Development of a collagen and chitosan bioink

PEREZ RECALDE, M; POLENTA JULIETA; GONZALEZ WUSENER, A; HERMIDA ELIDA

Mar del Plata, Buenos Aires

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

Sociedad Latinoamericana de Biomateriales

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 widelyemployed for designing scaffolds, their combined use as a potential bioink is rarely reported [1]. In fact, theirpoor mechanical performance in comparison to other hydrogel formulations, as well as their dissolution at acidicpH, pose drawbacks for this application. Considering the importance of collagen in tissue regeneration and thebenefits of chitosan [2], our goal is to create a printable ink from these biopolymers, that allows for the additionof 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 timesaccording the ink volume, as well as the rests between steps, measuring pH each time. Ink features were analysedby rheology. Printability was determined using an extrusion 3D bioprinter (LIFE SI, Argentina), through filamentwidth and gap areas. We assessed cellular viability by printing the ink containing DMEM (added 10x) and 1.105fibroblasts/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 285Pa.s at 0.015 s-1, determined by flow sweeps. By employing nebulisation in 3-4 steps separated each one by 10minutes of rest, we achieved mixtures with a pH of 6.80-6.90. We intentionally avoided reaching a pH of 7.00due to the pH-dependent gelation of chitosan. In addition to a pH increase, these nebulised inks showed anincrease in viscosity, reaching 24.300 Pa.s on day 1 post-nebulisation. The pH remained stable over the following5 days, but the viscosity continued increasing. So, for the assessment as a possible ink, we decided to evaluate italways 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 conditionaffected 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 shapesthat were placed in a 24-wells plate under sterility conditions. By culture in complete DMEM at 37 C and CO2atmosphere, cell viability was estimated at days 1, 7 and 14 after extrusion. By image analysis we estimated theviability to be > 80% in the three times.Conclusions: Due to collagen and chitosan properties, pH neutralisation was a mandatory step to include cellsmaking up a bioink. In this sense, we developed a mild method to obtain a homogeneous neutral ink. Medicalneeds for soft tissue, such as skin regeneration, could benefit from such a bioink. Further research is beingconducted to improve printability, by adding crosslinkers or a third polymer, as well as to explore the propertiesof the produced scaffolds.References[1] Decante G. et al., Biofabrication 13, 032001, 2021.[2] Pahlevanzadeh F. et al., Materials 13(11), 2663, 2020. Williams K. et al., Eur. Polym J. 2, 8-16, 2017.