RESUMO
In tissue engineering, a uniform cell occupation of scaffolds is crucial to ensure the success of tissue regeneration. However, this point remains an unsolved problem in 3D scaffolds. In this study, a direct method to integrate cells into fiber scaffolds was investigated by combining the methods of electrospinning of fibers and bioelectrospraying of cells. With the associating of these methods, the cells were incorporated into the 3D scaffolds while the fibers were being produced. The scaffolds containing cells (SCCs) were produced using 20% poly(lactide-co-glycolide) solution for electrospinning and mesenchymal stem cells from deciduous teeth as a suspension for bioelectrospraying. After their production, the SCCs were cultivated for 15 days at 37°C with an atmosphere of 5% CO2. The 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide test demonstrated that the cells remained viable and were able to grow between the fibers. Scanning electron microscopy showed the presence of a high number of cells in the structure of the scaffolds and confocal images demonstrated that the cells were able to adapt and spread between the fibers. Histological analysis of the SCCs after 1 day of cultivation showed that the cells were uniformly distributed throughout the thickness of the scaffolds. Some physicochemical properties of the scaffolds were also investigated. SCCs exhibited good mechanical properties, compatible with their handling and further implantation. The results obtained in the present study suggest that the association of electrospinning and bioelectrospraying provides an interesting tool for forming 3D cell-integrated scaffolds, making it a viable alternative for use in tissue engineering.
Assuntos
Polpa Dentária/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco/patologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Proliferação de Células , Sobrevivência Celular , Humanos , Ácido Láctico/química , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de Varredura , Poliésteres/química , Poliglactina 910/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Solventes/química , Estresse Mecânico , Dente/citologiaRESUMO
We studied the effect of different concentrations of diphenyl ditelluride (PhTe)(2) on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and neurofilament (NF) subunits from cerebral cortex and hippocampus of rats during development. (PhTe)(2)-induced hypophosphorylation of GFAP and NF subunits only in cerebral cortex of 9- and 15-day-old animals but not in hippocampus. Hypophosphorylation was dependent on ionotropic glutamate receptors, as demonstrated by the specific inhibitors 10 µM DL-AP5 and 50 µM MK801, 100 µM CNQX and 100 µM DNQX. Also, 10 µM verapamil and 10 µM nifedipine, two L-voltage-dependent Ca(2+) channels (L-VDCC) blockers; 50 µM dantrolene, a ryanodine channel blocker, and the intracellular Ca(2+) chelator Bapta-AM (50 µM) totally prevented this effect. Results obtained with 0.2 µM calyculin A (PP1 and PP2A inhibitor), 1 µM Fostriecin a potent protein phosphatase 2A (PP2A) inhibitor, 100 µM FK-506 or 100 µM cyclosporine A, specific protein phosphatase 2B inhibitors, pointed to PP1 as the protein phosphatase directly involved in the hypophosphorylating effect of (PhTe)(2). Finally, we examined the activity of DARPP-32, an important endogenous Ca(2+)-mediated inhibitor of PP1 activity. Western blot assay using anti-DARPP-32, anti-pThr34DARPP-32, and anti-pThr75DARPP-32 antibodies showed a decreased phosphorylation level of the inhibitor at Thr34, compatible with inactivation of protein kinase A (PKA) by pThr75 DARPP-32. Decreased cAMP and catalytic subunit of PKA support that (PhTe)(2) acted on neuron and astrocyte cytoskeletal proteins through PKA-mediated inactivation of DARPP-32, promoting PP1 release and hypophosphorylation of IF proteins of those neural cells. Moreover, in the presence of Bapta, the level of the PKA catalytic subunit was not decreased by (PhTe)(2), suggesting that intracellular Ca(2+) levels could be upstream the signaling pathway elicited by this neurotoxicant and targeting the cytoskeleton.
