Ae31e

The murine myoblast cell line C2C12 was purchased from National Biomedical Experimental Cell Resource Bank (Beijing, China). Myoblasts were grown at 37 °C in DMEM (high glucose, HyClone) culture medium containing 10% fetal bovine serum (Biological Industries), 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere (5% CO₂). When the cells reached 85–90% confluence, the differentiation of myoblasts was induced to form myotubes by changing the culture medium to DMEM medium containing 2% horse serum, 100 units/mL penicillin and 100 μg/mL streptomycin. Fresh differentiation medium was changed daily until the cells were harvested for analysis. Sodium lactate (#71718, Sigma–Aldrich, Shanghai, China) was added in either the culture medium or the differentiation medium at a final concentration of 5 mM. The observation of morphologies characteristics was carried out with an inverted microscope at 20× magnification (Motic, AE31E, Xiamen, China).
The cells cultured in the DMEM media with and without lactate supplementation were defined as the Lac and Con groups of myoblasts, respectively. Similarly, the cells differentiated in the differentiation media with and without lactate supplementation were defined as the Lac and Con groups of myotubes, respectively.

Fiber-spun membranes of PLA 10% (w/v) were synthesized by air-jet spinning, as reported previously [26 (link)], onto the 12 mm cover glass. Previously, to seed the 3D microspheres, the fiber membrane was sterilized by immersion in ethanol/antibiotic solution for 30 min and air-dried under the flow cabinet. For evaluating the colonization, after 24 h of microsphere formation by a magnetic levitation technique, two microspheres of DP-MSC and hFOB were seeded in the center of the fiber-spun membrane and incubated for 3 and 7 days. At the prescribed time, the evaluation of the interaction of the 3D microsphere above the surface was evaluated by a Live/Dead Cell Imagining kit (Invitrogen) following the manufacturer’s instructions and was observed under an epifluorescence microscope (model AE31E; MOTIC, Schertz, TX, USA). DAPI was used for observing the nucleus of the cells. The experiments were conducted in triplicate.

To identify the presence of eDNA and eRNA in the biofilm matrix, selected fluorescent dyes were used: Sytox Green (Molecular Probes, Eugene, OR) at a final concentration of 1 μM for DNA and RNA staining, Quant-iT PicoGreen (Invitrogen, Waltham, MA) at a 200-fold dilution of concentrated dye for dsDNA staining, and SYTO RNA Select (Invitrogen, Waltham, MA, USA) at a concentration of 500 nM for RNA identification. Biofilms were imaged using AE31E (Motic, Barcelona, Spain), IX73 (Olympus, Tokyo, Japan), and DMI6000B (Leica, Wetzlar, Germany) fluorescence microscopes. Additionally, the presence of eDNA and eRNA in the biofilm was confirmed by nuclease treatments. After 48 hours of biofilm culturing in 96-well plate (10⁵ cells/ml), DNase I (a final concentration of 20 U/ml; Thermo Scientific, Waltham, MA) or RNase A (a final concentration of 0.2 mg/ml; Thermo Scientific, Waltham, MA) in a reaction buffer containing MgCl₂ (Thermo Scientific, Waltham, MA) were added to RPMI 1640 medium. The incubation with nucleases was carried out for 1.5 h. After the digestion, extracellular nucleic acids were stained with Sytox Green and the fluorescence was measured using a Biotek Synergy H1 microplate reader (excitation: 485 nm, emission: 528 nm). Biofilms not treated with nucleases served as a reference.