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Many different biofabrication approaches as well as a variety of bioinks have been developed by researchers working in the field of tissue engineering. A main challenge for bioinks often remains the difficulty to achieve shape fidelity after printing. In order to overcome this issue, a homogeneous pre-crosslinking technique, which is universally applicable to all alginate-based materials, was developed. In this study, the Young’s Modulus after post-crosslinking of selected hydrogels, as well as the chemical characterization of alginate in terms of M/G ratio and molecular weight, were determined. With our technique it was possible to markedly enhance the printability of a 2% (w/v) alginate solution, without using a higher polymer content, fillers or support structures. 3D porous scaffolds with a height of around 5 mm were printed. Furthermore, the rheological behavior of different pre-crosslinking degrees was studied. Shear forces on cells as well as the flow profile of the bioink inside the printing nozzle during the process were estimated. A high cell viability of printed NIH/3T3 cells embedded in the novel bioink of more than 85% over a time period of two weeks could be observed.
In 3D bioprinting for cartilage regeneration, bioinks that support chondrogenic development are of key importance. Growth factors covalently bound in non-printable hydrogels have been shown to effectively promote chondrogenesis. However, studies that investigate the functionality of tethered growth factors within 3D printable bioinks are still lacking. Therefore, in this study, we established a dual-stage crosslinked hyaluronic acid-based bioink that enabled covalent tethering of transforming growth factor-beta 1 (TGF-β1). Bone marrow-derived mesenchymal stromal cells (MSCs) were cultured over three weeks in vitro, and chondrogenic differentiation of MSCs within bioink constructs with tethered TGF-β1 was markedly enhanced, as compared to constructs with non-covalently incorporated TGF-β1. This was substantiated with regard to early TGF-β1 signaling, chondrogenic gene expression, qualitative and quantitative ECM deposition and distribution, and resulting construct stiffness. Furthermore, it was successfully demonstrated, in a comparative analysis of cast and printed bioinks, that covalently tethered TGF-β1 maintained its functionality after 3D printing. Taken together, the presented ink composition enabled the generation of high-quality cartilaginous tissues without the need for continuous exogenous growth factor supply and, thus, bears great potential for future investigation towards cartilage regeneration. Furthermore, growth factor tethering within bioinks, potentially leading to superior tissue development, may also be explored for other biofabrication applications.