R3 insulin like growth factor 1

Cells were suspended in 500 μl EBM2 media supplemented with human epidermal growth factor (EGF; 5 ng/mL), vascular endothelial growth factor (VEGF; 0.5 ng/mL), R3-insulin like growth factor-1 (20 ng/mL), ascorbic acid (1 μg/mL), hydrocortisone (0.2 μg/mL), human basic fibroblast growth factor (bFGF; 10 ng/mL), heparin (22.5 μg/mL) (Lonza, Switzerland) and 2% FBS, and were seeded at a density of 7.5 × 10⁴ cells/well in 12-well plates coated with Matrigel. Twenty-four hours after seeding, tube structures formed by the cells were photographed with an IX81 inverted microscope.

BECs were obtained from Lonza and maintained in endothelial basal medium (EBM-2) supplemented with SingleQuots™ Kit containing 5% fetal bovine serum (FBS), human endothelial growth factor, hydrocortisone, vascular endothelial growth factor, human fibroblast growth factor-basic, ascorbic acid, R³-insulin like growth factor-1, gentamicin and amphotericin-B (Lonza). For experiments the cells were used at passages 7–10.

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).