Polysaccharide based hydrogels have been predominantly utilized as ink materials for 3D bioprinting due to biocompatibility and cell responsive features. However, most hydrogels require extensive crosslinking due to poor mechanical properties leading to limited printability. To improve printability without using cytotoxic crosslinkers, thermoresponsive bioinks could be developed. Agarose is a thermoresponsive polysaccharide with upper critical solution temperature (UCST) for sol-gel transition at 35-37°C. Therefore, we hypothesized that a triad of carboxymethyl cellulose(C)–agarose(A)–gelatin(G) could be a suitable thermoresponsive ink for printing since they undergo instantaneous gelation without any addition of crosslinkers after bioprinting. The blend of agarose-carboxymethyl cellulose was mixed with 1% w/v, 3% w/v and 5% w/v gelatin to optimize the triad ratio for hydrogel formation. It was observed that a blend (C2-A0.5-G1 and C2-A1-G1) containing 2% w/v carboxymethyl cellulose, 0.5% or 1% w/v agarose and 1% w/v gelatin formed better hydrogels with higher stability for up to 21 days in DPBS at 37°C. Further, C2-A0.5-G1 and C2-A1-G1hydrogels showed higher storage modulus 762 ±182 Pa & 2452 ± 430 Pa, higher porosity of 96.98 ± 2 % & 98.2 ± 0.8 % and swellability of 1518 ± 68% & 1587 ± 25 % respectively. To evaluate the in vitro potential of these bioink formulations, indirect and direct cytotoxicity were determined using NCTC clone 929 (mouse fibroblast cells) and HADF (primary human adult dermal fibroblast) cells as per the ISO 10993-5 standards. Importantly, the printability of these bioinks was confirmed using extrusion bioprinting by successfully printing different complex 3D patterns.
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