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.

Engineering a pro-regenerative immune response following scaffold implantation is integral to functional tissue regeneration. The immune response to implanted biomaterials is determined by multiple factors, including biophysical cues such as material stiffness, topography and particle size. In this study we developed an immune modulating scaffold for bone defect healing containing bone mimetic nano hydroxyapatite particles (BMnP). We first demonstrate that, in contrast to commercially available micron-sized hydroxyapatite particles, in-house generated BMnP preferentially polarize human macrophages towards an M2 phenotype, activate the transcription factor cMaf and specifically enhance production of the anti-inflammatory cytokine, IL-10. Furthermore, nano-particle treated macrophages enhance mesenchymal stem cell (MSC) osteogenesis in vitro and this occurs in an IL-10 dependent manner, demonstrating a direct proosteogenic role for this cytokine. BMnPs were also capable of driving pro-angiogenic responses in human macrophages and HUVECs. Characterization of immune cell subsets following incorporation of functionalized scaffolds into a rat femoral defect model revealed a similar profile, with micron-sized hydroxyapatite functionalized scaffolds eliciting pro-inflammatory responses characterized by infiltrating T cells and elevated expression of M1 macrophages markers compared to BMnP functionalized scaffolds which promoted M2 macrophage polarization, tissue vascularization and increased bone volume. Taken together these results demonstrate that nanosized Hydroxyapatite has immunomodulatory potential and is capable of directing antiinflammatory innate immune-mediated responses that are associated with tissue repair and 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.

Incorporating therapeutic genes into 3D biomaterials is a promising strategy for enhancing tissue regeneration. Alginate hydrogels have been extensively investigated for cartilage and bone tissue engineering, including as carriers of transfected cells to sites of injury, making them an ideal gene delivery platform for cartilage and osteochondral tissue engineering.The objective of this study was to develop gene-activated alginate hydrogels capable of supporting nanohydroxyapatite (nHA)-mediated non-viral gene transfer to control the phenotype of mesenchymal stem cells (MSCs) for either cartilage or endochondral bone tissue engineering.To produce these gene-activated constructs, MSCs and nHA complexed with plasmid DNA (pDNA) encoding for TGF-β3 (pTGF-β3), BMP2 (pBMP2), or a combination of both (pTGF-β3/pBMP2), were encapsulated into alginate hydrogels. Initial analysis using reporter genes showed effective gene delivery and sustained overexpression of the transgenes was achieved.Confocal microscopy demonstrated that complexing the plasmid with nHA prior to hydrogel encapsulation led to transport of the plasmid into the nucleus of MSCs, which did not happen with naked pDNA. Gene delivery of TGF-β3 and BMP2 and subsequent cell-mediated expression of these therapeutic genes resulted in a significant increase in sGAG and collagen production, particularly in the pTGF-β3/pBMP2 co-delivery group in comparison to the delivery of either pTGF-β3 or pBMP2 in isolation. In addition, stronger staining for collagen type II deposition was observed in the pTGF-β3/pBMP2 co-delivery group. In contrast, greater levels of calcium deposition were observed in the pTGF-β3 and pBMP2 only groups compared to co-delivery, with strong staining for collagen type X deposition, suggesting these constructs were supporting MSC hypertrophy and progression along an endochondral pathway. Together these results suggest that the developed gene-activated alginate hydrogels were able to support transfection of encapsulated MSCs and directed their phenotype towards either a chondrogenic or osteogenic phenotype depending on whether TGF-β3 and BMP2 were delivered in combination or in isolation.