Standard curve

The IAA production by the selected strains was estimated with a colorimetric assay using Salkowski’s reagent (0.5 M FeCl₃ in 35% HClO₄ aqueous solution) that is able to reveal in the presence of indole compounds such as IAA [16 (link)]. In particular, the strains were grown in 10 mL of R5A [17 (link)] and incubated for 24, 48, and 72 h (30 °C, 180 rpm). After incubation, the cultures were centrifuged, and the supernatants were mixed with Salkowski’s reagent (1:1). The optical density (OD) was recorded at 530 nm after 30 min of incubation, and a standard curve with known concentrations (0.5–100 μg mL⁻¹) of IAA (Sigma-Aldrich) was used to determine the amount of IAA produced.

The in vivo angiogenesis assay was performed as previously described [20 (link)]. Eight C57 black mice (20–25 g) were kept in temperature- and humidity-controlled rooms (22 °C, 50%) with lights on from 07:00 to 19:00 h, and water and food available ad libitum. The investigation was conducted in accordance with the ethical standards and according to the Declaration of Helsinki and the Italian law (Legislative Decree no.26, 4 March 2014), which acknowledges the European Directive 2010/63/UE, being approved by the authors’ institutional review board and the Italian Ministry of Health (authorization n. 55/2017-PR).
The mice were SC injected in the dorsal midline region with 0.4 mL of Bioregen^® or Regenyal Idea Bioexpander^®. In some of the experiments, the HA preparations were mixed with Matrigel (Becton Dickinson, Waltham, MA, USA) (1:1, v:v) on ice. At different times, compatible with the biomaterial half-life once injected (1–9 days for the absorbable Bioregen^® and 1–4 weeks for Bioexander^®), mice were sacrificed by CO₂ inhalation and implants were harvested. The implants were re-suspended in 1 mL of Drabkin’s reagent (Sigma-Aldrich, St. Louis, MO) for 18 h on ice, and haemoglobin concentration was determined by absorbance at 540 nm and compared with a standard curve (Sigma-Aldrich, St. Louis, MO). Some plugs were stored at −80 °C for protein extraction and Western blot analysis.

The DCFH-DA probe was used for ROS determination as described somewhere else 33 (link). Treated and control cells were lysed and diluted 1:10 with Tris 40 mM (pH 7.4) plus DCFH-DA (2′,7′-dichlorofluorescein diacetate; Molecular Probes, Eugene, OR, USA) 5 µM in methanol for 15 min at 37 °C. Fluorescence intensity was measured before and after 60 minutes of incubation in an LS50-B luminescence spectrometer (Perkin-Elmer, Boston, MA, USA). The reaction with superoxide anion, hydrogen peroxide, and hydroxyl radical to produce an oxidized, fluorescent DCFH derivative named DCF was monitored at an excitation wavelength of 525 nm (slit width = 5 nm). The bucket holder was thermostatically kept at 37 °C. Autofluorescence of the cellular lysate was less than 6% at any time. The fluorescent signals of both methanol (vehicle) and the substrates were recorded at the baseline, before DCF formation. DCF was quantified from a standard curve (Sigma Aldrich, St. Louis, MO, USA) in methanol.