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Description
The development of physiologically relevant vascular models remains a major challenge in tissue engineering, particularly when using extrusion-based 3D bioprinting with soft, liquid-like biomaterials. To overcome limitations related to structural instability and gravity, the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) method enables mid-air bioprinting within a temporary gelatin-based support bath. However, achieving high shape fidelity while maintaining biological function remains an open issue. This study aims to optimize FRESH bioprinting for vascular model fabrication by systematically analyzing key parameters affecting shape fidelity, mechanical stability, and cell viability.
A composite hydrogel of sodium alginate (SA) and gelatin (GEL) was prepared following a controlled heating and sterilization protocol. The FRESH method was applied using a CELLINK INKREDIBLE+ bioprinter, extruding the hydrogel into a gelatin-based slurry. Variables such as nozzle diameter (0.4 mm vs. 0.6 mm), SA concentration (4% vs. 6%), printing pressure (8–30 kPa), and speed (10 mm/s) were tested to identify optimal printing conditions. After printing, constructs were incubated at 37°C to liquefy the support bath and coated with 1% SA crosslinked in CaCl₂ to improve mechanical integrity.
Shape fidelity was evaluated using micro-CT imaging through qualitative and quantitative analysis. Tubular constructs with varying diameters (5, 15, 25 mm) were scanned before and after support removal. Dimensional parameters—including internal and external diameters and wall thickness—were compared to CAD models. Constructs printed with 6% SA showed low standard deviations and high dimensional accuracy. Nozzle size had minimal impact, while coating improved shape fidelity and construct stability, despite introducing slight geometric changes. Larger constructs exhibited shrinkage post-incubation, likely due to calcium-mediated crosslinking, whereas 5 mm structures showed negligible deformation.
Mechanical stability was assessed via uniaxial compression tests on cylindrical samples (9 mm diameter, 10 mm length). Results revealed high intra-batch consistency and moderate inter-batch variability. The mechanical response of bioprinted structures was comparable to native porcine aorta, especially for thinner-walled constructs (~0.8 mm). A minor reduction in mechanical strength was observed after 21 days at 37°C, likely caused by hydrogel swelling and partial degradation. Weekly re-crosslinking is proposed as a strategy to reduce this effect.
Biocompatibility was preliminarily assessed using AG01522 dermal fibroblasts embedded in the bioink. Cell viability was monitored over 21 days via immunofluorescence imaging. Viability remained consistently above 80%, confirming the cytocompatibility of the SA-GEL system and the FRESH process. However, a gradual decrease in cell density was observed, likely due to the limited mechanical strength and retention capacity of the bioink. Future improvements could include enhanced crosslinking and the incorporation of ECM components such as collagen or fibrin to support adhesion and proliferation.
In conclusion, this study validates the use of FRESH bioprinting combined with micro-CT analysis as a viable approach for creating stable and biologically compatible vascular models. Further optimization of bioink formulation and co-culture strategies will be crucial for functional tissue development.
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