Calcium sulphate

After 42 days of in vitro culture, the primed aggregates were prepared for implantation. A dual syringe approach, previously described by Kolambkar et al. [59 (link)], was adapted to imbed the cellular aggregates within hydrogels. Briefly, functionalised alginate (FMC Biopolymer; Sandvik, Norway) containing bone morphogenetic protein (BMP)-2 (Pfizer, MA, USA) at a concentration of 1.6 μg/100 μL was cross-linked by adding calcium sulphate (Sigma) to a final concentration of 8.4 mg/mL. Constructs were prepared by injecting 100 μL of cross-linked alginate into an electrospun, PCL nanofibre mesh tube [59 (link)], and two cellular aggregates from each group were placed within each alginate/mesh construct (Fig. 1). One group, which contained no aggregates within the mesh, was used as an acellular group (known as the Alginate group). These constructs were then incubated in culture medium within a 24-well ultralow-attachment plate (Corning, Lowell, MA, USA) for 2–6 h prior to implantation.

Zebrafish were maintained at the Garvan Institute of Medical Research and Victor Chang Cardiac Research Institute in Sydney, Australia. All procedures were approved by the Garvan Institute of Medical Research/St Vincent’s Hospital Animal Ethics Committee under Animal Research Authorities 15_13 (4 May 2015), 17_21 (7 August 2017) and 18_14 (18 July 2018). Larval zebrafish were housed in 1 L tanks (Tecniplast, Chester, PA, USA) and adult zebrafish in 3.5 L tanks (Tecniplast) with a maximum of 30 fish per tank. Fish were grown in 28 ± 1 °C recirculating chlorine-free water and were exposed to a day-night cycle of 14.5:9.5 hours light: dark and fed three times daily. Experimental animals were generated from weekly group matings. Fertilized embryos were incubated at 28.5 °C (Memmert, Büchenbach, Germany) in Embryo Media (0.03% (w/v) ocean salt (Aquasonic, Wauchope, Australia), 0.0075% (w/v) calcium sulphate (Sigma, St. Louis, MO, USA), 0.00002% (w/v) methylene blue (Thermo Fisher Scientific, Waltham, MA, USA) in water) at a density of 60 embryos per 25 mL dish (Thermo Fisher Scientific). At 3 days post-fertilization (dpf), zebrafish were screened for transgene expression using fluorescence microscopy (Leica DFC450 C), and at 1 wpf, larvae were placed into 1 L housing tanks in chlorine-free water, with the flow turned at 2 wpf.

All chemicals used were of analytical grade or highest purity available. All buffers and aqueous solutions were prepared with deionised water (>17 MΩ). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and yeast nitrogen base were from Amresco LLC (OH, USA). 2,6-dimethoxyphenol (DMP), acetic acid, boric acid, magnesium chloride, manganese sulphate, peptone from casein, potassium hydroxide, potassium sulphate, sodium hydrogen phosphate, sodium molybdate and sulfuric acid were from Fluka (St. Gallen, CH). Zeocin was from Invitrogen (Carlsbad, CA, USA). Sodium hydroxide was from Merck (Darmstadt, Germany). Agar, ammonium sulphate, glucose, glycerol, mEthanol, phosphoric acid (85%), potassium sulphate, potassium dihydrogen phosphate, potassium hydrogen phosphate and sodium chloride were from Roth (Karlsruhe, Germany). Agarose, biotin, calcium sulphate, citric acid, cobalt chloride, copper sulphate, EDTA, ferrous sulphate, magnesium sulphate, sodium iodide, Tris base, yeast extract and zinc chloride were from Sigma Aldrich (St. Louis, USA). Ethanol was from VWR (Radnor, USA). Escherichia coli NEB5α (New England Biolabs, Ipswich, MA, USA) was used for subcloning and Pichia pastoris × 33 (Invitrogen) for heterologous expression^{9 (link)}. A modified pGAPZ A vector (Invitrogen) under the control of the GAP promotor was used for expression in P. pastoris^{43 (link)}.