Introduction: To encourage proliferation of encapsulated cells, a biomaterial scaffold should mimic the natural cell environment – the extracellular matrix (ECM)[1]-[3]. Methacrylamide modified gelatin (gel-MOD) is a photopolymerizable hydrogel derived from collagen, the main ECM component. Therefore, it is the perfect choice as a scaffolding material. However, the polymerized hydrogel is very soft and has a high degree of swelling. Consequently, the CAD reproducibly for high resolution 3D printing, such as 2-Photon Polymerization (2PP), is limited. Recently, methacrylamide modified gelatin – further functionalized with additional crosslinkable functionalities (gel-MOD-AEMA) has been developed. In this work both types of modified gelatin, gel-MOD and gel-MOD-AEMA have been compared at three different concentrations (5, 10, and 15wt%).
Materials and Methods: For sample preparation gel-MOD(-AEMA) (5/10/15wt%), DMEM cell culture media, and 1mM water solvable photoinitiator (P2CK[4]) are mixed and put in an ultrasonic bath for 30min at 37°C. The formulations were dropped on glass bottom petri dishes and sealed with a coverslip to prevent evaporation. Structuring was performed with a femtosecond oscillator operating at 800nm and delivering 70fs pulses at a repetition rate of 80MHz. The laser beam was focused with a 32x water immersion objective (Zeiss, Germany) in the sample and guided by a galvo scanner with a velocity of 100mm/s. After structuring the sample was developed in cell culture media at 37°C to remove uncrosslinked material. All samples have been imaged 2h after structuring with a LSM700 confocal microscope (Zeiss, Germany).
Results and Discussion: For the formulation containing 5wt% gel-MOD no structures could be produced, even at reduced structuring velocity and higher power. At higher concentrations, gel-MOD samples exhibit extensive swelling, this can be somewhat reduced by using higher gelatin concentrations. However, at the highest concentration of 15wt% swelling still leads to deformations of the structure. Gel-MOD-AEMA, on the other hand exhibits very little swelling combined with an excellent structuring performance (see Figure 1). In contrast to gel-MOD it was even possible to fabricate structures at 5wt%. Further, the average power for structuring was lower for gel-MOD-AEMA, which allows higher structuring velocities and therefore shorter time for fabrication.
Conclusion: Our results indicate that the highly modified gel-MOD-AEMA derivative is a suitable material for 3D printing techniques, including 2PP. The high degree of functionalization results in a high structure quality, excellent CAD model replication and short time for fabrication. Since the material is derived from collagen, the major ECM component, and an excellent structuring performance and quality can be obtained, it is a perfect material for scaffold based tissue engineering.
We would like to acknowledge the financial support by the European Research Council (Starting Grant-307701)
References:
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