Chitin nanocrystal-assisted 3D bioprinting of gelatin methacrylate scaffolds

Abstract In recent years, there has been an increasing focus on the application of hydrogels in tissue engineering. The integration of 3D bioprinting technology has expanded the potential applications of hydrogels. However, few commercially available hydrogels used for 3D biological printing exhibit both excellent biocompatibility and mechanical properties. Gelatin methacrylate (GelMA) has good biocompatibility and is widely used in 3D bioprinting. However, its low mechanical properties limit its use as a standalone bioink for 3D bioprinting. In this work, we designed a biomaterial ink composed of GelMA and chitin nanocrystal (ChiNC). We explored fundamental printing properties of composite bioinks, including rheological properties, porosity, equilibrium swelling rate, mechanical properties, biocompatibility, effects on the secretion of angiogenic factors and fidelity of 3D bioprinting. The results showed that adding 1% (w/v) ChiNC to 10% (w/v) GelMA improved the mechanical properties and printability of the GelMA hydrogels, promoted cell adhesion, proliferation and vascularization and enabled the printing of complex 3D scaffolds. This strategy of incorporating ChiNC to enhance the performance of GelMA biomaterials could potentially be applied to other biomaterials, thereby expanding the range of materials available for use. Furthermore, in combination with 3D bioprinting technology, this approach could be leveraged to bioprint scaffolds with complex structures, further broadening the potential applications in tissue engineering.


Introduction
In recent years, there has been a burgeoning interest in hydrogelbased research, with its primary applications encompassing tissue engineering [1][2][3][4][5], cancer models [6][7][8][9][10], drug-controlled release [11][12][13] and biosensors [14][15][16][17].Among these research areas of study, tissue engineering has emerged as the most prominent direction [18].Traditional tissue engineering has predominantly utilized medical metals, thermoplastics, bio-ceramics, hydrogels and other materials.Tissue engineering is an emerging discipline that combines cell biology and material science to construct tissues or organs in vitro or in vivo.Hydrogels, which can mimic the extracellular matrix microenvironment, have demonstrated benefits in promoting nutrient penetration and facilitating cellular communication, migration and growth [19].These advantages have established hydrogels as a research focal point in the tissue engineering domain in recent years.Despite the numerous benefits of hydrogels, challenges persist in accomplishing complex tissue and organ repair and reconstruction, as traditional methods struggle to accurately repair and reconstruct intricate tissues and organs.Fortunately, the advent of 3D bioprinting technology presents new possibilities for tissue and organ repair and reconstruction [20][21][22].The integration of hydrogels with 3D bioprinting technology in tissue engineering offers several advantages over conventional methods.3D bioprinters can generate micron-level structures with high precision and facilitate personalized customization.Moreover, 3D bioprinting technology can create complex structural models that are challenging to accomplish using traditional techniques.This technological advancement may introduce novel approaches and hold significant clinical practical value for the repair and reconstruction of tissues and organs.
Numerous studies have been conducted on hydrogels for 3D bioprinting [23][24][25][26].In our study, we utilized medium substitution gelatin methacrylate (GelMA, 55-65%) as the primary component of the mixed bioinks, which helped achieve a balance between mechanical strength and cell compatibility.The rapid crosslinking advantages with 405 nm blue light under the photo-initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), along with excellent 3D printing performance, have rendered this material one of the most commonly used materials in 3D bioprinting.However, the relatively inferior mechanical properties of GelMA cannot fulfill the mechanical requirements of tissue engineering [27].To address this issue, a considerable number of articles have been published on incorporating inorganic or organic particles to enhance the mechanical properties of GelMA and regulate the rate of GelMA degradation.For instance, Gao et al. [28] added polyN-acryll glycine (PACG) to GelMA and demonstrated that the inclusion of PACG improved the compression and tensile strength of GelMA.Similarly, Gao et al. [27] showed that the addition of nanoclays to GelMA improved the material's porosity, enhanced its mechanical strength, reduced its degradation rate and maintained its excellent biocompatibility.
Chitin is known to exist in two main crystal forms: a crystal form and b crystal form.The molecular chains of a-chitin are arranged in an anti-parallel manner, which is conducive to the formation of strong intermolecular hydrogen bonds.In contrast, the molecular chains in b-chitin are arranged in parallel.a-Chitin exists in the exoskeletons of various arthropods, such as shrimp, crabs and other insects, while b-chitin is less abundant and is found in squid pen and cuttlefish bones.Due to its wide range of sources and stable structure, we chose a-chitin as the experimental material.Chitin nanocrystal (ChiNC) is an organic nanocrystal obtained from chitin acid solution.It is also a natural, non-toxic material with cell compatibility and has been shown to enhance the mechanical properties of various materials [29].For example, Gu et al. [30] found that ChiNC could regulate the natural polymer flow behavior and act as an organic nanofiller to improve the scaffold's mechanical properties.Similarly, Liu et al. [31] discovered that ChiNC could increase the stress resistance of poly(Llactide), facilitating cell adhesion, proliferation and upregulation of alkaline phosphatase activity, promoting calcium deposition.Hassanzadeh et al. [32] added ChiNC to GelMA to enhance its mechanical properties, this study showed that the addition of ChiNC increased the elastic modulus of GelMA by 1000 times and the strain by more than 200%.However, this study did not integrate 3D bioprinting technology and did not include a comprehensive investigation of vascularization.
In this study, we investigated the influence of incorporating various concentrations of ChiNC into GelMA on the physical and chemical properties of GelMA.In conjunction with 3D bioprinting, human umbilical vein endothelial cells (HUVECs) were seeded into the GelMA/ChiNC bioink.Utilizing 3D bioprinting technology, we subsequently fabricated a cell-laden scaffold, enabling us to examine the adhesion, proliferation, printability and vascularization expression of HUVECs within hydrogel scaffolds.Moreover, we assessed the 3D printing fidelity of both the 0% ChiNC group and the 1% ChiNC group at varying temperatures.

