologia óssea e em testes de compatibilidade de biomateriais que estaräo em contato constante com o tecido ósseo