Materials and Reagents

Luer lock syringe cap seal (Adhesive Dispensing, catalog number: 7015LLPK)
10cc clear syringe barrel (Adhesive Dispensing, catalog number: 7100LL1NPK)
10cc syringe piston (Adhesive Dispensing, catalog number: 7100009WPK)
27 G needle, 0.25'' long (Adhesive Dispensing, catalog number: TE727025PK)
5 ml syringe (VWR, catalog number: 613-2043)
RGD γ-irradiated alginate (Manufactured in house by collaborators in Alsberg’s Lab)
Commercially available NOVATACH^TM peptide-coupled alginate could also be used, but the crosslinking ratios would need to be modified depending on the MW of the alginate used.
Sodium Triphosphate (Sigma-Aldrich, catalog number: 72061)
Darvan 821AVR (Vanderbilt Minerals, DARVAN^® 821-A)
Sodium hydroxide (Sigma-Aldrich, catalog number: S8045)
Vascular Endothelial Growth Factor (Peprotech, catalog number: 100-20)
Bone Morphogenic Protein-2 (Peprotech, catalog number: 120-02)
Methylcellulose (Sigma-Aldrich, catalog number: M7027)
Dulbecco’s modified Eagle medium (GlutaMAX^TM; ThermoFisher, catalog number: 61965026)
Fetal bovine serum (FBS; Thermo Scientific, catalog number: 10270-106)
Penicillin streptomycin (Sigma-Aldrich, catalog number: 15070063)
Laponite (Laponite XLG, BYK Additives & Instruments)
Polycaprolactone (PCL; CAPA 6500D, Perstorp, Mn = 50 kDa)
Calcium Chloride (Sigma-Aldrich, catalog number: C5670)
Calcium sulfate (Sigma-Aldrich, catalog number: 255696)
3.5% RGD γ-irradiated alginate solution (see Recipes)
100 mM calcium chloride solution (see Recipes)
60 mM calcium sulphate solution (see Recipes)
Sodium triphosphate solution (see Recipes)

Equipment

Sterile stirrer bar and stirrer plate
3D bioplotter (RegenHU, 3D Discovery, Generation 1)

Software

BioCAD (RegenHU, 3D Discovery, Generation 1)
GraphPad (GraphPad Software, La Jolla, CA, USA; www.graphpad.com)

Procedure

3D printing process for PCL backbone
1. Use a 3D bioplotter from RegenHU to print all the scaffolds.
2. Print constructs of 4 mm in diameter and 5 mm in height, with both lateral and horizontal porosity and a fibre spacing of 1.2 mm, with polycaprolactone using a 30G needle, Figure 1.
  
  Figure 1. Geometrical Features of the PCL Framework
3. The printing parameters of the PCL are listed in Table 1.
4. Sterilise the scaffolds using ethylene oxide (ETO) sterilisation prior to hydrogel printing.
  
  Table 1. Parameters for printing the scaffolds with PCL using the RegenHU bioprinter
  Heat of the thermopolymer Tank 69°C
  Heat of the thermopolymer Head 72°C
  Pressure 1 bar
  Screw Speed 30 rev/min
  Feed Rate (Translational Speed) 3 mm/s

Generation of vascular and osteoinductive bioinks
Note: Each solution should be freshly made just prior to printing.
1. Prepare the 3.5% RGD γ-irradiated alginate solution (see Recipes).
2. Add methylcellulose (2:1) (w/w) particles and mix into solution using a sterile stirrer bar and stirrer plate. For 5 g of alginate, add 2.5 g of sterile methylcellulose.
3. Prepare nHA in sterile conditions following a previously described protocol (Cunniffe et al., 2010). Briefly:
  1. Prepare the 100mM calcium chloride solution (see Recipes).
  2. Prepare the sodium triphosphate solution (see Recipes).
  3. Once fully dissolved, filter sterile both solutions using a filter with a pore size of 0.45 µm.
  4. Slowly add the sodium triphosphate solution to the 100 mM calcium chloride solution and leave stirring for 2 min.
  5. Centrifuge solution at maximum speed for 60 min.
  6. Pour off supernatant and weigh pellet. Add the appropriate amount of nHA wet weight to bioink.
4. Add either nHA (2:1:2) (for vascular bioink) or Laponite (6:3:1) (for osteoinductive bioink) (w/w) to the alginate methylcellulose blend and mix into solution using a sterile stirrer bar and stirrer plate for 2-3 h. For example, for every 5 g of alginate, add 5 g of nHA or 0.83 g of Laponite.
5. Using a dual syringe approach linked via a luer lock, load bioinks with either VEGF (100 ng/ml) or BMP-2 (10 µg/ml) and pre-crosslink with the 60 mM calcium sulphate solution at a v/v ratio of 7:3 (see Figure 2). Allow bioinks to crosslink for 30 min before being printing.
  
