*2.6. Cell Cultures*

PLLA-SBA2 fibres were disinfected under an ultraviolet lamp for 1 h. Murine-derived stromal cells ST-2 (obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany), were cultured to confluence in 75 cm2 culture flasks in Roswell Park Memorial Institute medium (RPMI 1640) (GibcoTM, Thermo Fisher Scientific, Schwerte, Germany) containing 10% foetal bovine serum (FBS; Lonza) and 1% penicillin/streptomycin (Lonza) at 37 ◦C and 5 % CO2. Before seeding, cells were detached using Trypsin in DPBS (Sigma Aldrich, Munich, Germany), stained with 0.4% (*v*/*v*) trypan blue solution and counted using a Neubauer chamber (VWR). Then, ST-2 cells were seeded onto the electrospun scaffolds (including neat PLLA fibres as control) with a density of 2 × <sup>10</sup><sup>4</sup> cells/cm2. All samples were assayed in triplicate and each sample was incubated in the same RPMI medium described above. Cells were cultured for 7 days renewing the culture medium once after 3 days of culture. Viability analyses of ST-2 cells on composite fibres was assessed after a cultivation period of 1 day and 7 days by using a WST-8 assay (Cell Counting Kit-8, Sigma Aldrich, Munich, Germany), which is based on the conversion of tetrazolium salt to highly water-soluble formazan by mitochondrial enzymes of viable cells. At each time point, the culture medium was removed from each sample and each well with samples and cells was washed with DPBS and added with a solution of 10% WST-8 reagent in colourless medium. After an incubation period of 3 hours at 37 ◦C and 5% CO2, the solution was transferred into a 96 well plate to measure absorbance at 450 nm by using a microplate Elisa reader (PHOmo Elisa reader, Autobio Diagnostics Co. Ltd., Zhengzhou, China).

To investigate cell morphology, a preliminary evaluation was provided by SEM analysis after 1 day and 7 days of culture. Samples were fixed by using a solution containing paraformaldehyde, glutaraldehyde, sodium cacodylate trihydrate and sucrose (Sigma Aldrich, Munich, Germany). Subsequently, samples dehydration was achieved by using a series of aqueous ethanol solutions. The samples were then dried in a critical point drier (Leica EM CPD 300, Leica, Wetzlar, Germany) and

sputtered with gold. The cytoskeleton organization and nucleus morphology of cells on PLLA-SBA2 fibres were studied after 1 day and 7 days following seeding by staining with rhodamine phalloidin and DAPI (ThermoFisher Scientific, Schwerte, Germany). Briefly, samples were fixed by using a fixation solution containing 1,4-piperazinediethanesulfonic acid buffer, ethylene glycol tetraacetic acid, polyethylene glycol, paraformaldehyde, DPBS and sodium hydroxide (Sigma Aldrich, Munich, Germany), washed with DPBS and immersed in a permeabilization buffer for intracellular staining. 400 μL of a 8 μL/mL DPBS solution of rhodamine phalloidin was added in each well containing samples, then kept for 1 hour at 37◦C. After the removal of the dye, samples were vigorously washed with DPBS and 400 μL of a 1 μL/mL DPBS solution of DAPI was added to each well. Then, samples were washed in DPBS and analysed with a fluorescent microscope (Axio Scope A1, Zeiss, Jena, Germany).
