The ability to print defined patterns of cells and extracellular-matrix components in three dimensions has enabled the engineering of simple biological tissues, however bioprinting functional solid organs is beyond the capabilities of current biofabrication technologies. An alternative approach would be to bioprint the developmental precursor to an adult organ, using this engineered rudiment as a template for subsequent organogenesis in vivo. Here we demonstrate that developmentally inspired hypertrophic cartilage templates can be engineered in vitro using stem cells within a supporting gamma-irradiated alginate bioink incorporating Arg-Gly-Asp (RGD) adhesion peptides.Furthermore, these soft tissue templates can be reinforced with a network of printed polycaprolactone fibres, resulting in a ~350 fold increase in construct compressive modulus providing the necessary stiffness to implant such immature cartilaginous rudiments into load bearing locations. As a proofof-principal, multiple-tool biofabrication was used to engineer a mechanically reinforced cartilaginous template mimicking the geometry of a vertebral body, which in vivo supported the development of a vascularized bone organ containing trabecular-like endochondral bone with a supporting marrow structure. Such developmental engineering approaches could be applied to the biofabrication of other solid organs by bioprinting pre-cursors that have the capacity to mature into their adult counterparts over time in vivo.3

The ability of nano-hydroxyapatite (nHA) particles developed in-house to act as non-viral delivery vectors is assessed. These nHA particles are combined with collagen to yield bioactive, biodegradable collagen nano-hydroxyapatite (coll-nHA) scaffolds. Their ability to act as gene-activated matrices for BMP2 delivery is demonstrated with successful transfection of mesenchymal stem cells (MSCs) resulting in high calcium production.

Bone tissue engineering strategies that recapitulate the developmental process of endochondral ossification offer a promising route to bone repair. Clinical translation of such endochondral tissue engineering strategies will require overcoming a number of challenges, including the engineering of large and often anatomically complex cartilage grafts, as well as the persistence of core regions of avascular cartilage following their implantation into large bone defects. Here 3D printing technology is utilized to develop a versatile and scalable approach to guide vascularisation during endochondral bone repair. First, a sacrificial pluronic ink was used to 3D print interconnected microchannel networks in a mesenchymal stem cell (MSC) laden gelatin-methacryloyl (GelMA) hydrogel. These constructs (with and without microchannels) were next chondrogenically primed in vitro and then implanted into critically sized femoral bone defects in rats. The solid and microchanneled cartilage templates enhanced bone repair compared to untreated controls, with the solid cartilage templates (without microchannels) supporting the highest levels of total bone formation. However, the inclusion of 3D printed microchannels was found to promote osteoclast/immune cell invasion, hydrogel degradation, and vascularisation following implantation. In addition, the endochondral bone tissue engineering strategy was found to support comparable levels of bone healing to BMP-2 delivery, whilst promoting lower levels of heterotopic bone formation, with the microchanneled templates supporting the lowest levels of heterotopic bone formation. Taken together, these results demonstrate that 3D printed hypertrophic cartilage grafts represent a promising approach for the repair of complex bone fractures, particularly for larger defects where vascularisation will be a key challenge.

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