OBJECTIVES:Gelatin, a degraded collagen, has been widely used as a scaffolding material in tissue engineering applications. In this work, we aimed at the development of in situ, cross-linking, cytocompatible hydrogels by the use of transglutaminase as a cross-linker for potential application in the regeneration of tissues. METHODS:Hydrogels were prepared from gelatin of different concentrations and bloom degree (175 (G175) or 300 (G300) bloom gelatin) and cross-linked with various amounts of microbial transglutaminase (mTG) at 37°C. Mechanical properties and cross-linking degree were studied by rheology and swelling experiments. Four hydrogels with different stiffness were selected for studies with embedded human adipose-derived stem cells (hASCs). RESULTS:Hydrogels were obtained with storage modulus (G') values between 11 (±1) Pa and 1,800 (±200) Pa with gelation times between 80 (±6) and 450 (±36) seconds. G300 cross-linked gelatin hydrogels displayed higher gel stiffness, lower swelling ratio and gelled more rapidly compared to the hydrogels prepared from G175. Stiffer hydrogels (50 and 200 Pa) showed greater ability to support the proliferation of hASCs than softer ones (11 and 30 Pa). The highest cell proliferation was observed with a hydrogel of 200 Pa modulus. CONCLUSIONS:Overall, transglutaminase cross-linked gelatin hydrogels might be suitable as injectable hydrogels for the engineering of musculoskeletal and other types of connective tissues.