Three-dimensional printing of cell-laden hydrogels has evolved as a promising approach on the route to patient-specific or complex tissue-engineered constructs. However, it is still challenging to print structures with both, high shape fidelity and cell vitality. Herein, we used a synthetic nanosilicate clay, called Laponite, to build up scaffolds utilising the extrusion-based method 3D plotting. By blending with alginate and methylcellulose, a bioink was developed which allowed easy extrusion, achieving scaffolds with high printing fidelity. Following extrusion, approximately 70%-75% of printed immortalised human mesenchymal stem cells survived and cell viability was maintained over 21 days within the plotted constructs. Mechanical properties of scaffolds comprised of the composite bioink decreased over time when stored under cell culture conditions. Nevertheless, shape of the plotted constructs was preserved even over longer cultivation periods. Laponite is known for its favourable drug delivery properties. Two model proteins, bovine serum albumin and vascular endothelial growth factor were loaded into the bioink. We demonstrate that the release of both growth factors significantly changed to a more sustained profile by inclusion of Laponite in comparison to an alginate-methylcellulose blend in the absence of Laponite. In summary, addition of a synthetic clay, Laponite, improved printability, increased shape fidelity and was beneficial for controlled release of biologically active agents such as growth factors.

Due to their characteristic resemblance of the mineral component of bone, calcium phosphates are widely accepted as optimal bone substitute materials. Recent research focused on the development of pasty calcium phosphate cement (CPC) formulations, which can be fabricated into various shapes by low-temperature extrusion-based additive manufacturing, namely 3D plotting. While it could be demonstrated that sensitive substances like growth factors can be integrated in such printed CPC scaffolds without impairment of their biological activity live cells cannot be suspended in CPC as they may not be functional when enclosed in a solid and stiff matrix. In contrast, 3D bioprinting of soft cell-laden hydrogels (bioinks) enables the fabrication of constructs with spatially defined cell distribution, which has the potential to overcome problems of conventional cell seeding techniques-but such objects lack mechanical stability. Herein, we combine 3D plotting of CPC and bioprinting of a cell-laden bioink for the first time. As model bioink, an alginate-methylcellulose blend (alg/mc) was used, previously developed by us. Firstly, a fabrication regime was established, enabling optimal setting of CPC and cell survival inside the bioink. As the cells are exposed to the chemical changes of CPC precursors during setting, we studied the compatibility of the complex system of CPC and cell-laden alg/mc for a combined extrusion process and characterized the cellular behavior of encapsulated human mesenchymal stroma cells within the bioink at the interface and in direct vicinity to the CPC. Furthermore, biphasic scaffolds were mechanically characterized and a model for osteochondral tissue grafts is proposed. The manuscript discusses possible impacts of the CPC setting reaction on cells within the bioink and illustrates the advantages of CPC in bioprinting as alternative to the commonly used thermoplasts for bone tissue engineering.

For the generation of multi-layered full thickness osteochondral tissue substitutes with an individual geometry based on clinical imaging data, combined extrusion-based 3D printing (3D plotting) of a bioink laden with primary chondrocytes and a mineralized biomaterial phase was introduced. A pasty calcium phosphate cement (CPC) and a bioink based on alginate-methylcellulose (algMC) – both are biocompatible and allow 3D plotting with high shape fidelity – were applied in monophasic and combinatory design to recreate osteochondral tissue layers. The capability of cells reacting to chondrogenic biochemical stimuli inside the algMC-based 3D hydrogel matrix was assessed. Towards combined osteochondral constructs, the chondrogenic fate in the presence of CPC in co-fabricated and biphasic mineralized pattern was evaluated. Majority of expanded and algMC-encapsulated cells survived the plotting process and the cultivation period, and were able to undergo redifferentiation in the provided environment to produce their respective extracellular matrix (ECM) components (i.e. sulphated glycosaminoglycans, collagen type II), examined after 3 weeks. The presence of a mineralized zone as located in the physiological calcified cartilage region suspected to interfere with chondrogenesis, was found to support chondrogenic ECM production by altering the ionic concentrations of calcium and phosphorus in in vitro culture conditions.