3D printable and injectable lactoferrin-loaded carboxymethyl cellulose-glycol chitosan hydrogels for tissue engineering applications

In this study, carboxymethyl cellulose (CMC)-glycol chitosan (GC) hydrogel, a potential three-dimensional (3D) printing biomaterial ink for tissue engineering applications was synthesized using simple, biocompatible in situ-gelling Schiff's base reaction and ionic interactions. Different grades of hydrogels (C70G30, C50G50 and C30G70) were synthesized at physiological conditions. The oxidation of CMC and imine bond formation in the hydrogel were confirmed spectroscopically. Scanning electron microscopic images revealed the crosslinked interconnected pores in the cross-sectioned hydrogels (dried). Swelling (equilibrium: 1 h), porosity (~75%), in vitro degradation (>30 days) and thermal gravimetric analyses of the dried gels were studied. Initially, cytotoxicity assay was evaluated using mouse osteoblastic cells (MC3T3). These experiments revealed that CMC-GC gels formed stable hydrogel networks and were biocompatible. Particularly, C50G50 gels showed high printability (continuous extrusion) and post-printing stability (without secondary crosslinking). Gel 3D printing was optimized by varying the air pressure, temperature, needle size and nozzle speed, to obtain stable lattice structures (2 to 16 layers). The printed (2 and 5 layers) hydrogels showed high stability in phosphate buffer saline (PBS) solution (1 h), under UV light (1 h) and after autoclaving. The strut dimensions and porosity of the printed gels before and after the stability tests were analyzed. The hydrogel stability may be attributed to both the imine bond and ionic interaction between the cationic and anionic polymer side chains. Lactoferrin (glycoprotein) incorporated C50G50 gels showed sustained release up to 21 days in PBS (pH 7.4) solution and demonstrated increased biocompatibility (>80%) during in vitro cytotoxicity assays (MC3T3 cells and bone marrow mesenchymal stem cells) and Live/Dead assay (MC3T3 cells). A higher number of live osteoblast cells on the C50G50 hydrogels with increasing lactoferrin concentration was observed. These results show that the CMC-GC gels are promising bio-ink candidates for 3D printing and loading proteins or drugs for tissue engineering applications.