Crosslinkable gelatins for 3D printing: from material derivatization towards biomedical application

INTRODUCTION: Two Photon polymerization (2PP) is a rapid prototyping technique, ideal for the production of ultra-precise 3D scaffolds with nearly no structural limitations. In this respect, gelatin is an ideal scaffolding material because it contains cell-interactive RGD peptide sequences. However, currently applied photo-crosslinkable gelatin derivatives lack mechanical integrity for 2PP processing in adequately diluted conditions to yield optimal metabolite and nutrient transfer. To overcome these limitations, a derivative was synthesized which exhibits superior mechanical properties compared to currently applied methacrylamide-modified gelatin (gel-MOD). RESULTS AND DISCUSSION: The free amines present in gelatin B were completely methacrylated to obtain photo-crosslinkable gel-MOD. Additional crosslinkable functionalities were introduced via carbodiimide coupling of 2-amino-ethylmethacrylate (AEMA) to the carboxylic acids present in gel-mod. DSC measurements revealed that, in contrast to non-modified gelatin and gel-MOD, gel-MOD-AEMA remains soluble at room temperature. Furthermore, a drop in UCST alongside a reduction in the associated energy with this phase transition was observed. Both gel fraction tests as well as HR-MAS 1H NMR spectroscopy indicated higher crosslinking efficiencies for the produced AEMA derivatives. Real-time rheological monitoring of the crosslinking further supported the proposed hypothesis. The gel-MOD-AEMA derivative exhibited a far higher storage modulus compared to gel-MOD. Furthermore, live/dead staining experiments indicated similar biocompatibility for both materials. Finally, preliminary 2PP experiments illustrated the potential of the materials with respect to reproducibility and structural integrity of the produced scaffolds as gelatin solutions with concentrations of 10 w/v% and below could be processed. CONCLUSIONS: The incorporation of additional crosslinkable functionalities through reaction of the carboxylic acids present in gel-MOD provides an excellent method to improve the mechanical properties of gelatin-based hydrogels. Furthermore, the derivatization does not influence the cytocompatibility. These results furtherincrease the potential of 2PP starting from gelatin-based hydrogel building blocks to yield optimal, tailorable hydrogel scaffolds for soft tissue engineering applications