Implantation of Primed Cell Aggregates

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.