In situ cross-linkable hybrid hydrogels composed of gelatin and 4-arm-polypropylene oxide-polyethylene oxide (Tetronic) was developed as an injectable scaffold for tissue regeneration. The gelatin was modified by hydroxyphenyl propionic acid (HPA) and the Tetronic was conjugated with tyramines (Tet-TA). The hydrogels were rapidly formed by mixing the polymer solutions containing horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). The gelation time and mechanical properties of the hydrogels could be controlled by varying the HRP and H(2)O(2) concentrations. In vitro degradation study of the hybrid hydrogels was carried out using collagenase and the prolonged proteolytic degradation was obtained due to the presence of the Tetronic. Human dermal fibroblast (hDFB) was cultured in the hydrogel matrices to evaluate the cyto-compatibility. The encapsulated cells were shown to be highly viable and spread over the gel matrices, suggesting that the hybrid hydrogels have an excellent cyto-compatibility. The hydrogels were also subcutaneously injected in the back of mice and the results demonstrated that the hydrogels were rapidly formed at the injected site. From these results, we demonstrate that the in situ cross-linkable hydrogels formed by hybridization of gelatin and Tetronic via enzyme-mediated reactions hold great promise for use as injectable matrices for tissue regenerative medicine due to their tunable physico-chemical properties and excellent bioactivity.