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While some clinical advances in cartilage repair have occurred, osteochondral (OC) defect repair remains a significant challenge, with current scaffold-based approaches failing to recapitulate the complex, hierarchical structure of native articular cartilage (AC). To address this need, we fabricated bilayered extracellular matrix (ECM)-derived scaffolds with aligned pore architectures. By modifying the freeze-drying kinetics and controlling the direction of heat transfer during freezing, it was possible to produce anisotropic scaffolds with larger pores which supported homogenous cellular infiltration and improved sulfated glycosaminoglycan deposition. Neo-tissue organization in vitro could also be controlled by altering scaffold pore architecture, with collagen fibres aligning parallel to the long-axis of the pores within scaffolds containing aligned pore networks. Furthermore, we used in vitro and in vivo assays to demonstrate that AC and bone ECM derived scaffolds could preferentially direct the differentiation of mesenchymal stromal cells (MSCs) towards either a chondrogenic or osteogenic lineage respectively, enabling the development of bilayered ECM scaffolds capable of spatially supporting unique tissue phenotypes. Finally, we implanted these scaffolds into a large animal model of OC defect repair. After 6 months in vivo, scaffold implantation was found to improve cartilage matrix deposition, with collagen fibres preferentially aligning parallel to the long axis of the scaffold pores, resulting in a repair tissue that structurally and compositionally was more hyaline-like in nature. These results demonstrate how scaffold architecture and composition can be spatially modulated to direct the regeneration of complex interfaces such as the osteochondral unit, enabling their use as cell-free, off-the-shelf implants for joint regeneration. STATEMENT OF SIGNIFICANCE: The architecture of the extracellular matrix, while integral to tissue function, is often neglected in the design and evaluation of regenerative biomaterials. In this study we developed a bilayered scaffold for osteochondral defect repair consisting of tissue-specific extracellular matrix (ECM)-derived biomaterials to spatially direct stem/progenitor cell differentiation, with a tailored pore microarchitecture to promote the development of a repair tissue that recapitulates the hierarchical structure of native AC. The use of this bilayered scaffold resulted in improved tissue repair outcomes in a large animal model, specifically the ability to guide neo-tissue organization and therefore recapitulate key aspects of the zonal structure of native articular cartilage. These bilayer scaffolds have the potential to become a new therapeutic option for osteochondral defect repair.

Black Ti (bTi) surfaces comprising high aspect ratio nanopillars exhibit a rare combination of bactericidal and osteogenic properties, framing them as cell-instructive meta-biomaterials. Despite the existing data indicating that bTi surfaces induce osteogenic differentiation in cells, the mechanisms by which this response is regulated are not fully understood. Here, we hypothesized that high aspect ratio bTi nanopillars regulate cell adhesion, contractility, and nuclear translocation of transcriptional factors, thereby inducing an osteogenic response in the cells. Upon the observation of significant changes in the morphological characteristics, nuclear localization of Yes-associated protein (YAP), and Runt-related transcription factor 2 (Runx2) expression in the human bone marrow-derived mesenchymal stem cells (hMSCs), we inhibited focal adhesion kinase (FAK), Rho-associated protein kinase (ROCK), and YAP in separate experiments to elucidate their effects on the subsequent expression of Runx2. Our findings indicated that the increased expression of Runx2 in the cells residing on the bTi nanopillars compared to the flat Ti is highly dependent on the activity of FAK and ROCK. A mechanotransduction pathway is then postulated in which the FAK-dependent adhesion of cells to the extreme topography of the surface is in close relation with ROCK to increase the endogenous forces within the cells, eventually determining the cell shape and area. The nuclear translocation of YAP may also enhance in response to the changes in cell shape and area, resulting in the translation of mechanical stimuli to biochemical factors such as Runx2.