  Figure 2. Schematic of dual-syringe crosslinking approach

3D printing process of the implants
1. Place the piston in the 5cc barrel and load each bioink, either vascular or osteoinductive, into the piston using the luer lock.
2. Place each barrel in the two hydrogel heads of the 3D printer.
3. The printing parameters of the PCL are listed in Table 2.
  
  Table 2. Printing parameters for printing the bioinks within the PCL framework using the RegenHU bioprinter
  Pressure 1 bar
  Needle Size 27 G
  Feed Rate (Translational Speed) 3 mm/s
4. Via manipulation of the G-code (see Supplementary file) of the printer, z-stack each hydrogel at room temperature into the PCL framework to generate the following experimental groups: (1) VEGF Gradient, the vascular bioink loaded with 500 ng/ml of VEGF in the centre of the implant and base bioink in the periphery; (2) BMP-2 gradient, the osteoinductive bioink loaded with 10 µg/ml BMP-2 in the implant periphery (2 µg/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 3A in Freeman et al., 2020), see Figure 3.
  
  Figure 3. Schematic of the bioink pattern for each group and Marco image of the final implant
5. Crosslink post-printing constructs again in a bath Petri-dish filled with the 100 mM calcium chloride solution using a sterile tweezer and allowed to further crosslink for 1 min.
6. Constructs must be used immediately; if being implanted, the constructs should be printed the morning of surgeries.

Data analysis

Description of all in vivo analyses can be found in the original publication (Freeman et al., 2020). Statistics were performed using the following variables: (i) when there were two groups and one time point, a standard two-tailed t-test was performed; (ii) when there were more than two groups and one time point, a one-way analysis of variance (ANOVA) was performed; and (iii) when there were more than two groups and multiple time points, a two-way ANOVA was performed. All analyses were performed using GraphPad. For all comparisons, the level of significance was P ≤ 0.05.

Recipes

3.5% RGD γ-irradiated alginate solution
Dissolve the RGD γ-irradiated alginate in growth medium consisting of high-glucose Dulbecco’s modified Eagle medium (DMEM), 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin to make up a final concentration of 3.5% (w/v) alginate.
100 mM calcium chloride solution
Mix 0.735 g of CaCl₂ into 50 ml of sterile water.
Vortex and place on a rotator to dissolve.
60 mM calcium sulphate solution
Mix 0.408g of CaSO₄ into 50 ml of sterile water (note that this solution does not fully dissolve and needs to be mixed directly before use each time).
Sodium triphosphate solution
Mix 1.135 mg Na₃PO₄·12H₂O, 10 mg of NaOH pellets, and 100 µl of Darvan into 50 ml of sterile ultra-pure water.

Acknowledgments

This publication has emanated from research supported by a research grant from European Research Council (ERC) under Grant Number 647004 and the Irish Research Council (GOIPD/2016/324). The protocol was adapted from the previous publication “3D bioprinting spatiotemporally defined patterns of growth factors to tightly control tissue regeneration.” Published in Science Advances 2020 (Freeman et al., 2020).

Competing interests

There are no conflicts of interest or competing interests.

References

Cunniffe, G. M., O'Brien, F. J., Partap, S., Levingstone, T. J., Stanton, K. T. and Dickson, G. R. (2010). The synthesis and characterization of nanophase hydroxyapatite using a novel dispersant-aided precipitation method. J Biomed Mater Res A 95(4): 1142-1149.
Freeman, F. E., Pitacco, P., van Dommelen, L. H. A., Nulty, J., Browe, D. C., Shin, J. Y., Alsberg, E. and Kelly, D. J. (2020). 3D bioprinting spatiotemporally defined patterns of growth factors to tightly control tissue regeneration. Sci Adv 6(33): eabb5093.