Trypsin egta

Cytocompatibility studies of the Cs/n-HAp scaffolds were performed using the mouse embryonic fibroblast cell line (NIH-3T3, ATCC CTRL-1658™) as an in vitro model. NIH-3T3 cells were cultured in sterile T75 cm² cell culture flasks in Dulbecco’s Modified Eagle Medium low glucose (DMEM; Sigma-Aldrich, Milan, Italy) supplemented with 10% (v/v) fetal serum bovine (FBS, Sigma-Aldrich, Milan, Italy), 100 μg/mL penicillin-streptomycin solution (Pen/Strep, Sigma-Aldrich, Milan, Italy), and 2 mM of L-glutamine (L-Glu, Sigma-Aldrich, Milan, Italy) in a water-saturated atmosphere of 5% v/v CO₂ and 95% v/v air at 37 °C. Adherent cells were washed twice with Dulbecco’s Phosphate-Buffered Saline (D-PBS, Sigma-Aldrich, Milan, Italy), and then detached from the flask’s surface by adding D-PBS containing 2 mM trypsin-EGTA (Sigma-Aldrich, Milan, Italy) for 1–5 min at 37 °C, once reaching 70–90% confluence (every 2–3 days). Trypsin action was blocked by adding a suitable volume of culture medium (containing α2-antitrypsin). Cell suspensions were harvested by centrifugation (1200 rpm for 7 min, Beckman Coulter Allegra 6R centrifuge with the GH-3.8 swing bucket rotor). The obtained pellets were re-suspended in fresh culture medium and transferred to new flasks. For all experiments, cultures between passages 3 and 7 of propagation were used.

For DM and DM/n-HA nanostructures synthesis, iron (II) chloride tetrahydrate (FeCl₂·4H₂O, ≥99%), iron (III) chloride hexahydrate (FeCl₃·6H₂O, ≥97%) and Dextran from LeuconostocMesenteroids (Mw 6000 Da) were purchased by Alfa Aesar. Sodium hydroxide anhydrous pellets (NaCl, ≥99%), hydrochloric acid (≥37% wt. in water), phosphoric acid (≥85% wt. in water), ammonium hydroxide ((NH₄)OH, ≥30% wt. in water), and calcium acetate hydrate (Ca(CH₃COO)₂·XH₂O, ≥99%) were purchased from Sigma–Aldrich. Ultrapure water (18.2 MΩ/cm, obtained by a Milli-Q^® Direct Water Purification System, Merck Millipore, Darmstadt, Germany) has been used in all the experiments.
For the cell-based experiments, Eagle’s Minimum Essential Medium (E-MEM), fetal bovine serum (FBS), L-glutammine, penicillin/streptomycin antibiotic mix, Dulbecco’s phosphate buffer saline (D-PBS), trypsin-EGTA, paraformaldehyde (PFA), Triton X-100, as well as the fluorescent dye phalloidin-FITC were all purchased from Sigma–Aldrich (Milan, Italy). The Vectashield Anti-fade Mounting Medium with 4′,6-diamidino-2-phenylindole DAPI (Vector Laboratories, Peterborough, UK) was used for nuclear staining. All reagents, media supplements, and plastic-ware were supplied as cell-culture tested.