hBM-MSC-Laden 3D Bioprinted Gelatin–Alginate Hydrogels: Physicochemical Characterisation and Osteogenic Lineage Commitment

Gelatin–alginate composite hydrogels are some of the most prevalent bioinks used for extrusion-based three-dimensional (3D) bioprinting because of their combined bioactivity and ability to ionically crosslink. Ionically crosslinked gelatin–alginate constructs containing human bone marrow–derived mesenchymal stem cells (hBM-MSCs) were characterised over time under standardised in vitro conditions to assess physicochemical properties and resultant cell behaviour. Water uptake and degradation were quantified over time in phosphate-buffered saline (PBS) and collagenase type II media for up to 21 days. Cell viability and metabolic activity were quantified, and osteogenic gene expression (RUNX2, COL1A1, OCN) was assessed. Raman spectroscopy and compressive mechanical characterisation were performed. Collagen and glycosaminoglycan-related peaks were observed from extracellular matrix (ECM)-associated components, with an increased presence of protein-associated signatures later in culture. Hydrogels displayed nonlinear elastic behaviour with increased stress after longer incubation times, suggesting no degradation of mechanical integrity over the duration of the study. Hydrogels experienced rapid hydration followed by decreased swelling over time, with a maximum swelling ratio at 24 h. Degradation rates significantly increased over longer incubation times (p < 0.001) and in collagenase media compared to PBS (p < 0.001). Observed differences were likely due to both ion-exchange-mediated network disassembly and the dissolution of gelatin components. Cell metabolic activity decreased under osteogenic culture conditions, while changes in osteogenic marker expression were sequential, suggesting a transition from proliferation to early osteogenic commitment in this 3D system. This work provides both physicochemical and biological characterisation of a commonly utilised gelatin–alginate bioink system, to provide future optimisations within the field of extrusion-based bone tissue engineering, a reproducible baseline for future optimisation of bioink systems in extrusion-based bone tissue engineering.