Research on the characteristics and printing effect of chitin nanocrystal-gelatin methacrylate new bioink / 中华泌尿外科杂志

ABSTRACT

Objective:This study aimed to investigate the physical properties, biocompatibility, and 3D printing performance of a novel hybrid bioink composed of gelatin methacrylated (GelMA) and chitin nanocrystal (ChiNC).Methods:The study was conducted from May 2021 to December 2022, four different bioinks were prepared by adding varying amounts of ChiNC to GelMA bioink. The GelMA concentration in all four bioinks was 100 mg/ml, while the ChiNC concentrations were 0 mg/ml (no ChiNC added), 5 mg/ml, 10 mg/ml, and 20 mg/ml, respectively, named as GC0, GC5, GC10, and GC20 bioinks. The cross-sectional morphology of the hydrogels formed after photocuring the four bioinks was observed using scanning electron microscopy, and the porosity was calculated. Weighing the hydrogels before and after swelling, and then calculate the equilibrium swelling rate. HUVECs were seeded on the surfaces of the hydrogels prepared from the four bioinks and cultured in medium. Cell proliferation was assessed using CCK-8 assays at 1d, 3d, and 7d to compare the proliferation rates of cells on the four hydrogels. HUVECs were added to the four bioinks, and grid-like scaffolds were printed and cultured in medium. Live-Dead staining was performed at 1d and 7d to observe cell viability. Compare the printing effect of bioinks by observing its forming continuous threads properties during extrusion. Finally, tissue-engineered bladder patches simulating the mucosal layer, submucosal layer, and muscular layer anatomical structures of the bladder wall were 3D bioprinted using the optimized bioink composition, and the stability and fidelity of the printed structures were observed to further validate the feasibility of printing multi-layered complex structures with the bioink.Results:Scanning electron microscopy revealed that the porosity of the GC0, GC5, GC10, and GC20 hydrogels were (51.43±6.23)%, (51.85±6.47)%, (50.55±4.59)%, and (42.49±2.20)%, respectively. The differences in porosity between the GC0 group and the other three groups were not statistically significant ( P=0.9994, P=0.9948, P=0.1200). The equilibrium swelling ratio of the other three groups [(8.81±0.41), (7.95±0.19), (7.71±0.14)] was significantly lower than that of the GC0 group (9.37 ± 0.49), and the differences were statistically significant ( P=0.0457, P<0.01, P<0.01). CCK-8 assay showed no significant difference in absorbance value between the GC10 group (0.360±0.009) and the GC0 group (0.357±0.007), GC5 group (0.350±0.012), and GC20 group (0.345±0.018) on the first day ( P=0.9332, P=0.5464, P=0.4937). However, on the third day, the absorbance value of the GC10 group (0.755±0.012) was significantly higher than that of the GC0 group (0.634±0.010), GC5 group (0.704±0.009), and GC20 group (0.653±0.015) ( P<0.01, P=0.0033, P=0.0002). On the seventh day, the absorbance value of the GC10 group (1.001±0.031) was significantly higher than that of the GC0 group (0.846±0.026), GC5 group (0.930±0.043), and GC20 group (0.841±0.024)( P=0.0012, P=0.1390, P=0.0010). The addition of human umbilical vein endothelial cells (HUVECs) into the four groups of hydrogels enabled the printing of grid-like scaffolds, and Live-Dead staining was performed on day 1 and day 7. The cell viability of HUVECs in the four groups on day 1 was (90.13±1.63)%, (90.6±2.45)%, (92.58±2.15)%, and (91.40±3.17)%, respectively. There were no statistically significant differences between the GC0 group and the other three groups ( P=0.9869, P=0.3093, P=0.8008). On day 7, the cell viability was (89.97±3.10)%, (92.18±2.21)%, (92.05±2.25)%, and (90.12±1.97)% for the four groups, respectively. There were no statistically significant differences between the GC0 group and the other three groups ( P=0.3965, P=0.4511, P=0.9995). Bioink extrusion test showed that the GC0 hydrogel could be extruded continuously and form threads at temperatures between 24℃ and 25℃, while the GC10 hydrogel could be extruded continuously and form threads at temperatures between 24℃ and 27℃. Printing tissue engineered bladder patches simulating the anatomical structure of the bladder mucosal layer, submucosal layer, and muscular layer using GC10 bioink, and the printed patches were stable, without collapse, and had high fidelity. Conclusions:Adding ChiNC to GelMA promotes cell adhesion, proliferation, and expands the printing window of GelMA bioink. The biocompatibility of the mixed bioink prepared by adding 10 mg/ml ChiNC in GelMA is good, capable of printing tissue-engineered bladder patches that mimic the anatomical structure of natural bladder walls.