RESUMEN
The aim of this work was to determine the influence of the biomaterial environment on human mesenchymal stem cell (hMSC) fate when cultured in supports with varying topography. Poly(vinylidene fluoride) (PVDF) culture supports were prepared with structures ranging between 2D and 3D, based on PVDF films on which PVDF microspheres were deposited with varying surface density. Maintenance of multipotentiality when cultured in expansion medium was studied by flow cytometry monitoring the expression of characteristic hMSCs markers, and revealed that cells were losing their characteristic surface markers on these supports. Cell morphology was assessed by scanning electron microscopy (SEM). Alkaline phosphatase activity was also assessed after seven days of culture on expansion medium. On the other hand, osteoblastic differentiation was monitored while culturing in osteogenic medium after cells reached confluence. Osteocalcin immunocytochemistry and alizarin red assays were performed. We show that flow cytometry is a suitable technique for the study of the differentiation of hMSC seeded onto biomaterials, giving a quantitative reliable analysis of hMSC-associated markers. We also show that electrosprayed piezoelectric poly(vinylidene fluoride) is a suitable support for tissue engineering purposes, as hMSCs can proliferate, be viable and undergo osteogenic differentiation when chemically stimulated.
Asunto(s)
Diferenciación Celular , Electricidad , Células Madre Mesenquimatosas/citología , Microesferas , Osteogénesis , Polivinilos/farmacología , Fosfatasa Alcalina/metabolismo , Materiales Biocompatibles/farmacología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula , Supervivencia Celular , Células Cultivadas , Medios de Cultivo , Citometría de Flujo , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/enzimología , Células Madre Mesenquimatosas/metabolismo , Osteocalcina/metabolismo , Osteogénesis/efectos de los fármacos , Coloración y EtiquetadoRESUMEN
This study investigates the effect of electroactivity and electrical charge distribution on the biological response of human bone marrow stem cells (hBMSCs) cultured in monolayer on flat poly(vinylidene fluoride), PVDF, substrates. Differences in cell behaviour, including proliferation, expression of multipotency markers CD90, CD105 and CD73, and expression of genes characteristic of different mesenchymal lineages, were observed both during expansion in basal medium before reaching confluence and in confluent cultures in osteogenic induction medium. The crystallisation of PVDF in the electrically neutral α-phase or in the electroactive phase ß, both unpoled and poled, has been found to have an important influence on the biological response. In addition, the presence of a permanent positive or negative surface electrical charge distribution in phase ß substrates has also shown a significant effect on cell behaviour.