Preparation and characterization of the ChiNC
ChiNC was prepared with slight modifications according to the method described by Wang [33].First, 3 mol/l hydrochloric acid solution was added to chitin, which was then placed on a magnetic stirrer and heated to 60 C while stirring at a speed of 300 r/min for 6 h.The residue was washed with deionized water and centrifuged.The obtained suspension was filtered through a 100-mesh sieve and transferred to a dialysis bag (12KD-14KD) for dialysis in deionized water for 48 h.After dialysis, the suspension was sonicated (Scientz-950E, China) at 100 W of power for 20 min and then freeze-dried using a freeze-drying machine (SCIENTZ-10N, China) to obtain ChiNC powder.The resulting ChiNC powder was sterilized by cobalt-60 irradiation.The ChiNC were redispersed in a solution using an ultrasonic machine before use.We characterized ChiNC using transmission electron microscopy (TEM, JEM-F200, Japan).Using a pipette, 100 ll of ChiNC suspension was dropped onto a 230-mesh carbon, naturally dried and then the sample was observed and photographed under a TEM.The images were processed using Image J, and the length frequency distribution of ChiNC was analyzed using GraphPad Prism 8.0.2 software.

Hydrogel porosity
Four bioinks (0%, 0.5%, 1% and 2% ChiNC) were cured with 405 nm blue light for 2 min, lyophilized in a cold lyophilizer (SCIENTZ-10N, China) and the lateral pores were observed by cutting the samples along the longitudinal section.The samples were attached to sample brackets, and after the samples were coated with gold, the microscopic pores were observed using a scanning electron microscope (SEM, QUANTA FEG 250, USA).Porosity was defined as the ratio of the surface area occupied by the pores in the SEM images and analyzed using Image J software 1.53c.The data were statistically analyzed and graphed using GraphPad Prism 8.0.2 software.

Hydrogel equilibrium swelling rate
Four bioinks (0%, 0.5%, 1% and 2% ChiNC) were added to 48-well plates with four replicate wells for each group (500 ll per well).These structures were exposed to UV light (405 nm) for 5 min and freeze-dried for 48 h, after which the dry weight of each sample was recorded (Wd).The samples were then soaked in 1.5 ml PBS at 37 C for 48 h.Finally, we gently wiped the PBS on the sample surface with filter paper and recorded the weight (Ws) of each sample.The equilibrium swelling rate was calculated using the formula (WsÀWd)/Wd.

