Bioink Platform Utilizing Dual‐Stage Crosslinking of Hyaluronic Acid Tailored for Chondrogenic Differentiation of Mesenchymal Stromal Cells

3D bioprinting often involves application of highly concentrated polymeric bioinks to enable fabrication of stable cell‐hydrogel constructs, although poor cell survival, compromised stem cell differentiation, and an inhomogeneous distribution of newly produced extracellular matrix (ECM) are frequently observed. Therefore, this study presents a bioink platform using a new versatile dual‐stage crosslinking approach based on thiolated hyaluronic acid (HA‐SH), which not only provides stand‐alone 3D printability but also facilitates effective chondrogenic differentiation of mesenchymal stromal cells. A range of HA‐SH with different molecular weights is synthesized and crosslinked with acrylated (PEG‐diacryl) and allylated (PEG‐diallyl) polyethylene glycol in a two‐step reaction scheme. The initial Michael addition is used to achieve ink printability, followed by UV‐mediated thiol–ene reaction to stabilize the printed bioink for long‐term cell culture. Bioinks with high molecular weight HA‐SH (>200 kDa) require comparably low polymer content to facilitate bioprinting. This leads to superior quality of cartilaginous constructs which possess a coherent ECM and a strongly increased stiffness of long‐term cultured constructs. The dual‐stage system may serve as an example to design platforms using two independent crosslinking reactions at one functional group, which allows adjusting printability as well as material and biological properties of bioinks. A versatile bioink platform based on hyaluronic acid is designed for cartilage biofabrication. The novel dual‐stage crosslinking approach allows adjustment of bioink 3D printability and material properties of the final constructs. Selected bioink compositions are used for long‐term cell culture and support differentiation of human mesenchymal stromal cells and the distribution of newly produced extracellular matrix.