The use of allografts for the treatment of critical size cartilage defects holds disadvantages including the limited number of donors and the compromised chondrocyte viability. These limitations have prompted researchers to explore different options, such as cartilage engineering. Bioprinting, a promising tissue engineering technique, could be used to fabricate biomimetic constructs that can potentially replace allografts. The present study explores the generation of a biomimetic chondrocyte density gradient in full-thickness bioprinted Alginate/NFC scaffolds with a PCL framework and investigates the effect of this zonal distribution of cells on the production of cartilage matrix within the scaffolds. To this end, two types of scaffolds were bioprinted; one with a graded three-zone distribution of cells (bottom zone: 5×106 cells/ml, middle zone: 10×106 cells/ml, top zone: 20×106 cells/ml), and another with a homogeneous cell density (10×106 cells/ml) and cultured for 25 days. Furthermore, the mechanical properties of the multi-material scaffolds were evaluated. The results showed that a three-zone cell density gradient could be achieved using extrusion-based bioprinting. The gradient was maintained for 25 days in scaffolds cultured in non-chondrogenic media (absence of ascorbic acid) but was transformed into a two-zone gradient within the first 14 days, in cultures with chondrogenic media (presence of ascorbic acid in the medium), and maintained as such until the end of the experiment. The zonal distribution of cells led to a zonal distribution of sGAGs, after 14 days of culture, with the sGAGs deposition being increased in the areas of high cell density. However, there was no significant Collagen deposition within the scaffolds at any time point during the experiment. This study attempts to shed light into one of the gradients of the native cartilage tissue (cell density gradient), with the hope of contributing to the development of a biomimetic fully functional engineered cartilage scaffold in the future.

Three-dimensional (3D) bioprinting studies the initial condition of printed constructs, which makes construct static, and neglects the dynamic changes in natural tissue conformation during tissue repair, disease progression and regeneration processes. Therefore, the concept of four-dimensional (4D) bioprinting has been introduced that allows for the fabrication of scaffolds that can transform in time. The extracellular matrix stiffness is one component involved in the in vivo dynamic microenvironment of tissues such as cartilage. Nonetheless, the implementation of dynamic stiffness in in vitro 3D cell culture has not been intensively investigated yet, in particular not in 4D bioprinting for cartilage applications. Therefore, the aim of this study is to create a method for the temporal stiffening of a human mesenchymal stem cell-laden scaffold in 4D bioprinting for cartilage applications. To do this, inks made of tyramine-modified hyaluronic acid (THA), and THA combined with sodium alginate (SA) (THA-SA) were studied on their ability to stiffen days after being printed through photocrosslinking or ionic crosslinking (secondary crosslinking). The method that generated the greatest increase in stiffness was evaluated on the effect of time on stiffening and the effect of stiffening on the swelling behaviour and the internal structure of the scaffold. Next, the 4D printing method was translated to 4D bioprinting and the influence of the temporal stiffening method on the mechanical properties, cell viability, gene expression and matrix deposition was determined. This study demonstrated that in 4D printing, secondary photocrosslinking of THA and secondary ionic crosslinking of THA-SA resulted into scaffolds with a significant increase in stiffness. The method for secondary ionic crosslinking of THA-SA (2.5% w/v THA, 1.5% w/v SA) scaffolds achieved the largest increase in stiffness and was able to temporally stiffen at day 1, 3 or 5. This increase in stiffness was associated with a decrease in swelling ratio and a maintained internal microstructure. For the translation to 4D bioprinting, the method was adjusted for cell culture conditions and resulted in an increase in stiffness. However, the stiffening was not yet reproducible and therefore optimalization of the method will be required. Nevertheless, in the first week of cell culture, the bioprinted scaffolds achieved high cell viability and an equal cell distribution. At day 7, the gene for collagen type II was not expressed and cartilage-specific matrix deposition was not detected. With future studies focusing on optimizing the introduced 4D bioprinting protocol, this novel method for the ionic secondary crosslinking of THA-SA may function as a platform for studying the effect of temporal matrix stiffening on the behaviour of the embedded cells in fundamental research or as a stimulus for tissue formation in tissue engineering.

Bioprinting is a promising technique that has the ability to generate complex tissue structures for articular cartilage (AC) repair and regeneration. However, most biomaterials used for bioprinting are incapable of representing the complexity of native extracellular matrix (ECM). In this study, a novel cartilage specific bioink has been developed with tyramine modified hyaluronic acid (THA) and decellularized ECM (dECM) of AC for threedimensional bioprinting, combining the printable qualities of THA with AC dECM to create an optimized microenvironment. First, the influence of increasing concentrations of AC dECM on the rheological and printability properties was assessed. Next, human mesenchymal stem cells (hMSCs) were incorporated into bioinks composed of 2.5% w/v THA and AC dECM (0.0%-1.0% w/v). The THA-AC dECM bioinks have been successfully bioprinted into stable scaffolds with viable cells and were cultured for 28 days in chondrogenic differentiation medium. Subsequently, the effect of combining AC dECM with THA was evaluated by measuring the cell viability, cell differentiation and AC tissue deposition. The ink compositions with AC dECM (0.5% and 1.0% w/v) caused an increase in ink viscosity, yield stress and damping factor compared to the THA ink, but still showed shear thinning behavior and provided good printability. Spread cell morphology was observed for the scaffolds with addition of AC dECM compared to AC dECM free control with only THA, indicating enhanced cell adhesion to the hydrogel network. Moreover, a higher production of sulfated glycosaminoglycans (sGAGs) was observed for the scaffolds where AC dECM was added. The THA and AC dECM bioink composition showed promising results for AC tissue engineering and warrants further research into the development of bioinks with tissue-specific inductive properties.