Hydrogel rheological properties
The rheological properties of hydrogels were tested using a rheometer (Anton Paar MCR 302, AntonPaar, Australia) with a parallel-plate geometry (25 mm in diameter, a gap height of 50 mm).The rheometer assessed the shear rate and viscosity of the four bioinks (0%, 0.5%, 1% and 2% ChiNC) in the shear rate range of 10 0 -10 3 (s À1 ) at a temperature of 37 C.The storage modulus versus frequency was tested at 0.5% strain, a frequency range of 10 À1 -10 1 (Hz) and at 37 C.

Hydrogel mechanical properties
Four bioinks (0%, 0.5%, 1% and 2% ChiNC) were prepared and cured using 405 nm blue light for 5 min to form cylindrical hydrogels with a diameter of 12.6 mm and a thickness of 9.5 mm.The stress-strain behavior of each sample was measured using a universal mechanical compressor (EUT6502, SASTest, China) at a temperature of 25 C and a compression speed of 0.1 mm/s.Three replicates were assessed for each of the four groups.The differences in fracture strain among the four hydrogel groups were analyzed using GraphPad Prism software.

Cell viability
HUVECs were added to four groups of bioinks (0%, 0.5%, 1% and 2% ChiNC), with a cell density of 8 Â 10 6 cells/ml.3D bioprinted grid scaffolds were cultured in ECM, and cell viability within the 3D bioprinted scaffolds was assessed using a fluorescent live/ dead activity assay kit (KGAF001, KeyGEN Bio tech, China) on Days 1 and 7 according to the protocol.The scaffolds were visualized under a fluorescence microscope (NIKON Ti-A1, Japan), and images were processed with Image J software.

Cell proliferation
Four groups of bioinks (0%, 0.5%, 1% and 2%) were added to a 48well plate, each group consisted of four replicate wells, to which 200 ll of hydrogel was added.The hydrogel in each well was cured using 405 nm blue light for 2 min.Subsequently, 2 Â 10 5 HUVECs were seeded on the surface of each hydrogel, and 600 ll of culture medium was added to each well.The samples were incubated at 37 C in 5% CO 2 , with the culture medium being replaced every 2 days.On Days 1, 3 and 7, the samples from the different groups were washed with PBS three times.Following this, 180 ll of ECM culture medium and 20 ll of Cell Counting Kit-8 solution (CCK-8, Dojindo, Japan) were added to each well.After a 2-h incubation period, the samples were transferred to a 96well plate (100 ll/well), and the absorbance [optical density (OD) 450 nm] was measured using a microplate photometer (VersaMax, USA).

Immunofluorescence staining
HUVECs were cultured on the surface of '10% GelMA þ 1% ChiNC' hydrogel scaffolds.Immunofluorescence staining for CD31 was performed on Days 7 and 14 according to the manufacturer's protocol.Three random fields were selected and photographed, and the images were analyzed using Image J software.

Enzyme-linked immunosorbent assay
HUVECs were seeded in the four scaffold groups, and culture supernatants were collected on Days 3 and 7.The concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the culture supernatant were measured using VEGF (PV963, Cloud-Clone, China) and bFGF enzyme-linked immunosorbent assay (ELISA) kits (ab246531, Abcam, UK), respectively, following the manufacturer's protocol.This allowed for the evaluation of the impact of ChiNC concentration on the secretion of vascular secretory factors from HUVECs.

Comparison of bioink printing effects
To better observe the printing effect of bioink, we added a 0.5% (v/v) concentration of pigment (FLEUR COULEUR, China) to the bioink in both the 0% ChiNC and 1% ChiNC group.The printing effects of bioinks (0% ChiNC and 1% ChiNC groups) were evaluated at temperatures between 22 C and 29 C. In this study, we adjusted the printing temperature and allowed a 10-min waiting period each time to ensure accurate printing effects before testing.All scaffolds were printed with four layers and had dimensions of 15 mm Â 15 mm Â 1.0 mm.

