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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Cafarelli, Andrea (Scuola Superiore Sant'Anna)


Skeletal muscle (SM) tissue engineering aims to achieve a mature SM tissue in vitro, by combining precursor cells, suitable substrates and the right stimuli to facilitate cell growth and differentiation into the desired phenotype. In this field, polymeric thin films are attractive due to their small thickness (from hundreds of nm to some μm), flexibility, adhesiveness, permeability and similarity to natural biomembranes. [1]
Moreover, their tunable stiffness and the possibility to create micropatterns on their surface by nano and micro-fabrication techniques make them promising substrates to reproduce the mechanical and geometrical environment typical of natural SM tissue. In this work, we developed novel nanocomposite microgrooved polymeric thin films able to boost the skeletal muscle differentiation of C2C12 cells.

Solutions of poly(styrene-block-butadiene-block-styrene) (SBS) dissolved in tetrahydrofuran and polycaprolactone (PCL) dissolved in dichloromethane were prepared at 40 mg/mL. Barium titanate nanoparticles (BTNPs) (diameter: 300 nm) were added at different concentrations (0.01% w/v, 0.1% w/v, 1% w/v) and the final solutions were spin-coated (2000 rpm, 20 s) over a microgrooved (10 μm parallel channels) PDMS mold. A solution of PVA was cast over the thin films as supporting layer to detach the films and then dissolved in deionized water, thus obtaining freestanding microgrooved thin films.
The films were characterized in terms of topographical (stylus based profilometer and optical profilometer) and mechanical (nanoindenting through AFM) properties and used as substrates for adhesion and differentiation of murine myoblasts (C2C12, seeding density: 50000 cells/cm2).
During the differentiation period (7 days), the differentiation medium (DMEM + 1% FBS + 1% P/S +1% ITS) was renewed every day. At the end-point, the samples were stained for F-actin and nuclei and analyzed through a confocal microscope, to quantify myotubes dimensions and fusion index.

Results and conclusions
The thin films obtained showed an average thickness of 700 nm (SBS) and 1.3 μm (PCL). Their channels were 10 μm-wide and 1 μm-high. Their elastic modulus was 60 MPa (SBS) and 26 MPa (PCL), respectively.
Immunostaining evidenced the development of aligned myotubes that uniformly covered the sample area. PCL films showed a fusion index from 38.065±5.982% (BTNPs 0%) to 31.824±7.592% (BTNPs 1%) and SBS films from 62.018±3.382% (BTNPs 0%) to 65.146±7.942% (BTNPs 1%). These high values confirmed a very good level of differentiation, without significant differences between doped and not-doped films of the same polymer.
As a perspective, the piezoelectric properties of barium titanate nanoparticles will be exploited in combination with ultrasound stimulation to further boost cell differentiation, especially in SBS samples.

This work received support by INAIL (MIO-PRO project)

[1] Fujie, T., Polym J 48, 773–780 (2016)
[2] Hasebe et al., ACS Biomater. Sci. Eng. 2019, 5, 11, 5734–5743 (2019)


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