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Tissue engineering approaches that recapitulate cartilage biomechanical properties are emerging as promising methods to restore the function of injured or degenerated tissue. However, despite significant progress in this research area, the generation of engineered cartilage constructs akin to native counterparts still represents an unmet challenge. In particular, the inability to accurately reproduce cartilage zonal architecture with different collagen fibril orientations is a significant limitation. The arrangement of the extracellular matrix (ECM) plays a fundamental role in determining the mechanical and biological functions of the tissue. In this study, it is shown that a novel light-based approach, Filamented Light (FLight) biofabrication, can be used to generate highly porous, 3D cell-instructive anisotropic constructs that lead to directional collagen deposition. Using a photoclick-based photoresin optimized for cartilage tissue engineering, a significantly improved maturation of the cartilaginous tissues with zonal architecture and remarkable native-like mechanical properties is demonstrated.

Microtia is a congenital disorder that manifests as a malformation of the external ear leading to psychosocial problems in affected children. Here, we present a tissue-engineered treatment approach based on a bioprinted autologous auricular cartilage construct (EarCartilage) combined with a bioengineered human pigmented and prevascularized dermo-epidermal skin substitute (EarSkin) tested in immunocompromised rats. We confirmed that human-engineered blood capillaries of EarSkin connected to the recipient’s vasculature within 1 week, enabling rapid blood perfusion and epidermal maturation. Bioengineered EarSkin displayed a stratified epidermis containing mature keratinocytes and melanocytes. The latter resided within the basal layer of the epidermis and efficiently restored the skin color. Further, in vivo tests demonstrated favorable mechanical stability of EarCartilage along with enhanced extracellular matrix deposition. In conclusion, EarCartilage combined with EarSkin represents a novel approach for the treatment of microtia with the potential to circumvent existing limitations and improve the aesthetic outcome of microtia reconstruction.

Achieving reproducibility in the 3D printing of biomaterials requires a robust polymer synthesis method to reduce batch-to-batch variation as well as methods to assure a thorough characterization throughout the manufacturing process. Particularly biomaterial inks containing large solid fractions such as ceramic particles, often required for bone tissue engineering applications, are prone to inhomogeneity originating from inadequate mixing or particle aggregation which can lead to inconsistent printing results. The production of such an ink for bone tissue engineering consisting of gellan gum methacrylate (GG-MA), hyaluronic acid methacrylate and hydroxyapatite (HAp) particles was therefore optimized in terms of GG-MA synthesis and ink preparation process, and the ink's printability was thoroughly characterized to assure homogeneous and reproducible printing results. A new buffer mediated synthesis method for GG-MA resulted in consistent degrees of substitution which allowed the creation of large 5 g batches. We found that both the new synthesis as well as cryomilling of the polymer components of the ink resulted in a decrease in viscosity from 113 kPa·s to 11.3 kPa·s at a shear rate of 0.1 s−1 but increased ink homogeneity. The ink homogeneity was assessed through thermogravimetric analysis and a newly developed extrusion force measurement setup. The ink displayed strong inter-layer adhesion between two printed ink layers as well as between a layer of ink with and a layer without HAp. The large polymer batch production along with the characterization of the ink during the manufacturing process allows ink production in the gram scale and could be used in applications such as the printing of osteochondral grafts.

Biofabrication techniques including three-dimensional bioprinting could be used one day to fabricate living, patient-specific tissues and organs for use in regenerative medicine. Compared to traditional casting and molding methods, bioprinted structures can be much more complex, containing for example multiple materials and cell types in controlled spatial arrangement, engineered porosity, reinforcement structures and gradients in mechanical properties. With this complexity and increased function, however, comes the necessity to develop guidelines to standardize the bioprinting process, so printed grafts can safely enter the clinics. The bioink material must firstly fulfil requirements for biocompatibility and flow. Secondly, it is important to understand how process parameters affect the final mechanical properties of the printed graft. Using a gellan-alginate physically crosslinked bioink as an example, we show shear thinning and shear recovery properties which allow good printing resolution. Printed tensile specimens were used to systematically assess effect of line spacing, printing direction and crosslinking conditions. This standardized testing allowed direct comparison between this bioink and three commercially-available products. Bioprinting is a promising, yet complex fabrication method whose outcome is sensitive to a range of process parameters. This study provides the foundation for highly needed best practice guidelines for reproducible and safe bioprinted grafts.

Biofabrication techniques including three-dimensional bioprinting could be used one day to fabricate living, patient-specific tissues and organs for use in regenerative medicine. Compared to traditional casting and molding methods, bioprinted structures can be much more complex, containing for example multiple materials and cell types in controlled spatial arrangement, engineered porosity, reinforcement structures and gradients in mechanical properties. With this complexity and increased function, however, comes the necessity to develop guidelines to standardize the bioprinting process, so printed grafts can safely enter the clinics. The bioink material must firstly fulfil requirements for biocompatibility and flow. Secondly, it is important to understand how process parameters affect the final mechanical properties of the printed graft. Using a gellan-alginate physically crosslinked bioink as an example, we show shear thinning and shear recovery properties which allow good printing resolution. Printed tensile specimens were used to systematically assess effect of line spacing, printing direction and crosslinking conditions. This standardized testing allowed direct comparison between this bioink and three commercially-available products. Bioprinting is a promising, yet complex fabrication method whose outcome is sensitive to a range of process parameters. This study provides the foundation for highly needed best practice guidelines for reproducible and safe bioprinted grafts.