Three-dimensional printing of chemically crosslinked gelatin hydrogels for adipose tissue engineering

Despite their outstanding potential and the success that has already been achieved with three-dimensional (3D) printed hydrogel scaffolds, there has been little investigation into their application in the regeneration of damaged or missing adipose tissue (AT). Due to their macroscopic shape, microarchitecture, extracellular matrix-mimicking structure, degradability and soft tissue biomimetic mechanical properties, 3D printed hydrogel scaffolds have great potential for use in aesthetic, structural and functional restoration of AT. Here, we propose a simple and cost-effective 3D printing strategy using gelatin-based ink to fabricate scaffolds suitable for AT engineering. The ink, successfully printed here for the first time, was prepared by mixing gelatin and methylenebisacrylamide (a crosslinker) to initiate the crosslinking reaction. The solution was then loaded into the cartridge (temperature T = 35 degrees C) of a pneumatic extrusion-based 3D printer and printed on a cooled surface (T = 4 degrees C) in the appropriate time window for ink printability as verified by rheological tests. Subsequently, the printed gelatin hydrogels were crosslinked at different temperatures to optimize their stability and fix the printed structure. The gelatin scaffolds crosslinked at 20 degrees C remained stable for 21 days at physiological temperature, with compressive mechanical properties mimicking those of AT (i.e. elastic modulus = 20 kPa). The 3D printed scaffolds showed no indirect cytotoxic effects on a 3T3-L1 pre-adipocyte cell line. Moreover, the printed scaffolds successfully promoted adhesion and proliferation of primary human pre-adipocytes, as demonstrated by LIVE/DEAD staining and Alamar Blue assay. The differentiation of primary human pre-adipocytes isolated from three different donors according to adipogenic phenotype was demonstrated by an increase in PPAR gamma gene expression detected by real-time PCR and accumulated lipid droplets stained by Oil Red O, thus proving the potential of the 3D printed gelatin hydrogels as scaffolds for AT engineering.