RESUMO
Several techniques, such as additive manufacturing, have been used for the manufacture of polymer-ceramic composite scaffolds for bone tissue engineering. A new extruder head recently developed for improving the manufacturing process is an experimental 3D printer Fab@CTI that enables the use of ceramic powders in the processing of composite materials or polymer blends. Still, the manufacturing process needs improvement to promote the dispersion of ceramic particles in the polymer matrix. This article addresses the manufacture of scaffolds by 3D printing from mixtures of poly(ϵ-caprolactone) (PCL) and a glass powder of same composition of 45S5Bioglass®, labeled as synthesized bioglass (SBG), according to two different methods that investigated the efficiency of the new extruder head. The first one is a single extrusion process in a Fab@CTI 3D printer, and the other consists in the pre-processing of the PCL-SBG mixture in a mono-screw extruder with a Maddock® element, followed by direct extrusion in the experimental Fab@CTI 3D printer. The morphological characterization of the extruded samples by scanning electron microscope showed an architecture of 0°/90° interconnected struts and suitable porosity for bone tissue engineering applications. Scaffolds fabricated by two methods shows compressive modulus ranging from 54.4 ± 14.2 to 155.9 ± 20.4 MPa, results that are compatible to use in bone tissue engineering. Cytotoxicity assays showed non-toxic effects and viability forin vitroMG-63 cell proliferation. Alizarin Red staining test showed calcium deposition in all scaffolds, which suggests PCL/SBG composites promising candidates for use in bone tissue engineering. Results of cell morphology suggest more cell growth and adhesion for scaffolds fabricated using the pre-processing in a mono-screw extruder.
Assuntos
Engenharia Tecidual , Alicerces Teciduais , Cerâmica , Poliésteres , Polímeros , Porosidade , Impressão Tridimensional , Engenharia Tecidual/métodosRESUMO
Breast cancer is characterized by a hypoxic microenvironment inside the tumor mass, contributing to cell metastatic behavior. Hypoxia induces the expression of hypoxia-inducible factor (HIF-1α), a transcription factor for genes involved in angiogenesis and metastatic behavior, including the vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and integrins. Integrin receptors play a key role in cell adhesion and migration, being considered targets for metastasis prevention. We investigated the migratory behavior of hypoxia-cultured triple-negative breast cancer cells (TNBC) and endothelial cells (HUVEC) upon αvß3 integrin blocking with DisBa-01, an RGD disintegrin with high affinity to this integrin. Boyden chamber, HUVEC transmigration, and wound healing assays in the presence of DisBa-01 were performed in hypoxic conditions. DisBa-01 produced similar effects in the two oxygen conditions in the Boyden chamber and transmigration assays. In the wound healing assay, hypoxia abolished DisBa-01's inhibitory effect on cell motility and decreased the MMP-9 activity of conditioned media. These results indicate that αvß3 integrin function in cell motility depends on the assay and oxygen levels, and higher inhibitor concentrations may be necessary to achieve the same inhibitory effect as in normoxia. These versatile responses add more complexity to the role of the αvß3 integrin during tumor progression.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células Endoteliais/metabolismo , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Hipóxia Tumoral , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Meios de Cultivo Condicionados/farmacologia , Desintegrinas/farmacologia , Células Endoteliais/patologia , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Metaloproteinases da Matriz/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Oxigênio , Subunidades Proteicas/metabolismo , Hipóxia Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: Integrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the interaction between αvß3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. DisBa-01, a recombinant His-tag fusion, RGD-disintegrin from Bothrops alternatus snake venom, binds to αvß3 integrin with nanomolar affinity blocking cell adhesion to the extracellular matrix. Here we present in vitro evidence of a direct interference of DisBa-01 with αvß3/VEGFR2 cross-talk and its downstream pathways. METHODS: Human umbilical vein (HUVECs) were cultured in plates coated with fibronectin (FN) or vitronectin (VN) and tested for migration, invasion and proliferation assays in the presence of VEGF, DisBa-01 (1000 nM) or VEGF and DisBa-01 simultaneously. Phosphorylation of αvß3/VEGFR2 receptors and the activation of intracellular signaling pathways were analyzed by western blotting. Morphological alterations were observed and quantified by fluorescence confocal microscopy. RESULTS: DisBa-01 treatment of endothelial cells inhibited critical steps of VEGF-mediated angiogenesis such as migration, invasion and tubulogenesis. The blockage of αvß3/VEGFR2 cross-talk by this disintegrin decreases protein expression and phosphorylation of VEGFR2 and ß3 integrin subunit, regulates FAK/SrC/Paxillin downstream signals, and inhibits ERK1/2 and PI3K pathways. These events result in actin re-organization and inhibition of HUVEC migration and adhesion. Labelled-DisBa-01 colocalizes with αvß3 integrin and VEGFR2 in treated cells. CONCLUSIONS: Disintegrin inhibition of αvß3 integrin blocks VEGFR2 signalling, even in the presence of VEGF, which impairs the angiogenic mechanism. These results improve our understanding concerning the mechanisms of pharmacological inhibition of angiogenesis.