3D Bioprinted GelMA/ChiNC scaffold
In this study, scaffolds were printed using a bioprinter (SunP BioMaker 4, China) under sterile conditions throughout the entire bioprinting process.The interior of the 3D printer was wiped with gauze soaked in 75% alcohol, and the printer's ultraviolet sterilization system was activated for 30 min.HUVECs were resuspended in 37 C hydrogels at a density of 8 Â 10 6 cells/ml and loaded into a 3D printer syringe.
The printing parameters for live/dead staining scaffolds were as follows: the inner diameter of the printing needle was 0.25 mm, the line spacing was 3.0 mm, the printing nozzle temperature was set to 25 C, and the cooling platform was maintained at 8 C. The printing speed was 2 mm/s, with an extrusion speed of 0.3 mm 3 /s.Blue light irradiation at 50% intensity was used, with a distance of 60 mm and a duration of 30 s.

Statistical analysis
All data were expressed as mean 6 standard deviation (SD).Statistical analysis was performed using ANOVA with GraphPad Prism 8.0.2 (GraphPad Software, USA), where P < 0.05 was considered statistically significant.The data were indicated with * for P < 0.05, ** for P < 0.01, *** for P < 0.001, **** for P < 0.0001 and NS for no statistical significance.

Characterization of GelMA/ChiNC bioinks
We prepared four groups of bioinks with varying concentrations of ChiNC (0%, 0.5%, 1% and 2% ChiNC) and cured them with blue light and lyophilization.The cross-sections of the hydrogels were examined under SEM (Fig. 2A), and the porosity of the four groups of hydrogels was $50%, with no statistically significant differences observed (Fig. 2B).The addition of 2% ChiNC to 10% GelMA reduced the average equilibrium swelling ratio of the hydrogel from 8.451-6.775.The 10% GelMA alone exhibited a statistically significant difference compared to the other three groups (Fig. 2C).
To assess whether the GelMA/ChiNC bioinks could be extruded for 3D bioprinting, we evaluated the shear rate-viscosity and storage modulus-angular frequency of the four groups of bioinks.As depicted in Fig. 2D, higher shear rates resulted in lower viscosities.Under the same conditions, the viscosity of the bioinks decreased with the addition of ChiNC.The incorporation of 1% ChiNC into 10% GelMA reduced the viscosity to 46.06% at a shear rate of 100 (1/s).Moreover, the viscosity significantly decreased to 1.81% when the ChiNC concentration reached 2%.
The storage modulus of the GelMA/ChiNC hydrogel increased with increasing angular frequency, with higher ChiNC concentrations resulting in a greater storage modulus.When the angular frequency was 10 Hz, and 2% ChiNC was added to 10% GelMA, the storage modulus increased from 2410 to 5460 Pa, a 126.6% increase (Fig. 2E).The addition of ChiNC improved the mechanical properties of the GelMA-based printed scaffold.
The stress of the hydrogel increased as the strain increased.A comparison of four different hydrogels showed that higher concentrations of ChiNC resulted in greater stress for the hydrogel, and when 2% ChiNC was added, the hydrogel's maximum stress reached 2.0 Â 10 5 Pa (Fig. 2F).
As illustrated in Fig. 2G, the addition of ChiNC increased the fracture strain of the hydrogel.Comparing the 0% ChiNC group to the 2% ChiNC group, there were statistically significant differences in fracture strain.However, the differences were not statistically significant between the 0% ChiNC group and the other groups.

The biocompatibility of GelMA/ChiNC hydrogel
As shown in Fig. 3A, the majority of cells in each group of hydrogels were viable HUVECs, indicating good biocompatibility.
To further investigate the effects of ChiNC on the hydrogel, four types of hydrogel surfaces were cultured with HUVECs, and CCK-8 metabolic activity was assessed on Days 1, 3 and 7.There was no statistical difference between the four groups on Day 1.However, on Days 3 and 7, the OD value of the 1% ChiNC group was significantly higher than that of the other three groups (Fig. 3B).
The HUVECs grown on the hydrogel surface (10% GelMA þ 1% ChiNC) were immunofluorescence stained with CD31 on Days 7 (Fig. 3C) and 14 (Fig. 3D).There was a significant increase in the number of cells on Day 14 compared to Day 7, with the cells forming tight junctions and expressing a considerable amount of CD31.

