06 3D Printing of High Strength Chitosan Hydrogel

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rsc.li/biomaterials-science
Biomaterials
 Science
rsc.li/biomaterials-science
ISSN 2047-4849
PAPER
Soo Hyun Kim et al.
Biodegradable vascular stents with high tensile and 
compressive strength, a novel strategy for applying 
monofilaments via solid-state drawing and shaped-annealing 
processes
Volume 5
Number 3
March 2017
Pages 343-602Biomaterials
 Science
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Ramezani, M. Sun, M. Xie, J. Nie, S. Lv, J. Fu, J. Cai and Y. He, Biomater. Sci., 2020, DOI:
10.1039/D0BM00896F.
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1
3D Printing of High Strength Chitosan Hydrogel 
Scaffolds without any Organic Solvents
Luyu Zhou1,2#, Hamed Ramezani1,2#, Miao Sun3, Mingjun Xie1,2, Jing Nie1,2, Shang Lv1,2, Jie 
Cai4, Jianzhong Fu*1,2, Yong He*1,2
1. State Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical 
Engineering, Zhejiang University, Hangzhou 310027, China
2. Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, School of 
Mechanical Engineering, Zhejiang University, Hangzhou 310027, China
3. Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital, School of 
Medicine, Zhejiang University, Hangzhou 310000, China
4. College of Chemistry & Molecular Sciences, Wuhan University, Wuhan 430072, China
Keywords: 3D printing, chitosan hydrogel, high strength hydrogel, tissue engineering
Abstract: 3D printing of chitosan hydrogel has attracted wide interests because of its excellent 
biocompatibility, antibacterial activities, biodegradability, none-toxicity and low cost. However, 
chitosan inks are often involved in toxic and organic solvents. Moreover, recently reported 3D 
printed chitosan scaffolds lack enough strength, also limiting their use in tissue engineering. 
Here, we reported a chitosan ink yielded by dissolving chitosan into alkali aqueous solution. This 
chitosan ink is a stable solution at low temperature (5 °C), while once heated, the chitosan chains 
will self-assemble to gelation. Based on this principle, corresponding direct ink writing (DIW) 
method was developed to print high strength chitosan hydrogel. Specifically, the chitosan ink 
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was extruded into heated deionized water to complete the in-situ gelation. The temperature of 
nozzle and hot water was well controlled to make printing stable. The rheological behavior of 
chitosan ink was investigated and the printing parameter was systematically studied to print 
chitosan hydrogel scaffolds with high quality and high strength. Based on these, high strength 
(2.31 MPa for compressive strength) and complex chitosan hydrogel structures can be directly 
printed. The cell culture and the wound healing results further show that printed chitosan 
scaffolds with this method have great potential in tissue engineering.
1. Introduction
3D printing of hydrogel1 has been widely used in areas as diverse as implant,2,3 medical 
equipment,4,5 regenerative medicine,6,7 drug delivery8 and tissue engineering.9–11 Natural derived 
hydrogels are capable for providing an aqueous environment of mimicking extracellular matrix 
(ECM),12 supporting cell attachment and proliferation.13 Recently, for the better 
cytocompatibility,14 chitosan, a polysaccharide material from deacetylation of chitin with unique 
properties such as biocompatibility, biological activity, biodegradability and none-toxicity,15,16 
has been viewed as a desirable material in tissue engineering and regenerative medicine.17 
Some tries have been reported to print chitosan hydrogels. However, most 3D printed chitosan 
hydrogel in existing reports possessed poor mechanical properties. While in order to regenerate 
tissues with tissue engineering, one needs to provide the proper environment for tissue 
regeneration including media (i.e., scaffolds, gels) that are mechanically strong enough to 
support the process of tissue regeneration.18 Numerous recent reports have proven that high 
strength hydrogel with good biocompatibility is ideal for tissue engineering.19–21 Ang et al.22 
presented the 3D printing of chitosan-hydroxyapatite scaffolds by extruding chitosan pre-gel 
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solution into NaOH coagulation bath. Gang et al.23 refined their method by using dual-nozzle to 
extrude chitosan and NaOH solution separately to achieve the in-situ crosslinking. Unlike the 
previous work that using in-situ crosslinking to achieve the printing, Wu et al.24,25 printed 
chitosan pre-solution in the air and utilized the solvent evaporation to maintain the printed shape. 
However scaffolds printed in this way were dry and uncross-linked that need additional NaOH 
coagulation bath, which may cause some shrink- induced shape deformation, affecting the 
intrinsic biocompatibility of chitosan.26,27 In addition, the printing and crosslinking process are 
separated, resulting in a long fabrication time. Overall, all these methods utilized the acid 
dissolving method to prepare chitosan pre-gel solution, which may affect chitosan’s intrinsic 
properties and increase toxicity.28–30 More importantly, numerous reports have showed that 
chitosan hydrogel prepared in this way possess poor mechanical properties.31–33
Here, in this work, we proposed a new strategy to design novel chitosan ink which is yielded 
by dissolving chitosan into alkali aqueous solution.34,35 There is no organic solvent in this system 
which will affect the intrinsic properties of chitosan and thus has been viewed as a “green” 
method.36,37 What’s more, this chitosan ink possesses unique temperature-induced gelation 
property that once heated the dissolved chitosan chains will aggregate to form a high strength 
homogeneous architecture. Based on this, a corresponding direct ink writing (DIW) method was 
designed to print high strength chitosan hydrogel. First, according the gelation principle of 
chitosan ink, a DIW printerwas developed to achieve the rapidly printing of high strength 
chitosan hydrogel without toxicity. More specifically, the alkaline chitosan ink was extruded into 
heated deionized water to complete the in-situ gelation. The temperature of nozzle was well 
controlled to keep printing process stable. And the influence of temperature of deionized water 
on mechanical properties of printed hydrogel was studied to get the best printing temperature. 
