3D Bioprinting of Developmentally Inspired Templates for Bone and Joint Regeneration

Abstract

There is an urgent need to develop alternatives to synthetic joint prosthesis to promote the regeneration of diseased osteoarthritic joints. Cell based therapies have shown promise for repairing cartilage and bone; however, existing approaches are designed to repair small focal defects, and are not suitable for treating large injuries or for regenerating osteoarthritic joints. The objective of this thesis was to bioprint cell laden constructs capable of recapitulating key aspects of limb development as implants for large bone defect healing and joint regeneration. To this end, a novel biofabrication strategy for engineering whole bone organs was first developed by bioprinting hypertrophic cartilage templates with the capacity to undergo endochondral ossification following implantation in vivo. These soft cartilaginous templates could be mechanically reinforced with a network of co-printed polycaprolactone (PCL) microfibers, resulting in a dramatic increase in construct compressive modulus, thereby providing the necessary stiffness and strength that in principle would allow the implantation of such immature cartilaginous rudiments into load bearing locations. Next, it was demonstrated that this developmentally inspired biofabrication framework could be used to successfully repair critically sized femoral defects in rats, supporting comparable levels of bone healing to recombinant BMP-2 delivery. The incorporation of 3D-printed microchannels into the cartilage templates was also shown to guide vascularisation and mineralisation during defect repair. To facilitate the in vitro engineering of a human scale biological joint prosthesis, a strategy to engineer large homogenous cartilaginous constructs, through dynamic bioreactor culture at defined oxygen conditions, was then investigated. At 20% pO2, dynamic culture significantly suppressed chondrogenesis compared to static conditions; whereas dynamic culture at 3% pO2 significantly enhanced the distribution and amount of cartilage matrix components compared to static conditions. A novel approach for engineering stratified cartilage tissues mimicking those found within synovial joints was then developed. By combining inkjet bioprinting and 3D-printed microchambers, it was possible to engineer a modular array of mesenchymal condensations that underwent chondrogenesis and fused to form larger cartilage tissues. By orientating the fusion of these condensations, it was possible to engineer stratified cartilage tissues with collagen fiber orientations that mimicked the structure of native articular cartilage. Finally, using multi-tool biofabrication, followed by bioreactor culture, it was also possible to tissue engineer anatomically accurate, human-scale osteochondral templates containing stratified articular cartilage in the chondral region, and mechanically reinforced hypertrophic cartilage designed to support endochondral bone regeneration in the osseous region. To conclude, this thesis presents a novel bioprinting framework for the regeneration of whole joint surfaces by spatially directing the formation of hypertrophic cartilage and stratified hyaline cartilage within a reinforcing polymeric framework. Such bioprinted implants could transform the treatment of diseases such as osteoarthritis.