Hydrogels are commonly employed as soft polymeric scaffolds to encapsulate cells and spheroids in organ-on-chip models. Although the viscoelasticity of hydrogels can have a significant effect on cell behavior, this aspect remains underexplored in organ-on-chip systems. In this study, we investigated the effect of hydrogel viscoelasticity on Human Mesenchymal Stem Cell (hMSC) spheroids for application in bone-on-chip systems.

To this end, alginate-based hydrogels were engineered with varied viscoelastic profiles (fast-relaxing vs. slow-relaxing).  hMSC spheroids were encapsulated in these hydrogels and cultured for up to 21 days in growth or osteogenic media. Osteogenic differentiation was assessed by quantifying cellular alkaline phosphatase (ALP) activity and calcium deposition. Additionally, cytological staining and microscopic imaging were carried out using bone-specific stains.

Our results revealed differences in cell behavior and mineralization at the system versus (sub-)spheroid levels. At the system level, the total ALP activity was mainly affected by the type of cell culture medium used. However, at the (sub-)spheroid level, hydrogel viscoelasticity had a significant impact on cell migration and localization of mineralization.  To investigate the localization of mineralization, the alizarin red (calcium) and hematoxylin (cell) stains were digitally deconvoluted, and their stain intensities were quantified as a function of distance from the spheroid center.

These quantifications revealed that the localization of mineralization was more pronounced in spheroids encapsulated in fast-relaxing than those in slow-relaxing gels, while no significant differences between the localization in growth vs. osteogenic medium were observed. Overall, our findings indicate that viscoelasticity may play a more important role than biochemical cues in the media in terms of localized mineralization of spheroids. These results can have significant implications for in vitro bone tissue models such as bone-on-chip systems.