Generation of VEGF gradients using vascular bioink

Generation of VEGF gradient using Vascular Bioink

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

2.      Nanohydroxyapatite was prepared, in sterile conditions following a previously described protocol (Cunniffe et al. Journal of Biomedical Materials Research Part A, 95a, 1142-1149, 2010) .  

3.      A 3.5% RGD γ-irradiated Alginate solution was prepared by dissolving the RGD γ-irradiated Alginate in growth medium which consisted of high-glucose Dulbecco’s modified eagle medium (DMEM, GlutaMAXTM;GIBCO, Biosciences, Ireland), 10 % fetal bovine serum (FBS; EU Thermo Scientific), 100 U/mL penicillin (Sigma Aldrich), 100 g/mL streptomycin (Sigma Aldrich) to make up a final concentration of 3.5% (w/v).

4.      Methylcellulose and nHA (2:1:2) (w/w) particles were added and mixed into the Alginate solution using a sterile stirrer bar and stirrer plate.

5.      Using a dual syringe approach linked via a luer lock, the bioinks were loaded with VEGF to generate 3 experimental groups: [1] Homogenous VEGF, bioink loaded with 100ng/ml of VEGF was used to print constructs 8mm in diameter and 4mm high; [2] Gradient 1, bioink loaded with 100ng/ml of VEGF was developed to print a central 4mm core with a VEGF free bioink developed for around the periphery of the 8mm diameter construct; [3] Gradient 2, 80 ng/mL of VEGF was developed for the core and 20ng/mL of VEGF was developed for the periphery.

6.      Using the dual syringe approach again, all the bioinks were crosslinked with 60mM CaSO₄ and allowed to crosslink for 30 minutes, as was previously deemed optimum (Freeman et al. Sci Rep 7, 17042, 2017), before being loaded into the 10cc syringe barrel.

7.      The syringe barrels were loaded into the RegenHU printer, in sterile conditions. The following constructs were printed using the RegenHU: (i)Homogenous VEGF 100ng/ml VEGF Bioinks was loaded alone, and constructs 8mm in diameter and 4mm high. (ii) Gradient 1. Two syringe barrels were loaded into the printer one with a Bioink loaded with VEGF (100 ng/ml), which was used to print a central 5-mm core. Then a second syringe with a VEGF-free bioink used to print around the periphery of the 8-mm-diameter construct. (iii) Similar method was used for Gradient 2, where a VEGF (80 ng/ml) bioink was used to print the core, and a second bioink loaded with VEGF (20 ng/ml) was used to print the periphery.

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

9.      Constructs were cultured in growth medium in normoxic conditions for 14 days in vitro.

10.  The centre and periphery of each construct were separated by coring out the centre from the periphery of the scaffold and then snap-frozen at −80°C, 1 hour after printing, and after 14 days in vitro.

11.  An enzyme-linked immunosorbent assay was used to quantify the levels of VEGF (Bio-Techne, MN, USA) released by the alginates, as well as what VEGF was contained within the bioinks following printing/culture.

12.  The alginate samples were depolymerized with 1 ml of citrate buffer (150 mM sodium chloride, 55 mM sodium citrate, and 20 mM EDTA in H2O) for 15 min at 37°C.

13.  The cell culture media and depolymerized alginate samples were analysed at the specific time points detailed above.

14.  Assays were carried out as per the manufacturer’s protocol and analysed on a microplate reader at a wavelength of 450 nm.

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