Construct design and 3D printing process

Construct design and 3D printing process

1.      A 3D bioplotter from RegenHU (3D Discovery) was used to print all of the scaffolds.

2.      Using a 30G needle constructs of Ø 4mm x 5mm high with both lateral and horizontal porosity and a fibre spacing of 1.2 mm were printed with polycaprolactone (PCL; Cappa, Perstop).

3.      The printing parameters of the PCL listed in Table 1. Scaffolds were sterilised using ethylene oxide (ETO) sterilisation prior to hydrogel printing.

Table 1: Printing parameters for printing the scaffolds with  PCL using the RegenHU bioprinter

For the VEGF gradient study:

1.      The vascular bioink was prepared (as described in manuscript), crosslinked with 60mM CaSO₄ using a dual-syringe approach, and allowed to crosslink for 30 minutes as was deemed optimum in our previous studies (Freeman et al. Sci Rep 7, 17042, 2017)

2.      Three experimental groups were prepared as follows: [1] No VEGF, bioink not loaded with VEGF; [2] Homogenous, bioink loaded with 100ng/mL of VEGF deposited (25ng/construct) throughout the construct, and [3] Gradient, bioink loaded with 500ng/ml of VEGF deposited in the centre (25ng/construct) and VEGF-free bioink deposited on the outside (see Figure 1 (A) in manuscript).

3.      Each experimental bioink was loaded into the RegenHU machine and the co-ordinates of the pores within the PCL framework were established. Manipulating the G-code of the printer each bioink was z-printed within the pores of the framework depending on the experimental group. This was all done within the sterile hood of the RegenHU.

4.      Post-printing constructs were crosslinked again in a bath of 100 mM CaCl₂ for 1 minute.

For the BMP-2 release study:

1.      Both a fast and slow release bioink (see methods in manuscript) were prepared and using the dual syringe approach 2x10⁶/mL porcine MSCs were mixed to both bioinks to have an overall seeding density of 500x10⁵ porcine MSCs/construct before being crosslinked with 60mM CaSO₄. Both inks were allowed to crosslink for 30minutes.

2.      Both bioinks were printed within the PCL framework (as descriped above) to generate 2 experimental groups; [1] Fast Release, fast release bioink loaded with 2µg/mL of BMP-2 (0.5 µg/construct) deposited only in the periphery with the fast release bioink not loaded with BMP-2 in the centre, and [2] Slow Release, slow release bioink loaded with 2µg/mL of BMP-2 (0.5 µg/construct) deposited only in the periphery with the fast release bioink not loaded with BMP-2 in the centre (see Figure 2(A)).

3.      Post-printing constructs were crosslinked again in a bath of 100 mM CaCl₂ for 1 minute.

For the rat femoral defect:

1.      The vascular bioink, the osteoinductive bioink, and a base bioink (3.5% RGD γ-irradiated Alginate and 1.75% methylcellulose) were prepared, pre-crosslinked with 60mM CaSO₄ for 30minutes

2.      Three experimental groups were printed within the PCL framework (using same method as described above); [1] VEGF Gradient, the vascular bioink loaded with 500ng/ml of VEGF in the centre of the implant and base bioink in the periphery; [2] BMP-2 gradient, the osteoinductive bioink loaded with 10µg/mL BMP-2 in the implant periphery (2µg/construct), with the base bioink in the centre; [3] Composite (VEGF+BMP-2), the osteoinductive bioink in the periphery with the vascular bioink in the centre, see Figure 3(A). Post-printing constructs were crosslinked again in a bath of 100 mM CaCl₂ for 1 minute.

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