3D Bioprinted Cartilaginous Templates as Developmentally Inspired Implants for Large Bone Defect Regeneration

Abstract

Whilst bone possesses an inherent capacity for regeneration, defects above a critical size cannot heal on their own. Damaged or diseased bone can be treated using autografts or a range of different bone grafting biomaterials, however limitations with such approaches has motivated increased interest in developmentally inspired bone tissue engineering (BTE) strategies that seek to recapitulate the process of endochondral ossification (EO) as a means of regenerating critically sized defects. The clinical translation of such strategies will require the engineering of scaled-up, geometrically defined hypertrophic cartilage grafts that can be rapidly vascularised and remodelled into bone in mechanically challenging defect environments. The overall goal of this thesis was to 3D bioprint mechanically reinforced cartilaginous templates as developmentally inspired implants for large bone defect regeneration. This required the selection of a biomaterial ink able to support chondrogenesis of human mesenchymal stem/stromal cells (hMSCs) in vitro, the realisation of a reinforcing polycaprolactone (PCL) frame, and the engineering of cartilaginous templates in vitro. Firstly, fibrin hydrogels were prepared, laden with hMSCs and cultured for 5 weeks in chondrogenic media. In addition, to facilitate nutrient transport in vitro and potentially vascularisation in vivo, we introduced micro-channels into these constructs, and we showed that they remained patent throughout the culture period. It was demonstrated that fibrin can support encapsulated hMSCs chondrogenesis and progression along an endochondral pathway in vitro. In addition, it was shown how the fibrinogen content within fibrin-based bioinks influences chondrogenesis of encapsulated hMSCs and that it was possible to mechanically reinforce 3D bioprinted cartilaginous templates with a 3D printed polymer network. Then it was demonstrated how, by culture priming 3D bioprinted reinforced constructs, cartilage and hypertrophic cartilage templates were obtained, which were capable of directing endochondral bone formation upon in vivo implantation. Finally, it was demonstrated that it is possible to decellularise the in vitro engineered cartilaginous grafts to produce off-the-shelf hypertrophic cartilage grafts with osteogenic potential that can be used in bone repair applications.