Assuntos
Derivados de Benzeno/farmacologia , Córtex Cerebral/efeitos dos fármacos , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Filamentos Intermediários/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Animais , Córtex Cerebral/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Filamentos Intermediários/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Ratos , Transdução de SinaisRESUMO
The studies of signaling mechanisms involved in the disruption of the cytoskeleton homeostasis were performed in a model of quinolinic acid (QUIN) neurotoxicity in vitro. This investigation focused on the phosphorylation level of intermediate filament (IF) subunits of astrocytes (glial fibrillary acidic protein - GFAP) and neurons (low, medium and high molecular weight neurofilament subunits - NFL, NFM and NFH, respectively). The activity of the phosphorylating system associated with the IFs was investigated in striatal slices of rat exposed to QUIN or treated simultaneously with QUIN plus glutamate receptor antagonists, calcium channel blockers or kinase inhibitors. Results showed that in astrocytes, the action of 100 µM QUIN was mainly due to increased Ca(2+) influx through NMDA and L-type voltage-dependent Ca(2+) channels (L-VDCC). In neuronal cells QUIN acted through metabotropic glutamate receptor (mGluR) activation and influx of Ca(2+) through NMDA receptors and L-VDCC, as well as Ca(2+) release from intracellular stores. These mechanisms then set off a cascade of events including activation of PKA, PKCaMII and PKC, which phosphorylate head domain sites on GFAP and NFL. Also, Cdk5 was activated downstream of mGluR5, phosphorylating the KSP repeats on NFM and NFH. mGluR1 was upstream of phospholipase C (PLC) which, in turn, produced diacylglycerol (DAG) and inositol 3,4,5 triphosphate (IP3). DAG is important to activate PKC and phosphorylate NFL, while IP(3) contributed to Ca(2+) release from internal stores promoting hyperphosphorylation of KSP repeats on the tail domain of NFM and NFH. The present study supports the concept of glutamate and Ca(2+) contribution in excitotoxic neuronal damage provoked by QUIN associated to dysfunction of the cytoskeleton homeostasis and highlights the differential signaling mechanisms elicited in striatal astrocytes and neurons.
Assuntos
Astrócitos/citologia , Corpo Estriado/citologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Neurônios/citologia , Ácido Quinolínico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de Variância , Animais , Astrócitos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
In the present report, we showed that diphenyl ditelluride (PhTe)(2) induced in vitro hyperphosphorylation of glial fibrillary acidic protein (GFAP), vimentin and neurofilament (NF) subunits in hippocampus of 21 day-old rats. Hyperphosphorylation was dependent on L-voltage dependent Ca(2+) channels (L-VDCC), N-methyl-d-aspartate (NMDA) and metabotropic glutamate receptors, as demonstrated by the specific inhibitors verapamil, DL-AP5 and MCPG, respectively. Also, dantrolene, a ryanodine channel blocker, EGTA and Bapta-AM, extra and intracellular Ca(2+) chelators respectively, totally prevented this effect. Activation of metabotropic glutamate receptors by (PhTe)(2) upregulates phospholipase C (PLC), producing inositol 1, 4, 5-trisphosphate (IP(3)) and diacylglycerol (DAG). Therefore, high Ca(2+) levels and DAG directly activate Ca(2+)/calmodulin-dependent protein kinase (PKCaMII) and protein kinase C (PCK), resulting in the hyperphosphorylation of Ser-57 in the carboxyl-terminal tail domain of the low molecular weight NF subunit (NF-L). Also, the activation of Erk1/2, and p38MAPK resulted in hyperphosphorylation of KSP repeats of the medium molecular weight NF subunit (NF-M). It is noteworthy that PKCaMII and PKC inhibitors prevented (PhTe)(2)-induced Erk1/2MAPK and p38MAPK activation as well as hyperphosphorylation of KSP repeats on NF-M, suggesting that PKCaMII and PKC could be upstream of this activation. Taken together, our results highlight the role of Ca(2+) as a mediator of the (PhTe)(2)-elicited signaling targeting specific phosphorylation sites on IF proteins of neural cells of rat hippocampus. Interestingly, this action shows a significant cross-talk among signaling pathways elicited by (PhTe)(2), connecting glutamate metabotropic cascade with activation of Ca(2+) channels. The extensively phosphorylated amino- and carboxyl- terminal sites could explain, at least in part, the neural dysfunction associated with (PhTe)(2) exposure.
Assuntos
Derivados de Benzeno/toxicidade , Cálcio/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Neurofilamentos/metabolismo , Compostos Organometálicos/toxicidade , Vimentina/metabolismo , Animais , Benzilaminas/farmacologia , Western Blotting , Canais de Cálcio Tipo L/metabolismo , Córtex Cerebral/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Eletroforese em Gel de Poliacrilamida , Hipocampo/metabolismo , Técnicas In Vitro , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Wistar , Estaurosporina/farmacologia , Sulfonamidas/farmacologiaRESUMO
Os autores tecem alguns comentários sobre os insucessos na implantologia oral, evidenciando que o principal deles é a má formação do profissional nessa nobre àrea da Odontologia