Hydrogel-polyester combination scaffolds for tissue engineering purposes

INTRODUCTION: The present work aims at the development of 3D scaffolds using both fused deposition modelling as well as a cryogenic treatment to create a fully interconnected pore network. Two material classes will be applied as starting compounds including polyesters (for their mechanical properties) and crosslinkable gelatin precursors (for their cell-interactive properties). MATERIALS AND METHODS: In a first part, poly(lactic acid) (PLA) was processed using fused deposition modelling (Ultimaker). Next, the porous PLA scaffolds were incubated in aqueous methacrylamide-modified gelatin (gel-MOD) solutions of varying concentrations (i.e. 2-10 w/v%) containing 2 mol% Irgacure 2959. The gel-MOD had a modification degree of 80%. Next, after vacuum application, the scaffolds were exposed to UV-light (365 nm) for 2 hours, placed in a cryo-unit and cooled from room temperature to -30°C during 5.5 hours, applying a temperature gradient of 30°C between top and bottom of the scaffolds. In a final step, the frozen hydrogel-PLA combinatory scaffolds were transferred to a freeze-dryer to remove the ice crystals. The scaffolds were characterized using among other micro-computed tomography (µCT) and scanning electron microscopy (SEM). In addition to assessing the water uptake capacity, the mechanical properties were determined using compression tests. In parallel, similar hydrogel precursor solutions containing MC3T3 cells were introduced into the 3D printed PLA scaffolds, followed by hydrogel crosslinking through UV irradiation. The obtained materials were characterized using confocal laser scanning microscopy and the metabolic activity of the seeded and encapsulated cells was assessed with respectively Presto Blue and gene expression studies. RESULTS AND DISCUSSION: In a first part, the efficiency of the UV crosslinking of gel-MOD was assessed after its introduction in PLA. Interestingly, the results indicated that relatively high gel fractions were obtained which were comparable to the ones obtained in the absence of the PLA. Furthermore, mechanical tests showed that the PLA structure determines the strength of the scaffolds as the elastic moduli of both the filled as well as the native structures were comparable. Currently, cell work is ongoing to determine the optimal cryogelation conditions for sufficient cellular infiltration and proliferation. However, the reference samples consisting of polymerized hydrogels encapsulating MC3T3 cells present in the PLA scaffolds were very promising. After 7 days, a 100% increase of the metabolic activity was observed indicating the occurrence of cell proliferation in the scaffolds. In addition, live-dead stains only showed a limited number of dead cells which did not survive the UV crosslinking. Moreover, an ALP stain came out positive indicating the potential of the scaffolds to support osteogenesis. CONCLUSIONS: The UV crosslinking of cells encapsulated into a cell-interactive hydrogel is a valuable route to introduce cells throughout a 3D PLA scaffold. The combination of a cryogel and a PLA scaffold is a potential candidate for tissue engineering. Yet, the applied polymer-related and cryogenic parameters are crucial aspects which determine the cell seeding efficiency and the possibility to enable cellular infiltration throughout the scaffold. REFERENCES 1. Van Vlierberghe S. et al., Biomacromolecules 8: 331-337, 2007