Page 142 - IJB-9-1

International Journal of Bioprinting BNC-reinforced GelMa enhances property of bioprinted cartilage

2.11. Cartilage regeneration in vivo bath because BNC is insoluble in water, and it would
[35]
Cell-laden constructs were cultivated in vitro for 1 day and form a colloid or suspension in hydrogel . With the
then implanted into nude mice subcutaneously to observe increase of BNC ratio, the composite hydrogel became
cartilage formation in vivo. The samples were taken out at more turbid (Figure 1A). The stress-strain curves of each
4, 8, 12, and 24 weeks after implantation for biomechanical composite hydrogel sample showed that with the increase
test, glycosaminoglycan (GAG) content test, total collagen of BNC content, the slope of the curve gradually increased
content test, and histological examination. (Figure 1B). By calculating the slope of 0.1 – 0.2 mm/mm
strain, Young’s moduli of 10% GelMA, 0.075% BNC,
2.12. Biomechanical and biochemical analysis of the 0.225% BNC, 0.375% BNC, and 0.525% BNC were
regenerated cartilage 28.19 ± 3.058 kpa, 28.72 ± 2.066 kpa, 35.34 ± 1.430 kpa,
The compression tests of regenerated cartilage (n = 3) were 49.94 ± 2.775 kpa, and 53.74 ± 3.844 kpa, respectively
performed by Instron 5967 with a 100 N pressure sensor as (Figure 1C). From the 0.225% BNC group, Young’s
described above, and Young’s modulus of the samples was modulus of the hydrogels was significantly higher than
measured based on the slope of the stress-strain curve set that of 10% GelMA (P < 0.001) and increased with the
between 10% and 20% strain. increment of BNC concentration. However, there was no
significant difference in Young’s modulus between 0.375%
The dimethylmethylene blue GAG test kit (GenMed BNC and 0.525% BNC (P > 0.05). In our opinion, when
Scientifics Inc., Shanghai, China) was used to detect the the BNC content is too high, its distribution in the solution
GAG content in the samples (n = 3). Being a unique becomes uneven, which may affect the full crosslinking
amino acid in collagen, hydroxyproline accounts for about of GelMA. Fourati et al. found that when nanocellulose
13.4% of the total content of collagen. The hydroxyproline was used as the additive phase, the mechanical properties
detection kit (Nanjing Jiancheng Bioengineering Institute, of the composites would initially increase, but decreased
Nanjing, China) was used to quantify the content of after exceeding a certain amount . In another study, Fan
[36]
hydroxyproline in samples (n = 3). All operations et al. also found that when cellulose nanocrystals (CNCs)
were carried out according to previously established were added to 15%, there was no significant difference
protocols . between 10% CNC and 15% CNC when CNCs were used
[34]
[37]
2.13. Histological examination to strengthen GelMA/HAMA hydrogels . Moreover, we
found that the hydrogel with 0.525% BNC was difficult
Regenerated cartilages were collected and subjected to to be extruded and the nozzle could be blocked easily.
histological examinations. The samples were fixed in 4% Therefore, in the subsequent experiment, 0.375% BNC
paraformaldehyde, dehydrated, and embedded in paraffin. was selected as the experimental group (BNC/GelMA
Hematoxylin and eosin (H&E), Alcian blue (Solarbio, group), and 10% GelMA without BNC was set as the
China), and Safranine-O (Solarbio, China) staining control group (GelMA group).
were performed, as previously described, to evaluate the
cartilage extracellular matrix deposition and histological 3.2. Characterization of hydrogels
structure in the regenerated cartilage .
[32]
3.2.1. Rheological test
2.14. Statistical analyses We detected the gelatinization with temperature change
GraphPad Prism 8.0 software was utilized for statistical and shear thinning behavior of the BNC/GelMA hydrogel
analyses. All experimental data were obtained from at by rheological analysis. We observed that at 0 – 40°C, G’
least three repeated experiments, and data are reported (storage modulus) and G” (loss modulus) of both hydrogels
as means ± standard deviation. Statistical significance decreased with the increase in temperature. At 0 – 23.5°C,
was evaluated by Student’s t-test or one-way analysis of when the storage modulus (G’) dominated (G’ > G”), both
variance. Values of P < 0.05 were considered statistically materials had gel-like properties. Conversely, at 23.5 –
significant. Data distribution should meet the normal 40°C, when the loss modulus (G”) dominated (G’ < G”),
distribution requirements. the materials had liquid-like properties. Thus, 23.5°C was
the common gel-forming point of these two materials
3. Results and discussion (Figure 2A and B). The gelling temperature of the hydrogel
3.1. Selection of BNC proportion in the did not change after BNC was added. In addition, it could
composite hydrogels be seen that both the BNC/GelMA group and the GelMA
group have obvious shear thinning behavior at 21°C
This study showed that BNC existed in suspension in 10% (Figure 2C). These results suggested that the BNC/GelMA
GelMA solutions after heating and mixing in the water hydrogel was suitable for 3D printing.

Volume 9 Issue 1 (2023) 134 https://doi.org/10.18063/ijb.v9i1.631