(23e) Evaluation of a Chitosan-Gelatin Thermogelling Hydrogel As a Bioprinter Ink Using an Inexpensive Platform

Tissue engineering focuses on producing tissues and organs in vitro, to meet the demand for transplantable tissue, using tissue templates called scaffolds. Cell population of scaffolds is random. Cells infiltrate from the surrounding environment into the scaffold and their arrangement, distribution, and orientation are either random, or factors of the scaffold’s properties (i.e. porosity). However, tissue morphologies are non-random and show distinct arrangement. Bioprinting, 3D printing of cells encapsulated in carrier inks, will allow cells to be printed similar to natural tissue morphologies.

Hydrogels are overwhelmingly used for bioprinting due to their dual phase of liquid and solid. Current hydrogels require damaging crosslinking agents (ultraviolet, pH or chemical) to gel. In contrast, gelatin-chitosan hydrogels gel when the temperature is raised to body temperature (37°C).  However, when using an injectable, thermogelling hydrogel a number of factors must be considered.  These factors include the preparation method, gelation characteristics, bed characteristics, feed rate, and layer height.  We obtained a $700 commercial 3D printer and modified it to print from a sterile syringe for $100. Custom print codes were generated in Python and then script was rendered using a software provided by the manufacturer.

We investigated a 2%, 3%, and 4% (wt/v) 1:1 chitosan-gelatin (CG) hydrogel.  We investigated various solution preparation steps (centrifugation, mixing, and degassing) that could help in obtaining consistent printability and fiber formation. Since gelation depends on the temperature of the print surface, we developed temperature profiles of the bed via IR imaging and grid-based assessment using thermocouples. We used agarose to create a uniform print surface to maintain a constant gap with the needle tip.  We evaluated the effect of feed rate and needle height on fiber size, the distribution of cells post-printing, and the viability of cells printed in media. We found that the fiber size decreased from 760µm, to 243µm as the feed rate increased from 10cm/min to 100cm/min, increased needle height may decrease fiber size to an optimal minimum.  Distinct fiber morphology was observed for the optimum height.  Finally, we were able to encapsulate well distributed cells and show cells printed in media with our processing parameters exhibited excellent viability and no contamination after 5 days.  Overall we printed 3D, sterile, cell laden structures with an inexpensive bioprinter. Temperature played the greatest role in fiber size, followed by feed rate. These findings will help in printing cell-laden structures at  expected to lead to more functional tissues.