Type 1 collagen from bovine achilles tendon

Type I collagen from Bovine Achilles tendon was purchased from Sigma Aldrich. Collagen films were produced using a solvent casting method, as previously described in Reference [9 (link)]. At the end of each reaction step, all the samples were washed with 70 mL H₂O (three times × 15 min) and 30 mL of EtOH (one time × 15 min). After the washes, all the samples were dried under a hood at room temperature for 30 min.
Sample (a): Collagen matrix (90 mg, 8 × 12 cm) was covered by 20 mL of PBS pH 7.4 and put under gentle agitation. Chloroaceton (290 μL) was added and stirred overnight. The sample was washed and dried and used for the subsequent reaction.
Sample (b): Sample (a) has been covered by 20 mL of PBS pH 8, and methoxylamine hydrochloride (284 mg) was added and the reaction was left under agitation overnight. After the wash, NaBH₃CN (207 mg) was added in 20 mL of citrate buffer of pH 6.00 and the reaction was stirred for 24 h. The obtained Sample was washed and dried.
Samples (3) and (4). Neoglycosylation reactions were performed covering Sample (b) with 20 mL of Acetate Buffer at pH 4.00, Glucose (617 mg), and Maltose (1.172 g). The reaction was stirred for 24 h and the obtained sample was washed and dried.

Type I collagen from bovine Achilles tendon (Sigma-Aldrich, St. Louis, MO, USA), Viscolma collagen suspension dissolved at 12% (Viscofan BioEngineering, Weinheim, Germany), nanohydroxyapatite (nanoXIM) aggregates (Fluidinova S.A., Maia, Portugal), 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (Fluka, Buchs, Switzerland), hydrochloric acid (HCl) (Merck KGaA, Darmstadt, Germany), Vancomycin (50 mg/mL, HIKMA Farmacêutica, S.A., Sintra, Portugal) and Gentamicin (40 mg/mL, Labesfal—Laboratórios Almiro, S.A., Portugal), were kindly provided by Artur Salgado S.A. (Maia, Portugal).
Alamar blue dye (resazurin), magnesium chloride (MgCl2), 4′-6-diamidine-2-phenylindole (DAPI), formaldehyde 4% and Triton X100 were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA); the dimethyl sulfoxide (DMSO) was obtained from Merck (Merck KGaA, Darmstadt, Germany). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), fungizone, penicillin-streptomycin and trypsin were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). The DC™ protein assay was purchased from Bio-Rad. The Alexa fluorconjugated phalloidin 594 and the Quant-iT™ Picogreen^® DNA assay kit were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA).