Sdf 1

Triculture tumor models were prepared as described previously [13 (link),14 (link)]. The heparin fraction was functionalized with 2 mol of a fibronectin-derived adhesion-mediating peptide sequence, H₂N-GCWGGRGDSP-CONH₂ (RGD-SP; M_w 990; synthesized in house) per mole of HM6. Afterwards, vascular endothelial growth factor (VEGF; PeproTech, Hamburg, Germany), stromal cell-derived factor 1 (SDF-1; Miltenyi Biotec, Bergisch Gladbach, Germany), and fibroblast growth factor 2 (FGF-2; Miltenyi Biotec) were added to stimulate HUVEC-tube formation, and mixed at a concentration which corresponds to a final concentration of each 5 µg/mL per casted gel. Breast-cancer cells (1 × 10⁴ cells/gel), MSCs (1.2 × 10⁴ cells/gel), and HUVECs (1.2 × 10⁵ cells/gel) were each added into the heparin–RGD-growth factor mixture. Finally, the HM6-cell suspension was mixed with PEG–MMP or PEG–MMP–GFOGER in a volume ratio of 1:1 and 20 µL gels were casted onto a Sigmacote-treated microscope slide. For angiogenesis triculture experiments, PEG–MMP hydrogels were created with a crosslinking degree of γ = 0.76 and PEG–MMP–GFOGER with γ = 1, which equated to equal stiffness, due to PEG–MMP–GFOGER having one less arm available for crosslinking. Tricultures were cultured in 1.5 mL of ECGM at 37 °C and 5% CO₂.

Solid PLA discs were coated with bovine (Viscofan, Weinheim, Germany) or rat tail collagen (Invitrogen, Karlsruhe, Germany). Collagens were diluted 1:100 with PBS and discs were incubated for one hour to assure even coating.
3D cages were filled with a collagen gel solution following an established protocol [56 (link),57 (link)]. Briefly, collagen type I (3 mg/mL bovine, Viscofan, Weinheim, Germany), aqua dest, M199 (10×), NaHCO₃ and NaOH and SDF-1 (500 ng, Miltenyi, Bergisch Gladbach, Germany) were combined within an ice bath to prevent polymerization of the solution. Next, 300 μL of the liquid collagen solution was pipetted into a PLA cage sitting in a 24-well ultra-low attachment plate (Corning, Wiesbaden, Germany) and allowed to polymerize.

A cylinder of PLA filament (Ultimaker silver metallic PLA, iGo3D, Hannover, Germany) with a diameter of 4 mm and a height of 7 mm was printed with the Ultimaker 2+. Mechanical, thermal and other properties are listed in the technical data sheet from Ultimaker. The cylinder was designed with a 3D modelling software (Autodesk Inventor Professional 2013, Autodesk, San Rafael, CA, USA) comparable to the size of the excised piece of femur during surgery (Figure 7E). Pores with a diameter of approximately 1 mm were included into the wall of the scaffold to facilitate bone ingrowth (Figure 7A–D). A collagen solution (10× M199, 6% NaHCO₃, 2.5%, 65% collagen solution (Viscofan; 5 mg/mL 16.5% water) was prepared. In total, 100 µl of this solution was supplemented with 100 ng SDF-1 or BMP-7 (both Miltenyi Biotec, Bergisch Gladbach, Germany), respectively, and pipetted into the cylinder and polymerized at room temperature.