Despite numerous advances in the field of tissue engineering and regenerative medicine, monitoring the formation of tissue regeneration and its metabolic variations during culture is still a challenge and mostly limited to bulk volumetric assays. Here, a simple method of adding capsules‐based optical sensors in cell‐seeded 3D scaffolds is presented and the potential of these sensors to monitor the pH changes in space and time during cell growth is demonstrated. It is shown that the pH decreased over time in the 3D scaffolds, with a more prominent decrease at the edges of the scaffolds. Moreover, the pH change is higher in 3D scaffolds compared to monolayered 2D cell cultures. The results suggest that this system, composed by capsules‐based optical sensors and 3D scaffolds with predefined geometry and pore architecture network, can be a suitable platform for monitoring pH variations during 3D cell growth and tissue formation. This is particularly relevant for the investigation of 3D cellular microenvironment alterations occurring both during physiological processes, such as tissue regeneration, and pathological processes, such as cancer evolution.

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The actin cytoskeleton plays a key role in differentiation of human mesenchymal stromal cells (hMSCs), but its regulation in 3D tissue engineered scaffolds remains poorly studied. hMSCs cultured on 3D electrospun scaffolds made of a stiff material do not form actin stress fibers, contrary to hMSCs on 2D films of the same material. On 3D electrospun-and 3D additive manufactured scaffolds, hMSCs also displayed fewer focal adhesions, lower lamin A and C expression and less YAP1 nuclear localization. Together, this shows that dimensionality prevents the build-up of cellular tension, even on stiff materials. Knock down of either lamin A and C or zyxin resulted in fewer stress fibers in the cell center. Zyxin knock down reduced lamin A and C expression, but not vice versa, showing that this signal chain starts from the outside of the cell. Our study demonstrates that dimensionality changes the actin cytoskeleton through lamin A and C and zyxin, an important insight for future scaffold design, as the actin network, focal adhesions and nuclear stiffness are all critical for hMSC differentiation.

Stanniocalcin-1 (STC1) secreted by mesenchymal stromal cells (MSCs) has antiinflammatory functions, reduces apoptosis, and aids in angiogenesis, both in vitro and in vivo. However, little is known about the molecular mechanisms of its regulation. Here, we show that STC1 secretion is increased only under specific cell-stress conditions. We find that this is due to a change in actin stress fibers and actin-myosin tension. Abolishment of stress fibers by blebbistatin and knockdown of the focal adhesion protein zyxin leads to an increase in STC1 secretion. To also study this connection in 3D, where few focal adhesions and actin stress fibers are present, STC1 expression was analyzed in 3D alginate hydrogels and 3D electrospun scaffolds. Indeed, STC1 secretion was increased in these low cellular tension 3D environments. Together, our data show that STC1 does not directly respond to cell stress, but that it is regulated through mechanotransduction. This research takes a step forward in the fundamental understanding of STC1 regulation and can have implications for cell-based regenerative medicine, where cell survival, anti-inflammatory factors, and angiogenesis are critical.