Detection of vascularization factors in GelMA/ ChiNC hydrogel
HUVECs were implanted in four groups of scaffolds, and the levels of VEGF and bFGF were measured using ELISA kits.
At Days 3 and 7, the 1% ChiNC group showed the highest levels of VEGF, with 704.92 6 14.55 and 798.74 6 8.58 pg/ml, respectively, exhibiting statistically significant differences compared to the other three groups (Fig. 4A).
The content of bFGF in the 1% ChiNC group was higher than that in the 0% ChiNC and 0.5% ChiNC groups at Days 3 and 7, with statistical significance.However, there was no statistically significant difference between the 2% ChiNC and 1% ChiNC groups at Days 3 and 7 (Fig. 4B).

Comparison of bioink printing effects
In this study, the printing effects of two groups of bioinks were compared at different bioink temperatures.The scaffolds were printed in four layers.The printing process for the 0% ChiNC group was discontinuous and irregular at temperatures ranging from 22 C to 23 C, indicating excessive viscosity.At temperatures ranging from 24 C to 25 C, the printing was continuous and filamentary.The adjacent printed filaments fused at temperatures ranging from 26 C to 27 C. Conversely, the printing process for the 1% ChiNC group was discontinuous and irregular at temperatures ranging from 22 C to 23 C, but the printing was continuous, and filamentation was favorable at temperatures ranging from 24 C to 27 C.The adjacent printed filaments fused at temperatures ranging from 28 C to 29 C (Fig. 5).

GelMA/ChiNC printed scaffolds
All structures were printed using 1% ChiNC group hydrogels.Fig. 6A illustrates the printed grid scaffold with a printed filament spacing of 2.2 mm and dimensions of 10 mm (length) Â 10 mm (width) Â 1.0 mm (thickness).The layer height was set at 0.25 mm, and four layers were printed.Fig. 6B presents the printed 'NKU' characters (the English abbreviation of Nankai University), with dimensions of 30 mm (length) Â 10 mm (width) Â 1.0 mm (thickness), and four layers were printed.Fig. 6C depicts the printed tube with a diameter of 10 mm and a layer height of 0.25 mm.A total of 80 layers were printed.

