Alginate is a commonly used bioink in 3D bioprinting. Matrix stiffness is a key determinant of mesenchymal stem cell (MSC) differentiation, suggesting that modulation of alginate bioink mechanical properties represents a promising strategy to spatially regulate MSC fate within bioprinted tissues. In this study, we define a printability window for alginate of differing molecular weight (MW) by systematically varying the ratio of alginate to ionic crosslinker within the bioink. We demonstrate that the MW of such alginate bioinks, as well as the choice of ionic crosslinker, can be tuned to control the mechanical properties (Young’s Modulus, Degradation Rate) of 3D printed constructs. These same factors are also shown to influence growth factor release from the bioinks. We next explored if spatially modulating the stiffness of 3D bioprinted hydrogels could be used to direct MSC fate inside printed tissues. Using the same alginate and crosslinker, but varying the crosslinking ratio, it is possible to bioprint constructs with spatially varying mechanical microenvironments. Moreover, these spatially varying microenvironments were found to have a significant effect on the fate of MSCs within the alginate bioinks, with stiffer regions of the bioprinted construct preferentially supporting osteogenesis over adipogenesis.

Significant progress has been made in the field of cartilage and bone tissue engineering over the last two decades. As a result, there is real promise that strategies to regenerate rather than replace damaged or diseased bones and joints will one day reach the clinic however, a number of major challenges must still be addressed before this becomes a reality. These include vascularization in the context of large bone defect repair, engineering complex gradients for bone‐soft tissue interface regeneration and recapitulating the stratified zonal architecture present in many adult tissues such as articular cartilage. Tissue engineered constructs typically lack such spatial complexity in cell types and tissue organization, which may explain their relatively limited success to date. This has led to increased interest in bioprinting technologies in the field of musculoskeletal tissue engineering. The additive, layer by layer nature of such biofabrication strategies makes it possible to generate zonal distributions of cells, matrix and bioactive cues in 3D. The adoption of biofabrication technology in musculoskeletal tissue engineering may therefore make it possible to produce the next generation of biological implants capable of treating a range of conditions. Here, advances in bioprinting for cartilage and osteochondral tissue engineering are reviewed.

Therapeutic growth factor delivery typically requires supraphysiological dosages, which can cause undesirable off-target effects. The aim of this study was to 3D bioprint implants containing spatiotemporally defined patterns of growth factors optimized for coupled angiogenesis and osteogenesis. Using nanoparticle functionalized bioinks, it was possible to print implants with distinct growth factor patterns and release profiles spanning from days to weeks. The extent of angiogenesis in vivo depended on the spatial presentation of vascular endothelial growth factor (VEGF). Higher levels of vessel invasion were observed in implants containing a spatial gradient of VEGF compared to those homogenously loaded with the same total amount of protein. Printed implants containing a gradient of VEGF, coupled with spatially defined BMP-2 localization and release kinetics, accelerated large bone defect healing with little heterotopic bone formation. This demonstrates the potential of growth factor printing, a putative point of care therapy, for tightly controlled tissue regeneration.

The regeneration of complex tissues and organs remains a major clinical challenge. With a view towards bioprinting such tissues, we developed a new class of pore-forming bioink to spatially and temporally control the presentation of therapeutic genes within bioprinted tissues. By blending sacrificial and stable hydrogels, we were able to produce bioinks whose porosity increased with time following printing. When combined with amphipathic peptide-based plasmid DNA delivery, these bioinks supported enhanced non-viral gene transfer to stem cells in vitro. By modulating the porosity of these bioinks, it was possible to direct either rapid and transient (pore-forming bioinks), or slower and more sustained (solid bioinks) transfection of host or transplanted cells in vivo. To demonstrate the utility of these bioinks for the bioprinting of spatially complex tissues, they were next used to zonally position stem cells and plasmids encoding for either osteogenic (BMP2) or chondrogenic (combination of TGF-β3, BMP2 and SOX9) genes within networks of 3D printed thermoplastic fibers to produce mechanically reinforced, gene activated constructs. In vivo, these bioprinted tissues supported the development of a vascularised, bony tissue overlaid by a layer of stable cartilage. When combined with multiple-tool biofabrication strategies, these gene activated bioinks can enable the bioprinting of a wide range of spatially complex tissues.