Orbital shaker

For the phage release assay, we followed the procedure described by Korehei and Kadla [31 (link)] with minor modifications. Briefly, ≈2 cm² of PCL_Fulbright nanofiber was soaked in 3 mL phage lysate for 1 h with gentle shaking using a VWR Orbital Shaker. The soaked fiber was washed in 3 mL of phage buffer, which was shaken at 60 rpm at room temperature. The fibers were transferred into a fresh 3 mL phage buffer solution every 10 min for 10 washes. The PCL fiber was left to air dry for 2 h and stored at 4 °C overnight. The fibers were then plated on the host bacterial lawn and incubated at 37 °C for 48 h. The plates were assessed for clear borders surrounding the phage dressing.
A viability assay was performed to determine the number of phages released from the phage-incorporated fiber. The protocol outlined by Salalha et al. (2006) was followed with few modifications [16 (link)]. PCL_Fulbright nanofiber cut into ≈2 cm² segments were placed in a microcentrifuge tube containing 1 mL of phage buffer. The tube was incubated for 60 min at room temperature to allow the phages to release into solution. After incubation, the tube was vortexed, and ten-fold dilutions of the buffer solution were plated using the agar-overlay method. The plates were incubated at 37 °C for 48 h, and PFU/mL was determined.

Cell viability was determined using the thiazolyl blue tetrazolium bromide (MTT) assay. After incubation with the indicated l-EPSs, all the media in 96-well plates were removed and replaced with serum-free media containing 0.5 mg/mL of MTT (Sigma-Aldrich). The plate was further incubated with 5% CO₂ for 4 h at 37 °C, then MTT solution was removed and cells were lysed with 200 μL dimethyl sulfoxide (DMSO) (Sigma-Aldrich). The plate was then placed on an orbital shaker (VWR) for 30 min before determining the absorbance at 570 nm using a microplate reader (Biotek Instruments). The absorbance values were expressed as a percentage of untreated control cells (control = 100%)^{28 (link)}.

The viability of cells was determined using cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kits (Cayman Chemical, Ann Arbor, MI, USA). Cells were seeded at a concentration of 5 × 10⁴ cells/100 μL in differentiation medium per well with 96-well black wall culture plates. The treatment of MSCs with various concentrations of H₂O₂ (50, 100, 200, 400, and 800 nM; H₂O₂, 30% (w/w) solution, Sigma-Aldrich) during culture was also examined. The H₂O₂ stock was diluted by PBS, and the same volume of diluted H₂O₂ solution was added to the culture medium. MSCs were cultured without H₂O₂ but PBS (osteogenic differentiation medium with 0 nM H₂O₂) as a control. Cells were incubated with different levels of H₂O₂ treatments for 6, 24, and 48 h in the dark. The MTT viability assay was not performed for longer periods of time because high cell density led to high absorbance readings that impaired the accuracy of detection. DMEM with 10% MTT was added and incubated for 4 h, after wich the culture medium was completely discarded. The formed formazan was dissolved with 100 µl dimethyl sulfoxide (DMSO; Sigma-Aldrich) to produce a purple color, and the plates were then placed on an orbital shaker (VWR) set at low speed for 5 min. The absorbance was measured at 570 nm using a microplate reader (BioTek, Winooski, VT, United states).

The highest-producer clone was selected and cultured in Luria–Bertani broth containing 50 µg/mL ampicillin (LB-Amp) in 2 L Erlenmeyer flasks. For the initial growth, 200 mL of LB-Amp broth was maintained overnight at 37 °C with 250 rpm agitation in an orbital shaker (VWR International, Radnor, PA, USA). After 12 h of culture, cells were harvested using a Z36 HK centrifuge (Hermle Labortechnik, Wehingen, Germany) at 5000× g for 10 min. The cell pellet was then resuspended in fresh LB-Amp broth containing 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) to induce RBD production. The induction was conducted at 30 °C with agitation at 250 rpm for 8–12 h. After induction, cells were recovered by centrifugation at 5000× g for 10 min at 4 °C. Cell pellets were kept at −20 °C until further processing.

For the preparation of our PCL_Fulbright nanofiber, polyethylene glycol (PEG 8000) purified phage lysate was used. The high titer phage lysate was centrifuged using a Thermo Scientific Sorvall Legend XTR centrifuge at 5500× g at 4 °C for 10 min. The supernatant was transferred to a new tube, and 1 M of NaCl and 10% of PEG were added. The solution was gently stirred at 15 rpm using a VWR Orbital Shaker at 4 °C overnight until the PEG and NaCl were in solution. The sample was then centrifuged at 5500× g at 4 °C for 10 min, and the supernatant was decanted, ensuring that most of the PEG has been removed. The pellet was gently re-suspended in fresh phage buffer and stirred overnight at 4 °C. Another centrifugation was performed, and the final cell pellet was re-suspended in phage buffer and stored at 4 °C until use. The phage titer in the PEG-purified lysate was 1 × 10¹³ PFU/mL. This lysate was used for all subsequent experiments unless otherwise noted.

Release studies were carried out using different media, including PBS (pH 7.4, Gibco), 100% FBS (Sigma, F1051), 1% FBS in PBS pH 7.4, and 3.6% BSA (Sigma, A7906) in PBS (pH 7.4, Gibco). Release buffer volumes, total drug loadings, and sample dimensions are indicated in the figure captions. Sink conditions were maintained across all of the in vitro release studies through frequent buffer changes, ensuring that the released drug concentration was always at least five times lower than the solubility limit of the drug in the release buffer being used in order to avoid changes in release kinetics caused by drug levels approaching their saturation limit. Samples were incubated in release medium at 37 °C under constant agitation on an orbital shaker at 115 rpm (VWR). At designated timepoints, sampling was performed by complete removal of medium and replenishment with fresh buffer. Sampling timepoints included 24 h and then every 3–4 days thereafter. For PBS release conditions, samples were analyzed directly. For samples with protein (100% FBS, 1% FBS, 3.6% BSA), protein was precipitated by addition of acetonitrile. Samples were vortexed and centrifuged to separate extracted drug from precipitated protein. The supernatant was then analyzed for drug concentration by HPLC.