3D bioprinting has emerged as a promising technology in the field of tissue engineering and regenerative medicine due to its ability to create anatomically complex tissue substitutes. However, it still remains challenging to develop bioactive bioinks that provide appropriate and permissive environments to instruct and guide the regenerative process in vitro and in vivo. In this study alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize an alginate-gelatin methacryloyl (GelMA) interpenetrating network (IPN) bioink to enable the bioprinting of cartilaginous tissues. The inclusion of alginate sulfate had a limited influence on the viscosity, shear-thinning and thixotropic properties of the IPN bioink, enabling high-fidelity bioprinting and supporting mesenchymal stem cell (MSC) viability post-printing. The stiffness of printed IPN constructs greatly exceeded that achieved by printing alginate or GelMA alone, while maintaining resilience and toughness. Furthermore, given the high affinity of alginate sulfate to heparin-binding growth factors, the sulfated IPN bioink supported the sustained release of transforming growth factor-β3 (TGF-β3), providing an environment that supported robust chondrogenesis in vitro, with little evidence of hypertrophy or mineralization over extended culture periods. Such bioprinted constructs also supported chondrogenesis in vivo, with the controlled release of TGF-β3 promoting significantly higher levels of cartilage-specific extracellular matrix deposition. Altogether, these results demonstrate the potential of bioprinting sulfated bioinks as part of a 'single-stage' or 'point-of-care' strategy for regenerating cartilaginous tissues. STATEMENT OF SIGNIFICANCE: This study highlights the potential of using sulfated interpenetrating network (IPN) bioink to support the regeneration of phenotypically stable articular cartilage. Construction of interpenetrating networks in the bioink enables unique high-fidelity bioprinting and provides synergistic increases in mechanical properties. The presence of alginate sulfate enables the capacity of high affinity-binding of TGF-β3, which promoted robust chondrogenesis in vitro and in vivo.

Successful osteochondral defect repair requires regenerating the subchondral bone whilst simultaneously promoting the development of an overlying layer of articular cartilage that is resistant to vascularization and endochondral ossification. During skeletal development articular cartilage also functions as a surface growth plate, which postnatally is replaced by a more spatially complex bone-cartilage interface. Motivated by this developmental process, the hypothesis of this study is that bi-phasic, fibre-reinforced cartilaginous templates can regenerate both the articular cartilage and subchondral bone within osteochondral defects created in caprine joints. To engineer mechanically competent implants, we first compared a range of 3D printed fibre networks (PCL, PLA and PLGA) for their capacity to mechanically reinforce alginate hydrogels whilst simultaneously supporting mesenchymal stem cell (MSC) chondrogenesis in vitro. These mechanically reinforced, MSC-laden alginate hydrogels were then used to engineer the endochondral bone forming phase of bi-phasic osteochondral constructs, with the overlying chondral phase consisting of cartilage tissue engineered using a co-culture of infrapatellar fat pad derived stem/stromal cells (FPSCs) and chondrocytes. Following chondrogenic priming and subcutaneous implantation in nude mice, these bi-phasic cartilaginous constructs were found to support the development of vascularised endochondral bone overlaid by phenotypically stable cartilage. These fibre-reinforced, bi-phasic cartilaginous templates were then evaluated in clinically relevant, large animal (caprine) model of osteochondral defect repair. Although the quality of repair was variable from animal-to-animal, in general more hyaline-like cartilage repair was observed after 6 months in animals treated with bi-phasic constructs compared to animals treated with commercial control scaffolds. This variability in the quality of repair points to the need for further improvements in the design of 3D bioprinted implants for joint regeneration. STATEMENT OF SIGNIFICANCE: Successful osteochondral defect repair requires regenerating the subchondral bone whilst simultaneously promoting the development of an overlying layer of articular cartilage. In this study, we hypothesised that bi-phasic, fibre-reinforced cartilaginous templates could be leveraged to regenerate both the articular cartilage and subchondral bone within osteochondral defects. To this end we used 3D printed fibre networks to mechanically reinforce engineered transient cartilage, which also contained an overlying layer of phenotypically stable cartilage engineered using a co-culture of chondrocytes and stem cells. When chondrogenically primed and implanted into caprine osteochondral defects, these fibre-reinforced bi-phasic cartilaginous grafts were shown to spatially direct tissue development during joint repair. Such developmentally inspired tissue engineering strategies, enabled by advances in biofabrication and 3D printing, could form the basis of new classes of regenerative implants in orthopaedic medicine.

Articular cartilage defects fail to heal spontaneously, typically progressing to osteoarthritis. Bone marrow stimulation techniques such as microfracture (MFX) are the current surgical standard of care; however MFX typically produces an inferior fibro-cartilaginous tissue which provides only temporary symptomatic relief. Here we implanted solubilised articular cartilage extracellular matrix (ECM) derived scaffolds into critically sized chondral defects in goats, securely anchoring these implants to the joint surface using a 3D-printed fixation device that overcame the need for sutures or glues.

