RESUMEN
Vascularization is crucial for bone regeneration after the transplantation of tissue-engineered bone grafts in the clinical setting. Growing evidence suggests that mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are potently pro-angiogenic both in vitro and in vivo. In the current study, we fabricated a novel EV-functionalized scaffold with enhanced pro-angiogenic and pro-bone regeneration activities by coating decalcified bone matrix (DBM) with MSC-derived EVs. EVs were harvested from rat bone marrow-derived MSCs and the pro-angiogenic potential of EVs was investigated in vitro. DBM scaffolds were then coated with EVs, and the modification was verified by scanning electron microscopy and confocal microscopy. Next, the pro-angiogenic and pro-bone regeneration activities of EV-modified scaffolds were evaluated in a subcutaneous bone formation model in nude mice. Micro-computed tomography scanning analysis showed that EV-modified scaffolds with seeded cells enhanced bone formation. Enhanced bone formation was confirmed by histological analysis. Immunohistochemical staining for CD31 proved that EV-modified scaffolds promoted vascularization in the grafts, thereby enhancing bone regeneration. This novel scaffold modification method provides a promising way to promote vascularization, which is essential for bone tissue engineering.
Asunto(s)
Matriz Ósea/fisiología , Regeneración Ósea , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Neovascularización Fisiológica , Andamios del Tejido , Animales , Matriz Ósea/irrigación sanguínea , Matriz Ósea/metabolismo , Calcificación Fisiológica , Células Cultivadas , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Ratones Desnudos , Osteogénesis , Ratas , Ratas Sprague-Dawley , Ingeniería de Tejidos/métodos , Microtomografía por Rayos XRESUMEN
In this article, gelatin (GT) and polycaprolactone (PCL) blended with a weight ratio of 50:50 were dissolved in the trifluoroethanol (TFE) or the acetic acid-doped TFE solvent system (0.2% relative to TFE) to prepare fibrous scaffolds of GT/PCL with different compositional and morphological homogeneities (denoted as the group 1 and the group 2 scaffolds) by electrospinning. The morphology and composition of the two groups of fibrous scaffolds were examined by scanning electron microscopy and Fourier transform infrared spectroscopy, respectively. Then, using green fluorescence protein-labeled mouse fibroblasts and HaCaT cells (a human keratinocyte cell line) as the model cells, cell adhesion, morphology, and proliferation were assessed by laser scanning confocal microscopy, scanning electron microscopy, and cell counting kit-8 assay, respectively. The results showed that the morphological and compositional inhomogeneity of the group 1 scaffolds had a remarkable influence on cell adhesion and proliferation. In contrast, there was no significant difference among the group 2 scaffolds because of their good consistency in fiber morphology and composition. Phase separation resultant GT content variance in the group 1 scaffolds is suggested as one of the major causes. This study highlighted the importance of producing morphologically uniform and compositionally homogeneous composite nanofibers while electrospinning natural and synthetic polymer blends.