Discussion
Hydrogels exhibiting excellent biocompatibility, mechanical properties, and suitability for cell proliferation have emerged as a prominent research focus [34][35][36].In this study, we incorporated varying concentrations of ChiNC into 10% GelMA to examine the influence of ChiNC concentration on the rheological and mechanical properties of the bioink.Furthermore, we assessed the biocompatibility of GelMA/ChiNC hydrogels and the impact of ChiNC on the adhesion, proliferation and vascularized secretions of HUVECs within the GelMA/ChiNC hydrogels.Our findings suggest that ChiNC enhanced the mechanical properties of GelMA, reduced hydrogel viscosity and improved 3D bioprinting efficiency.Additionally, ChiNC promotes HUVECs' adhesion, proliferation and expression of vascularization factors on the hydrogel surface.
The primary sources of ChiNC raw materials are the shells of crustaceans, such as shrimp and crabs.ChiNC nanoparticles typically exhibit a whisker-like shape, with diameters ranging from 160-320 nm, in accordance with the findings of Jung et al. [37].These whisker-shaped nanoparticles function analogously to the addition of steel reinforcement in concrete, bolstering the overall mechanical properties of the GelMA hydrogel.
Our study demonstrated that the incorporation of ChiNC into 10% GelMA did not alter the hydrogel's porosity, which is in line with previous research [30,31].Maintaining consistent porosity in hydrogels is advantageous for tissue engineering scaffolds, as it facilitates nutrient transport, cell attachment, migration and proliferation [38,39].
As a nanomaterial, the addition of ChiNC to GelMA hydrogel led to a decreased swelling rate, a finding corroborated by previous research on GelMA [40].With increasing ChiNC concentration, both the stiffness and interaction forces between ChiNC and GelMA rose, thereby inhibiting the swelling of GelMA/ChiNC hydrogels [29].The diminished swelling rate of the hydrogel is advantageous for preserving the scaffold's shape by minimizing the deformation of the printed scaffold in aqueous environments.
The shear-thinning property of a hydrogel is a crucial determinant of its suitability for 3D bioprinting.Our findings revealed that the viscosity of GelMA hydrogel diminished as the shear rate increased, a result consistent with numerous studies [41][42][43].A decrease in viscosity during extrusion through the print nozzle is beneficial, while viscosity recovery post-printing on the platform is favorable for retaining the original shape.Consequently, the shear-thinning characteristic of the hydrogel is advantageous for 3D bioprinting.Introducing ChiNC to 10% GelMA decreased the hydrogel's viscosity, with a substantial reduction in the viscosity of GelMA/ChiNC hydrogel observed at a ChiNC concentration of 2%.As printed scaffolds can deform more easily when the viscosity of the mixed hydrogel is too low, we think that the 2% ChiNC group bioink is unsuitable for 3D bioprinting.
Hydrogel scaffolds require good mechanical properties, including good elastic properties and mechanical strength to be suitable for in vivo transplantation.The storage modulus is an indicator of elasticity, and our results demonstrate that the incorporation of ChiNC increases the storage modulus of the hybrid hydrogel, this is due to the physical cross-linking between GelMA and ChiNC, resulting in an improvement in the storage modulus of the hydrogel network.A higher storage modulus enables the hydrogel to retain its original shape better, thus improving printing reliability [44,45].The increased elasticity of the hydrogel is beneficial for tissue engineering scaffolds.
We also noted that the mechanical strength of the hydrogels was substantially enhanced by incorporating ChiNC into 10% GelMA, with increased ChiNC concentrations resulting in higher stress in the hybrid hydrogels under identical strain.This phenomenon can be attributed to the high aspect ratio and modulus of ChiNC, which enables the dispersion of greater pressure loads for GelMA, as well as the bridging role of nanoparticles within the hydrogel network, improving network stability and stress resistance [31,46].This finding also elucidates the reason behind the improvement in the failure strain rate of the hybrid hydrogels upon ChiNC addition, as enhanced compression resistance would be beneficial for tissue engineering applications.
Biocompatibility is a fundamental criterion for biomaterials used in tissue engineering, as it underpins cell adhesion, growth and proliferation on hydrogel scaffolds.In this study, we assessed the biocompatibility of GelMA/ChiNC hydrogels by seeding HUVECs within the hydrogel and cultivating them for 7 days.We conducted live/dead staining, which demonstrated that the cells thrived within the hydrogel, Live/dead staining revealed that the cells exhibited high survival rates within the hydrogel, suggesting that the GelMA/ChiNC hydrogel is biocompatible and appropriate for bioink printing.Furthermore, we seeded HUVECs on the hydrogel surface and assessed cell metabolic viability using the CCK-8 assay.The CCK-8 results demonstrated no statistically significant differences in OD values among the four groups on Day 1.The OD values increased with the rising ChiNC concentration on Days 3 and 7, whereas they decreased when the ChiNC concentration reached 2%.This observation suggests that low concentrations of ChiNC might promote cell adhesion and proliferation.However, at a ChiNC concentration of 2%, the hydrogel surface morphology appeared unfavorable for cell adhesion and proliferation [47].
Since HUVECs express the endothelial cell-specific antigen CD31, a marker of vascularization, we used CD31 immunofluorescence staining to assess vascularization.The CD31 immunofluorescence results revealed that HUVECs adhered and grew effectively on the '10% GelMA þ 1% ChiNC' hydrogel surface, further demonstrating the suitability of '10% GelMA þ 1% ChiNC' hydrogel for cell adhesion and proliferation.
One of the challenges in tissue engineering is the absence of optimal vascularization.Presently, the incorporation of angiogenic growth factors such as VEGF and bFGF serves as a potential  Regenerative Biomaterials, 2023, Vol. 10, rbad058 | 7 solution to promote vascularization.Both VEGF and bFGF exhibit a synergistic effect in stimulating angiogenesis [48][49][50][51].Enhanced expression of these factors can prompt endothelial cells to degrade the basement membrane, enabling them to migrate to the defective site for proliferation.Eventually, new vessel lumens emerge, the capillary network becomes denser and new vessels mature [52].Thus, we assessed the expression of VEGF and bFGF vascularization factors to ascertain the role of ChiNC in promoting vascularization.In our study, we seeded HUVECs into four hydrogel groups with varying concentrations of ChiNC (0%, 0.5%, 1% and 2%) to examine the effect of ChiNC on the secretion of angiogenic growth factors by HUVECs.Our results indicated that VEGF secretion increased with the rising concentration of ChiNC but decreased when the concentration reached 2%.This observation was consistent with the CCK-8 results, which suggested that the increase in cell number may have contributed to the elevated VEGF secretion.However, other factors, such as differences in gene expression, could have also played a role.Similarly, our study revealed that bFGF secretion increased with escalating ChiNC concentration but ceased to increase when the concentration reached 2%.Although, theoretically, a decline in cell metabolic viability at 2% ChiNC should have led to a reduction in bFGF secretion, we did not observe this phenomenon.It is plausible that differences in gene expression accounted for this observation, warranting further investigation.
Printability is a fundamental prerequisite for hydrogels intended for use as bioinks.In this study, we assessed the physical properties, biocompatibility and ELISA results of four hydrogel groups to identify the most appropriate bioink for 3D bioprinting.Our findings indicate that the 1% ChiNC hydrogel is a superior candidate for 3D bioprinting due to its broader printable window compared to the control group (0% ChiNC).Incorporating nanoparticles into the GelMA hydrogel enhanced the hydrogel network's stability, improved its storage modulus and broadened the 3D bioprinting range.
We used the '10% GelMA þ 1% ChiNC' bioink for 3D bioprinting of grids, 'NKU' characters and tubes, suggesting that this bioink is a viable option for 3D bioprinting applications.Our bioink exhibited favorable biocompatibility, mechanical properties, cell proliferation and vascularization promotion and enhanced 3D printing outcomes.Consequently, we believe it may be applicable in various tissue engineering contexts, such as bladder and skin tissue engineering.
Currently, we are conducting further research on hydrogels based on GelMA and ChiNC for bladder defect repair.We posit that incorporating ChiNC into hydrogels could augment their efficacy and broaden their application scope.This strategy might also be applicable to other types of bioinks in tissue engineering.In conclusion, our study highlights the potential of our bioink for 3D bioprinting and its prospective applications in tissue engineering.

