Optimization of chitosan-gelatin-based 3D-printed scaffolds for tissue engineering and drug delivery applications

The combination of biocompatible materials and advanced three-dimensional (3D) additive manufacturing technologies holds great potential in the development of finely tuned complex scaffolds with reproducible macroand micro-structural characteristics for biomedical applications, such as tissue engineering and drug delivery. In this study, biocompatible printable inks based on chitosan, collagen and gelatin were developed and 3D-printed with a pneumatic-based extrusion printer. The printability of various chitosan-gelatin (CS-Gel) hydrogel inks was assessed by evaluating the quality of the printed constructs. The inks required an extrusion pressure of 150 +/- 40 MPa with G22 and G25 nozzles for optimal printing. Inks with low chitosan concentrations (<4% w/v) exhibited poor printability, while inks with 4 % w/v chitosan and 1 % w/v gelatin (CG) demonstrated satisfactory extrusion and printing quality. The addition of collagen (0.1 % w/v) to the optimized ink (CGC) did not compromise printability. Post-printing stabilization using KOH produced self-supporting scaffolds with consistent morphological integrity, while weaker bases like NaOH/EtOH and ammonia vapors resulted in lower pore sizes and reduced structural stability. Water evaporation studies showed that neutralized samples had slower evaporation rates due to the strong intermolecular interactions formed during the neutralization process, contributing to a stable crosslinked network. FTIR spectra confirmed the formation of polyelectrolyte complexes in the CS-Gel and CS-Gel-Collagen blends, further enhancing structural stability. Swelling tests indicated that neutralized constructs maintained stability in different pH environments, with KOH-treated samples exhibiting the lowest swelling ratios and the highest structural stability. After optimizing the ink composition, 10 wt% Levofloxacin was loaded in the constructs as a model antibiotic and it's in vitro release rate was quantified. Drug loading was approximately 4 % for both ink compositions GC and CGC. CG Levo released over 80 % of levofloxacin within the first hour, reaching full release in 24 h, indicating inadequate control, while CGK Levo exhibited slower initial release (55 % in 15 min) followed by stabilized release after 4 h, likely due to controlled diffusion from expanded constructs. These findings demonstrate that the developed hydrogel inks and optimized printing parameters can produce scaffolds suitable for tissue engineering applications. Finally, the cell compatibility of the 3D-printed constructs was confirmed with MTT assay on fibroblasts and the antimicrobial activity of the drug-loaded constructs was tested against E. coli and S. aureus, showing an increase of the bacteria free zone from 8 +/- 0.4 mm of the control against E. coli up to 16.4 +/- 0.37 mm in the presence of the KOH-treated CG Levo printed construct.