Introduction/Objectives This study aims to develop a tissue-responsive hydrogel scaffold to support skin tissue regeneration by comparing different crosslinkers, including a matrix-metalloproteinase (MMP)-sensitive crosslinker which facilitates cell-matrix remodeling. The different materials produced were characterized in an acellular and cellular context, aiming to enhance cell viability and proliferation in tissue regeneration applications. Methods Norbornene-functionalized polyvinyl alcohol (PVA-NB) was used as the hydrogel precursor and was rapidly crosslinked under UV-light (<60 s) with cell-cleavable and non-cleavable crosslinkers, such as an MMP sequence flanked by two cysteines, and a dithiolated Polyethylene glycol (HS-PEG-SH), respectively, in presence of 0.1 wt% of Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The final formulation concentration of PVA-NB was 5 wt%, and 50% of the norbornene groups were used for the crosslinking. A thiolated cell-adhesion promoting peptide sequence including the Arginine-Glycine-Aspartic Acid (RGD) motif was also incorporated in the hydrogels. The produced matrices were characterized in terms of swelling, long-term stability, degradation kinetics and photorheology. After all conditions were studied and optimized, fibroblasts were encapsulated within the matrix and the viability, adhesion and cell-matrix interactions were evaluated. Results A dithiolated PEG with a similar molecular weight to the MMP was used to better understand and compare the effects inflicted upon the material by the different crosslinkers. The two different hydrogels exhibited similar photorheological behavior, such as reactivity and storage modulus of approximately 2 kPa. Their swelling behavior was also very similar as well as their stability over time under physiological conditions, where the hydrogels were not dissolved after 45 days. However, a different trend was observed after investigation of their accelerated enzymatic degradation kinetics using collagenase. In fact, the PVA-MMP hydrogels had degraded within 24 hours whereas the non-cleavable PVA-PEG hydrogels were only 20% degraded after 5 days. Immortalized adult dermal fibroblasts were mixed in the formulation to enable observation of the cell-matrix interactions. The viability remained high after 14 days, proving that the matrices exhibit high biocompatibility. Further microscopy observation after 2 weeks in culture revealed that the cells had longer protrusions and a diversity of morphologies in the PVA-MMP hydrogel, whereas cells remained rounded when encapsulated in the PVA-PEG hydrogels, despite the presence of RGD in both formulations. Conclusions The cell-responsive hydrogel platform developed in this study provides a dynamic environment for tissue regeneration. This cleavable design is well-suited for clinical applications, as it provides mechanical integrity and the capacity for cellular remodeling. Furthermore, the material’s adaptability to numerous formats, including electrospinning, granular hydrogels, light-based high-resolution techniques (Two-photon polymerization or volumetric printing) and injectable hydrogels expands its potential utility in future regenerative therapies. Acknowledgements Christian Doppler Research Association, Austrian Federal Ministry for Digital & Economic Affairs, National foundation for Research, Technology & Development. Disclosure of interest: None declared