3D bioprinting is usually implemented on flat surfaces, posing serious limitations in the fabrication of multilayered curved constructs. 4D bioprinting, combining 3D bioprinting with time-dependent stimuli-induced transformation, enables the fabrication of shape-changing constructs. Here, a 4D biofabrication method is reported for cartilage engineering based on the differential swelling of a smart multi-material system made from two hydrogel-based materials: hyaluronan and alginate. Two ink formulations are used: tyramine-functionalized hyaluronan (HAT, high-swelling) and alginate with HAT (AHAT, low-swelling). Both inks have similar elastic, shear-thinning, and printability behavior. The inks are 3D printed into a bilayered scaffold before triggering the shape-change by using liquid immersion as stimulus. In time (4D), the differential swelling between the two zones leads to the scaffold's self-bending. Different designs are made to tune the radius of curvature and shape. A bioprinted formulation of AHAT and human bone marrow cells demonstrates high cell viability. After 28 days in chondrogenic medium, the curvature is clearly present while cartilage-like matrix production is visible on histology. A proof-of-concept of the recently emerged technology of 4D bioprinting with a specific application for the design of curved structures potentially mimicking the curvature and multilayer cellular nature of native cartilage is demonstrated.

Bone-to-soft tissue interfaces are responsible for transferring loads between tissues with significantly dissimilar material properties. The examples of connective soft tissues are ligaments, tendons, and cartilages. Such natural tissue interfaces have unique microstructural properties and characteristics which avoid the abrupt transitions between two tissues and prevent formation of stress concentration at their connections. Here, we review some of the important characteristics of these natural interfaces. The native bone-to-soft tissue interfaces consist of several hierarchical levels which are formed in a highly specialized anisotropic fashion and are composed of different types of heterogeneously distributed cells. The characteristics of a natural interface can rely on two main design principles, namely by changing the local microarchitectural features (e.g., complex cell arrangements, and introducing interlocking mechanisms at the interfaces through various geometrical designs) and changing the local chemical compositions (e.g., a smooth and gradual transition in the level of mineralization). Implementing such design principles appears to be a promising approach that can be used in the design, reconstruction, and regeneration of engineered biomimetic tissue interfaces. Furthermore, prominent fabrication techniques such as additive manufacturing (AM) including 3D printing and electrospinning can be used to ease these implementation processes. Biomimetic interfaces have several biological applications, for example, to create synthetic scaffolds for osteochondral tissue repair.

Successfully treating osteochondral defects involves regenerating both the damaged articular cartilage and the underlying subchondral bone, in addition to the complex interface that separates these tissues. In this study, we demonstrate that a cartilage template, engineered using bone marrow-derived mesenchymal stem cells, can enhance the regeneration of such defects and promote the development of a more mechanically functional repair tissue. We also use a computational mechanobiological model to understand how joint-specific environmental factors, specifically oxygen levels and tissue strains, regulate the conversion of the engineered template into cartilage and bone in vivo.

Extracellular matrix (ECM)-derived implants hold great promise for tissue repair, but new strategies are required to produce efficiently decellularized scaffolds with the necessary porosity and mechanical properties to facilitate regeneration. In this study, we demonstrate that it is possible to produce highly porous, elastic, articular cartilage (AC) ECM-derived scaffolds that are efficiently decellularized, nonimmunogenic, and chondro-permissive. Pepsin solubilized porcine AC was cross-linked with glyoxal, lyophilized and then subjected to dehydrothermal treatment. The resulting scaffolds were predominantly collagenous in nature, with the majority of sulphated glycosaminoglycan (sGAG) and DNA removed during scaffold fabrication. Four scaffold variants were produced to examine the effect of both ECM (10 or 20 mg/mL) and glyoxal (5 or 10 mM) concentration on the mechanical and biological properties of the resulting construct. When seeded with human infrapatellar fat pad-derived stromal cells, the scaffolds with the lowest concentration of both ECM and glyoxal were found to promote the development of a more hyaline-like cartilage tissue, as evident by increased sGAG and type II collagen deposition. Furthermore, when cultured in the presence of human macrophages, it was found that these ECM-derived scaffolds did not induce the production of key proinflammatory cytokines, which is critical to success of an implantable biomaterial. Together these findings demonstrate that the novel combination of solubilized AC ECM and glyoxal crosslinking can be used to produce highly porous scaffolds that are sufficiently decellularized, highly elastic, chondro-permissive and do not illicit a detrimental immune response when cultured in the presence of human macrophages.