Development of a collagen and chitosan bioink

MERCEDES PÉREZ-RECALDE; JULIETA POLENTA; ANA GONZALEZ WUSENER; ÉLIDA B. HERMIDA

Congreso; XII Latin-American Congress of Artificial Organs and Biomaterials; 2023

Introduction and objective: The development of bioinks for 3D bioprinting has been extensively researched, given the technique’s relevance in tissue engineering. While collagen and chitosan hydrogels are widely employed for designing scaffolds, their combined use as a potential bioink is rarely reported [1]. In fact, their poor mechanical performance in comparison to other hydrogel formulations, as well as their dissolution at acidic pH, pose drawbacks for this application. Considering the importance of collagen in tissue regeneration and the benefits of chitosan [2], our goal is to create a printable ink from these biopolymers, that allows for the addition of cells at neutral pH.Methodology: Type I collagen from rat tail, and low molecular weight chitosan were dissolved together at 1.25% w/v each in 0.05 M acetic acid. To increase pH, nebulisation with NaHCO3 0.8 M was employed, tunning the times according the ink volume, as well as the rests between steps, measuring pH each time. Ink features were analysed by rheology. Printability was determined using an extrusion 3D bioprinter (LIFE SI, Argentina), through filament width and gap areas. We assessed cellular viability by printing the ink containing DMEM (added 10x) and 1.105 fibroblasts/mL. Cell viability was determined using confocal microscopy and a LIVE/DEAD kit.Results and discussion: The collagen and chitosan blends had a pH of 5.30-5.40 and an apparent viscosity of 285 Pa.s at 0.015 s-1, determined by flow sweeps. By employing nebulisation in 3-4 steps separated each one by 10 minutes of rest, we achieved mixtures with a pH of 6.80-6.90. We intentionally avoided reaching a pH of 7.00 due to the pH-dependent gelation of chitosan. In addition to a pH increase, these nebulised inks showed an increase in viscosity, reaching 24.300 Pa.s on day 1 post-nebulisation. The pH remained stable over the following 5 days, but the viscosity continued increasing. So, for the assessment as a possible ink, we decided to evaluate it always on day 1 after nebulisation. Regarding printability, the ink showed a tendency to spread after extrusionwith a spreading ratio (measured filament width/needle diameter) of 2.8 in the first minutes; this condition affected the gap areas in grid designs. The fibroblasts-laden bioink was extruded at an estimated shear rate of 10 s-1. We printed solid circular shapes that were placed in a 24-wells plate under sterility conditions. By culture in complete DMEM at 37 C and CO2 atmosphere, cell viability was estimated at days 1, 7 and 14 after extrusion. By image analysis we estimated the viability to be > 80% in the three times.Conclusions: Due to collagen and chitosan properties, pH neutralisation was a mandatory step to include cells making up a bioink. In this sense, we developed a mild method to obtain a homogeneous neutral ink. Medical needs for soft tissue, such as skin regeneration, could benefit from such a bioink. Further research is being conducted to improve printability, by adding crosslinkers or a third polymer, as well as to explore the properties of the produced scaffolds.