48 well plate

The adapted cells in minimal media (explained in growth section) were used to prepare 48‐well plates (Sarstedt, Germany) with each well containing 250 µL of cell culture. The RFP fluorescence was measured relative to the optical density (OD₆₀₀) using a spectrofluorophotometer TECAN infinite M200 PRO reader (Mannedorf, Switzerland).
Similarly, GFP fluorescence and cell growth were measured simultaneously using a Synergy plate reader (BioTek Instruments, Inc, Winooski, VT, USA). In all cases, the appropriate amount of inducers (IPTG, ATc and arabinose) were added to induce reporter protein's expression from t = 0 h (in 48‐well plates only), if required. Following induction, cells were grown at 30°C with continuous shaking for the next 24 h on a spectrofluorophotometer, and fluorescence relative to OD_600nm was recorded at an interval of every 20 min. For sfGFP, an excitation wavelength of 485 nm and an excitation wavelength of 535 nm was used. For RFP, an excitation wavelength of 575 nm and an excitation wavelength of 620 nm were used. The optical density (OD_600nm) was measured simultaneously for each well to calculate GFP fluorescence intensity/OD₆₀₀ and RFP fluorescence intensity/OD₆₀₀ ratio for fluorescence assays. The strains not expressing GFP or RFP were used as a control and respective uninduced counterpart for determining background fluorescence.

Gelatin hydrogels (250 μL final volume was used) were prepared by dissolving gelatin products in 1× phosphate buffered saline (PBS) at 50 °C to form solutions of 100 mg/mL, mixing them with 1 mM PEG-4SG and letting them assemble at 25 °C for 1 h in Ace silicone O-rings (Z504165). Gelatin films (250 μL final volume was used) were prepared by dissolving gelatin products in 1× PBS at 50 °C to form a solution of 100 mg/mL, mixing them with 1 mM PEG-4SG at 25 °C in 48-well plates (Sarstedt, Nümbrecht, Germany) and allowing the liquid to evaporate overnight in a fume hood.

Cellular studies were conducted using human MG63 cells (APABCAM, Rio de Janeiro, Brazil), a human osteoblast cell line. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Cultilab, São Paulo, Brazil), supplemented with 10% fetal bovine serum (FBS, Cultilab, São Paulo, Brazil) and gentamicin (10 mg/mL) (Gibco™, Paisly, UK). Incubated at a temperature of 37 °C in a humid atmosphere containing 5% CO₂, when confluence was reached by occupying more than 80%, the cells were ready for seeding. Cells were separated by trypsinization (0.25% Trypsin-EDTA Gibco™, Paisly, UK) and centrifuged (Labnet Centrifuge—HERMLE Z 300K, Winooski, VT, USA), then resuspended with culture medium. Before seeding, scaffolds were placed in Petri dishes and sterilized under UV irradiation for 30 min. Cells were calculated using a hemocytometer (Prolab, São Paulo, Brazil) and were pipetted into each scaffold in 48-well plates (Sarstedt^®, Numbrecht, Germany) at a cell density of 15 × 10⁴ cells for the tests described below, except for the test of gene expression. Replicates were performed according to ISO 10993-5, with three repetitions of the experiments and five scaffolds for each test in each repetition.

For measuring in vitro migration, 1 × 10⁵ cells/well were seeded in 48-well plates (Sarstedt, Nümbrecht, Germany) and incubated for 24 h. To stop proliferation, cells were treated with mitomycin C (10 µg/mL, abcr GmbH, Karlsruhe, Germany) for 2 h; then, they were washed and incubated for 22 h at 37 °C, 5% CO₂. Circular scratches were created using a 100 µL pipette tip and measured after 0 h and 24 h. Scratch areas were analyzed using ImageJ-macro MRI_would_healing_tool (http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool, accessed on 14 December 2019).

The genome synthesis at early infection (1–48 h p.i.) was determined by inoculating cells in 48-well plates (Sarstedt) with each viral strain. The cells were infected at a m.o.i. of 0.01 and subsequently washed, overlain with fresh medium and incubated at 25 °C. At 6, 15, 24 and 48 h p.i. supernatants from three wells per strain were removed and the cells were scratched, suspended in 0.5 ml of L-15 medium and harvested separately. Quantitative real-time PCR (qPCR) was used to determine the number of genome copies from cell lysates at the different time points, as further indicated. Extension of the exponential phase was established using regression lines and/or significant increase in RNA levels. First, we assessed whether the growth of each strain adjusted to a line, using regression analysis from the first time point to the last; if the resultant regression line was not significant (R²>0.95), the regression analysis was performed from the second time point. When no significant regression line was found, Bonferroni’s multiple comparison was performed between consecutive time points to determine the existence of a significant increase in RNA load.