Integrating vascular and hypertrophic cartilage microtissues to fabricatescaled-up grafts for endochondral bone tissue engineering

ABSTRACTThe repair of large bone defects remains a major clinical challenge, in part due to inadequate vascularization and poor integration of graft materials. Tissue engineering strategies that recapitulate the developmental process of endochondral ossification, whereby a cartilage template remodels into bone, have shown significant potential in pre-clinical models of large bone defect healing. However, successfully scaling these approaches to clinically relevant sizes will require the development of strategies to support the rapid vascularization of the graft following implantation in vivo. Here, mechanically reinforced templates were first fabricated by integrating hypertrophic cartilage microtissues derived from human mesenchymal stem/stromal cells (MSCs) within an osteoconductive 3D-printed polycaprolactone (PCL) framework coated with nano-hydroxyapatite (nanoHA). In vitro the cartilage microtissues fused and generated an extracellular matrix rich in sulphated glycosaminoglycans and collagen. To prevascularize these constructs, vascular microtissues derived from a co-culture of endothelial cells and MSCs were incorporated into a central channel within the construct, which generated a microvascular network within the graft in vitro. Following subcutaneous implantation, hypertrophic cartilage templates with ( vascular-channel group) and without ( empty-channel group) this central vascularized channel supported endochondral bone formation. Quantitative microCT and histological analyses revealed significantly greater remaining bone in the empty-channel group, whereas the vascular-channel group supported enhanced vascularization and remodeling of the graft in vivo. These findings support the continued development and testing of a modular biofabrication strategy that combine self-organizing hypertrophic cartilage and vascular microtissues with osteoconductive 3D-printed architectures to generate scalable, prevascularised hypertrophic cartilage templates for endochondral bone repair. Key-words: spheroids, microtissues, hypertrophic cartilage, vascularization, endochondral ossification, bone tissue engineering.