PLLA surfaces with embedded ZnO nanoparticles that enhance cell differentiation

Researchers from our group have just published (paper link here) work with a biodegradable composite that can be used to drive and enhance cell differentiation. Murine C2C12 myoblasts were cultured on these composites and characterised at different timepoints. Poly(lactic acid) (PLLA) was used to create composites with ZnO nanoparticles. PLLA is used in biomedical research and clinical applications for its good biocompatibility properties and because it degrades by hydrolysis in biological environments. Zinc has been shown to promote myoblast proliferation and differentiation. PhD student Ms Sara Trujillo has prepared this post explaining their work.

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This work explores the idea of a dynamic system as a trigger of cell differentiation. We show that, initially, the ZnO particles are covered by a layer of PLLA but after around 8 days of hydrolytic degradation, this PLLA layer disappears and the particles become available (see figure 1 below).

We observed that, at short times, the differentiation of C2C12 myoblasts is similar between the neat PLLA surface and the nanocomposite. However, maintaining the culture for longer periods (around two weeks), significantly enhances the myogenic differentiation of C2C12 onto the composites compared to the bulk polymer (see figure 2 below). We characterized the system along time and showed that the release of zinc is too low to promote C2C12 differentiation and the changes in roughness measured are not enough to trigger differentiation. Therefore, we concluded that the sequential appearance of the particles on the surface, as time goes by, is the signal that boosts myogenic differentiation.

AFM height and phase images showing surface features of neat PLLA and PLLA/ZnO nanocomposite. (a) The initially non-degraded matrices (0 d) and (b, c) 8–15 days degraded surfaces are shown; roughness values are highlighted. Scale bar: 2 μm.

Figure 1: AFM height and phase images showing surface features of neat PLLA and PLLA/ZnO nanocomposite. (a) The initially non-degraded matrices (0 d) and (b, c) 8–15 days degraded surfaces are shown; roughness values are highlighted. Scale bar: 2 μm.

cell assays

Figure 2: Cell proliferation and cell differentiation. (a) Sketch showing the differences between the assays performed. C2C12 cells were culture on PLLA or PLLA/ZnO for 4 days with differentiation media or C2C12 cells were culture on PLLA or PLLA/ZnO for 14 days with growth media. (b) Representative images of sarcomeric myosin immunostaining (green) and DAPI (red) are presented at 4 days with differentiation media (scale bar: 200 μm) and (c) percentage of differentiation was calculated at 4 days as number of cells differentiated per total number of cells. (d) C2C12 proliferation curves (cells/cm2) during 2 weeks of culture in growth medium. (e) Representative images for sarcomeric myosin and DAPI at 14 days of culture with growth media (scale bar: 100 μm) and (f) percentage of differentiation was calculated at 14 days. (g) Fusion index distribution (number of nuclei per myotube) at 14 days was calculated and (h) myotube thickness distribution (μm) at 14 days is also shown. Statistical significance was observed in a t-test (***) p-value < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the paper.)