Ascorbic acid

Sulfonated-PRX triblock copolymers composed of sulfopropyl ether-modified α-CDs threaded onto a PEG chain as a middle PRX segment and poly(benzyl methacrylate) (PBzMA) at both terminals of the PEG as anchoring segments (SPE-PRXs) were prepared as described previously.^{33 (link)} SPE-PRXs with different numbers of threading CDs were obtained by altering the PEG/α-CD molar ratios. HBMSCs, an HBMSC Growth Medium BulletKit (HBMSC growth medium), HUVECs, endothelial growth medium-2 (HUVEC growth medium) supplemented with 0.1% VEGF, 0.1% human epidermal growth factor, 0.1% R3-insulin-like growth factor-1, 0.1% ascorbic acid, 0.04% hydrocortisone, 0.4% human fibroblast growth factor-2, 0.1% heparin, 2% fetal bovine serum, and 0.1% gentamicin were purchased from Lonza (Walkersville, MD, USA). Mesenchymal stem cell osteogenic differentiation medium (HBMSC differentiation medium) was purchased from Promo Cell (Heidelberg, Germany). Trypsin/ethylenediaminetetraacetic acid (EDTA) solution, phosphate buffered saline (PBS), 4% paraformaldehyde, alizarin red S, and dimethyl sulfoxide (DMSO) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Ammonia solution (28%) was purchased from Kanto Chemical Industry (Tokyo, Japan). A 24-well tissue culture polystyrene (TCPS) plate was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Primary Caviae porcellus vascular endothelial cells were derived from segments of aortic tissue isolated from normal animals (guinea pigs) as previously described by Wang JM. et al. (2017) [71 (link)]. Human umbilical vein endothelial cells of the HUVEC line were obtained commercially (C2517A, Lonza, Walkersville, MD, USA). Cells were cultured in endothelial growth medium (EGM-2) enriched with fetal bovine serum (FBS), hydrocortisone, fibroblast growth factor (fFGF), vascular endothelial growth actor (VEGF), Epidermal Growth Factor (EGF), ascorbic acid, gentamicin sulphate, amphotericin (GA-1000) and heparin (Lonza, Poland), at 37 °C in a humidified atmosphere containing 5% CO₂. Every 2–3 days, the medium was changed, and the cells were passaged at 80–90% confluence. Cells were trypsinized, washed with phosphate buffered saline (PBS), and their viability was then assessed by trypan blue staining and cell suspensions were adjusted to working density.

Pooled HUVEC (from 3 to 6 individual donors, Lonza, Cologne, Germany) and HUAEC (from 250 individual donors, PeloBiotech GmbH, Planegg, Germany) were cultivated in EGM-2 Bullet Kit containing endothelial cell culture medium supplemented with 2% (v/v) fetal bovine serum, vascular endothelial growth factor, basic fibroblast growth factor, human epithelial growth factor, insulin-like growth factor-1, hydrocortisone, heparin, ascorbic acid, gentamycin, and amphotericin B (Lonza) in a standard humidified incubator at 37 °C with 5% (v/v) CO₂. Cells of passage 5 were seeded in 4-well PCA chamber slides (Sarstedt, Nürnbrecht, Germany) at a cell density of 15,000 cells/well with 1 mL medium per well, and cultivated for up to 7 days. Medium was exchanged at day 2 and day 4 of cultivation.

The mouse preosteoblastic cell line MC3T3-E1 (ATCC, Manassas, VA, USA) was cultured in α-MEM + GlutaMAX (Gibco) supplemented with 10% fetal bovine serum (FBS, PAA Laboratories GmbH, Pasching, Austria), 100 U mL⁻¹ penicillin, 100 µg mL⁻¹ streptomycin (Gibco) and primary human osteoblasts (Cambrex Bio Science, Walkersville, MD, USA) from two donors (one from humerus and one from tibia) in osteoblast growth medium supplemented with 10% FBS, 0.1% gentamicin sulphate/amphotericin B and 0.1% ascorbic acid (Lonza, Walkersville, MD, USA) at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. As previously described [8 (link)], an agitated seeding method was used to ensure homogenous cell distribution throughout the scaffold. In brief, scaffolds were placed in non-adherent 48 well plates, a solution of 2 × 10⁵ cells in culture medium was pipetted against the wall of each sample well, and the plates were agitated for 3 h at 37 °C. The scaffolds were then moved to a new 48 well plate and incubated for 24 h at 37 °C in the corresponding culture medium.

Normal human osteoblasts, CC‐2538, and normal human bone marrow‐derived mesenchymal stem cells, PT‐2501 (Lonza, Allendale, NJ, USA), were used, grown in medium containing fetal bovine serum and gentamicin and amphotericin‐B. For differentiation, 10 mM glycerol‐2‐phosphate, 50 μg/mL ascorbic acid (Lonza), 2 mM CaCl₂, and 10 nM 1,25‐dihydroxy vitamin D₃ were added. Charcoal‐stripped fetal bovine serum was used to eliminate endogenous lipids. Cells were grown at 37°C with 5% CO₂. At 80% confluence, cells were detached with 0.05% trypsin in 0.02% EDTA. All experiments were performed after the third passage. Media were replaced at 2‐ to 3‐day intervals.