RESUMO
Glutamate released by osteoblasts sharing similarities with its role in neuronal transmission is a very new scientific concept which actually changed the understanding of bone physiology. Since glutamate release is a calcium (Ca(2+))-dependent process and considering that we have previously demonstrated that the dissolution of bioactive glass with 60% of silicon (BG60S) can alter osteoblast Ca(2+)-signaling machinery, we investigated whether BG60S induces glutamate secretion in osteoblasts and whether it requires an increase in intracellular Ca(2+). Here we showed that the extracellular Ca(2+) increase due to BG60S dissolution leads to an intracellular Ca(2+) increase in the osteoblast, through the activation of an inositol 1,4,5-triphosphate receptor (InsP(3)R) and a ryanodine receptor (RyR). Additionally, we also demonstrated that glutamate released by osteoblasts can be profoundly altered by BG60S. The modulation of osteoblast glutamate released by the extracellular Ca(2+) concentration opens a new window in the field of tissue engineering, since many biomaterials used for bone repair are able to increase the extracellular Ca(2+) concentration due to their dissolution products.
Assuntos
Cálcio/metabolismo , Ácido Glutâmico/farmacologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Animais Recém-Nascidos , Materiais Biocompatíveis/farmacologia , Osso e Ossos/metabolismo , Cálcio/farmacologia , Cálcio da Dieta/farmacologia , Espaço Extracelular/metabolismo , Vidro , Inositol 1,4,5-Trifosfato/farmacologia , Osteoblastos/efeitos dos fármacos , Ratos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/farmacologia , Silício/farmacologia , Engenharia TecidualRESUMO
We investigated the influence of extracellular calcium concentration, caused by the dissolution of a bioactive glass with 60% of silicon (BG60S), on intracellular calcium (Ca(i) (2 +)) signals and expression of inositol 1, 4, 5-triphosphate receptors (InsP(3)R) in primary culture of osteoblasts. We found that BG60S caused an increase in Ca(i) (2 +) signals in this cell type. Additionally, osteoblasts pre-incubated in the presence of BG60S showed an increase in Ca(i) (2 +) when cells were stimulated with vasopressin. On the other hand, a decrease in Ca(i) (2 +) signals were observed in osteoblasts pre-treated with BG60S and stimulated with KCl. We furher found that in osteoblasts, the type I InsP(3)R is preferentially distributed in the nucleus while the type II InsP(3)R in the cytoplasm. Preincubation of osteoblasts with BG60S altered the receptor expression level, increasing the type I InsP(3)R in the nucleus and decreasing type II InsP(3)R in the cytosol. Together, our results showed that in osteoblasts, BG60S increased Ca(i) (2 +)signals and altered Ca(i) (2 +) machinery.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Osteoblastos/fisiologia , Silício/administração & dosagem , Animais , Animais Recém-Nascidos , Materiais Biocompatíveis/administração & dosagem , Materiais Biocompatíveis/química , Células Cultivadas , Relação Dose-Resposta a Droga , Teste de Materiais , Osteoblastos/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
In this work, novel composites based on calcium phosphates (CaP)/collagen (COL) doped with Zn(+2) have been synthesized. They were characterized by SEM coupled to EDS microprobe in order to evaluate their morphology and chemical composition, respectively. The biocompatibility of these synthetic CaP/COL nanocomposites doped and undoped with Zn(+2) was investigated through osteoblast cell culture assay. Calcium phosphates were produced via aqueous precipitation routes where two different phases were obtained, hydroxyapatite (HAP) and biphasic hydroxyapatite-betatricalcium phosphate (HAPbetaTCP). In the sequence, the type-I collagen (COL) was added to the inorganic phase based on calcium phosphate and the mixture was blended until a homogenous composite was obtained. Zn(+2) aqueous solution (1.0 wt%) was used as the doping reagent. The cell viability and the alkaline phosphatase production of osteoblasts in the presence of the composites were evaluated and compared to control osteoblasts. Also, the biocompatibility of the composite was investigated through cell morphological analysis using optical microscopy of osteoblasts. All experiments were performed in triplicates (n = 3) from three different experiments. They were analyzed by variance test (ANOVA) and Bonferroni's post-test with differences statistically significant at p < 0.05. The results showed that the CaP/COL composites doped and undoped with Zn(+2) did not present alterations in cell morphology in 72 h and had similar cell viability and alkaline phosphatase activity to the control. All the tested CaP/COL composites showed adequate biological properties with the potential to be used in bone tissue replacement applications.
Assuntos
Substitutos Ósseos/química , Substitutos Ósseos/farmacologia , Colágeno/farmacologia , Durapatita/farmacologia , Nanoestruturas/administração & dosagem , Osteoblastos/efeitos dos fármacos , Zinco/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Colágeno/química , Durapatita/química , Teste de Materiais , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Osteoblastos/citologia , Osteoblastos/fisiologia , Tamanho da Partícula , Ratos , Ratos Wistar , Zinco/químicaRESUMO
Cartilage tissue has poor capability of self-repair, especially in the case of severe cartilage damage due to trauma or age-related degeneration. Cell-based tissue engineering using scaffolds has provided an option for the repair of defects in adult cartilage tissue. Mesenchymal stem cells (MSC) and chondrocytes are the two major cell sources for cartilage tissue engineering. The present study combined culture conditions of MSC in a chitosan-gelatin matrix in chondrogenic media to evaluate their effects on MSC viability and chondrogenesis for cartilage tissue engineering. MSC were harvested from rabbit bone marrows and cultured in chondrogenic media supplemented, or not, with dexamethasone in a chitosan-gelatin film (C-GF). The association of C-GF and dexamethasone promoted significant increase in cell adhesivity, viability and proliferation when compared to MCS cultured in media without dexamethasone or C-GF. In addition, dexamethasone promoted increase in the collagen concentration of MSC cultures. A reduction of alkaline phosphatase activity after three weeks of culture in chondrogenic media was verified. No influence of the C-GF or of dexamethasone was observed in this matter. Therefore, it is reasonable to suggest that biomaterial-based chitosan-gelatin and chondrogenic media supplemented with dexamethasone may stimulate the proliferation and differentiation of MSC according to the complex environmental conditions. The information presented here should be useful for the development of biomaterials to regulate the chondrogenesis of MSC suitable for cartilage tissue engineering.
Assuntos
Quitosana/química , Condrócitos/citologia , Condrogênese/fisiologia , Dexametasona/administração & dosagem , Matriz Extracelular/química , Gelatina/química , Células-Tronco Mesenquimais/citologia , Animais , Materiais Biomiméticos/química , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/fisiologia , Condrogênese/efeitos dos fármacos , Teste de Materiais , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Coelhos , Engenharia Tecidual/métodosRESUMO
Bioactive glass macroporous structures were developed in this work to be used as scaffolds for bone tissue engineering applications. A sol-gel route was used to obtain glass foams with the introduction of a gas phase in the solution and by vigorous agitation of the sol-gel solution that contains a foam agent. Stable and homogeneous foams were formed near the gelation point, which were than dried and heat-treated. Macroporous structures with interconnected pores of up to 500 mu m, porosity as high as 88% and specific surface area of 92 m(2)/g were obtained. The porous glasses were tested in osteoblast cultures to evaluate adhesion, proliferation, collagen and alkaline phosphatase production. Osteoblast proliferation was higher in the presence of the foams as well as was the collagen secretion, when compared to control. The alkaline phosphatase production was not altered. Viable osteoblasts could be seen inside the foams, suggesting that the produced porous glass foams are a promising materials for bone repair, since it provides a good environment for the adhesion and proliferation of osteoblasts.