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, fiber-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 fiber-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.

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.

Individual cells and multicellular systems respond to cell-scale curvatures in their environments, guiding migration, orientation, and tissue formation. However, it remains largely unclear how cells collectively explore and pattern complex landscapes with curvature gradients across the Euclidean and non-Euclidean spectra. Here, we show that mathematically designed substrates with controlled curvature variations induce multicellular spatiotemporal organization of preosteoblasts. We quantify curvature-induced patterning and find that cells generally prefer regions with at least one negative principal curvature. However, we also show that the developing tissue can eventually cover unfavorably curved territories, can bridge large portions of the substrates, and is often characterized by collectively aligned stress fibers. We demonstrate that this is partly regulated by cellular contractility and extracellular matrix development, underscoring the mechanical nature of curvature guidance. Our findings offer a geometric perspective on cell-environment interactions that could be harnessed in tissue engineering and regenerative medicine applications.

The existing 3D printing methods exhibit certain fabrication-dependent limitations for printing curved constructs that are relevant for many tissues. Four-dimensional (4D) printing is an emerging technology that is expected to revolutionize the field of tissue engineering and regenerative medicine (TERM). 4D printing is based on 3D printing, featuring the introduction of time as the fourth dimension, in which there is a transition from a 3D printed scaffold to a new, distinct, and stable state, upon the application of one or more stimuli. Here, we present an overview of the current developments of the 4D printing technology for TERM, with a focus on approaches to achieve temporal changes of the shape of the printed constructs that would enable biofabrication of highly complex structures. To this aim, the printing methods, types of stimuli, shape-shifting mechanisms, and cell-incorporation strategies are critically reviewed. Furthermore, the challenges of this very recent biofabrication technology as well as the future research directions are discussed. Our findings show that the most common printing methods so far are stereolithography (SLA) and extrusion bioprinting, followed by fused deposition modelling, while the shape-shifting mechanisms used for TERM applications are shape-memory and differential swelling for 4D printing and 4D bioprinting, respectively. For shape-memory mechanism, there is a high prevalence of synthetic materials, such as polylactic acid (PLA), poly(glycerol dodecanoate) acrylate (PGDA), or polyurethanes. On the other hand, different acrylate combinations of alginate, hyaluronan, or gelatin have been used for differential swelling-based 4D transformations. TERM applications include bone, vascular, and cardiac tissues as the main target of the 4D (bio)printing technology. The field has great potential for further development by considering the combination of multiple stimuli, the use of a wider range of 4D techniques, and the implementation of computational-assisted strategies.

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.

The treatment of articular cartilage defects remains a significant clinical challenge. This is partially due to current tissue engineering strategies failing to recapitulate native organization. Articular cartilage is a graded tissue with three layers exhibiting different cell densities: the superficial zone having the highest density and the deep zone having the lowest density. However, the introduction of cell gradients for cartilage tissue engineering, which could promote a more biomimetic environment, has not been widely explored. Here, we aimed to bioprint a scaffold with different zonal cell densities to mimic the organization of articular cartilage. The scaffold was bioprinted using an alginate-based bioink containing human articular chondrocytes. The scaffold design included three cell densities, one per zone: 20 × 10⁶ (superficial), 10 × 10⁶ (middle), and 5 × 10⁶ (deep) cells/mL. The scaffold was cultured in a chondrogenic medium for 25 days and analyzed by live/dead assay and histology. The live/dead analysis showed the ability to generate a zonal cell density with high viability. Histological analysis revealed a smooth transition between the zones in terms of cell distribution and a higher sulphated glycosaminoglycan deposition in the highest cell density zone. These findings pave the way toward bioprinting complex zonal cartilage scaffolds as single units, thereby advancing the translation of cartilage tissue engineering into clinical practice.