Conclusions
This study investigated the effects of different concentrations of ChiNC on 10% GelMA hydrogel concerning mechanical properties, rheological properties, porosity, equilibrium swelling rate, biocompatibility, cell adhesion and proliferation.Our findings demonstrated the impact of ChiNC addition to GelMA on cell adhesion and proliferation.The results revealed that incorporating ChiNC into GelMA enhanced the mechanical properties, printability, cell adhesion and proliferation.ELISA results of angiogenic factors and 3D printing indicated that the 1% ChiNC group was optimal for 3D bioprinting.Thus, we recommend the 1% ChiNC group as the most suitable for 3D bioprinting due to its improved printability, cell support and proliferation.Furthermore, the addition of ChiNC could be explored in conjunction with other bioinks to enhance their application potential in tissue engineering.In conclusion, ChiNC proves to be a promising biomaterial in the field of tissue engineering.

Figure 1 .
Figure 1.Preparation and characterization of ChiNC.(A) ChiNC obtained through the acidolysis of chitin with hydrochloric acid and subsequent freezedrying.(B) Optical image of ChiNC aqueous suspension and the structural formula of ChiNC.(C) TEM image of ChiNC in the aqueous suspension.(D) Length/frequency distribution of ChiNC.

Figure 3 .
Figure 3. Biocompatibility of the hydrogels.(A) Live/dead staining of hydrogels with varying ChiNC concentrations on Days 1 and 7. (B) Cell Counting Kit-8 assay for hydrogels with different ChiNC concentrations on Days 1, 3 and 7 (n ¼ 4).CD31 immunofluorescence staining of HUVECs on Day 7 (C) and Day 14 (D) on the surface of the 1% ChiNC group hydrogel (blue represents the nucleus and green represents CD31).

Figure 4 .
Figure 4. Secretion of angiogenic factors from human umbilical vein endothelial cells.Concentration of VEGF (A) and bFGF (B) in the HUVECs culture medium supernatant of GelMA hydrogels containing different concentrations of ChiNC on Days 3 and 7 (n ¼ 3).

Figure 5 .
Figure 5.Comparison of printing effects between 0% ChiNC group and 1% ChiNC group at different temperatures.