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Whilst, the rheology behaviors of the chitosan ink were investigated to prove its printability. 
Furthermore, to improve the printing resolution, the effects of printing parameters on the printing 
resolution were studied. With the optimist printing setting obtained by systematic experiments 
and analysis, complex chitosan hydrogel structures with high strength can be printed. Then, by 
testing the cell morphology and viability of 3D printed chitosan scaffolds, the biocompatibility of 
this method was verified, demonstrating its potential in tissue engineering. Ultimately, the 
wound healing results show that the printed chitosan scaffolds are ideal for tissue engineering. 
2. Results and Discussions
2.1. Printing principle of chitosan hydrogel
As shown in Figure 1(A), the dissolution of chitosan in alkali aqueous solution is based on the 
destruction of hydrogen bonds between chitosan molecular chains and the formation of new 
hydrogen bonds complexes between the macromolecules and alkali/urea under low 
temperature.35 The dissolution of chitosan was similar to that of cellulos and chitin in alkali/urea 
aqueous solution, in which these polysaccharides are associated with solvent molecules through 
the formation of new hydrogen bonds complexes between the macromolecules and 
alkali/urea.38,39 More specifically, at room temperature, the hydrogen bonds between chitosan 
chains hinder the dissolution of chitosan particles in alkali aqueous solution. However, once 
freezing, such hydrogen bonds would be destructed and at the same time, new hydrogen bonds 
complexes form between chitosan chains and alkali/urea micro molecules, leading to the 
dissolution of chitosan. Therefore, after repeating the freezing-thawing process for several times, 
all chitosan particles will dissolve and the chitosan ink is obtained. The phase change before and 
after freezing-thawing process of chitosan in alkali aqueous solution is shown in Figure 1(A). 
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This chitosan ink is stable at low temperature, but when increasing the temperature, such 
hydrogen bonds complexes will be destructed and the chitosan chains will self-assemble in 
parallel to form regenerated nanofibers and thereby construct the chitosan hydrogels.35,40 There is 
no organic solvent in the whole process, thus this method can be viewed as a “green” method. 
What is more, according to this unique temperature-induced gelation property, a corresponding 
DIW method is designed to print chitosan hydrogel, as shown in Figure 1(B). The chitosan ink 
was loaded in a syringe which was assembled into the printer with a nozzle shield. To avoid 
unexpected gelation of chitosan ink in syringe, a temperature controller equiped with a water 
cooling system was set around the syringe to ensure a homogeneous storing temperature. 
Furthermore, rapid in-situ gelation after extrusion was desirable for printing high strength 
filaments. Therefore, a heating system was implemented to maintain a stable temperature of 
deionized water for better in-situ gelation. Once the cold chitosan ink was extruded into hot 
deionized water, the temperature of chitosan ink would increase because of the huge temperature 
difference, leading to the gelation, thereby complete the in-situ printing of chitosan hydrogel.
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Figure 1. Preparation and DIW printing process of high strength chitosan hydrogel. (A) The 
schematic illustration of the material preparation of chitosan ink (B) The schematic illustration of 
the set up for printing high strength chitosan hydrogel. (C) The rheological behaviors of chitosan 
pre-gel solution: (i) Effect of temperature on storage modulus (G′) and loss modulus (G′′). (B) 
Viscosity as a function of shear rates.
To prove the in-situ gelation process during printing, the rheological behaviors of chitosan ink 
were investigated, as shown in Figure 1(C). The variation of storage modulus (G′) and loss 
modulus (G″) of chitosan ink was demonstrated with the change of temperature from 5 to 50 °C 
in Figure 1(Ci). The temperature at point of intersection of storage and loss modulus (G′= G″) is 
the gelation temperature. Below gelation temperature, the solution retains a stable sol-state for 
storing and printing. While as the temperature increases, G′ increased rapidly and crossed over 
G″. In the end, the G′ is almost 3 orders of magnitude larger than G′′, indicating the chitosan 
hydrogel is in a stable gel state. Meantime, viscosity is a key factor that influences the extrusion 
process. High viscosity can keep chitosan hydrogel as filaments before gelation instead of 
breaking or forming droplets. However, if the viscosity of printing ink is too high, it would be 
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hard to extrude through fine nozzles. Figure 1(Cii) shows the viscosity of chitosan ink as a 
function of shear rates. The viscosity of chitosan pre-gel solution at low shear rate is high, which 
is enough for printed filaments to keep continuous. Moreover, the chitosan ink showed a shear-
thinning behavior at high shear rate, which ensures it can flow readily through fine nozzles. 
Overall, these results indicate chitosan ink has appropriate rheological properties for in-situ 
gelation printing.
2.2 Printing settings for printing chitosan hydrogel 
To print chitosan hydrogel with high quality, the resolution of printed fiber under different 
printing settings were investigated. As the printing setup showed in Figure 2(A), the chitosan ink 
was extruded out from a nozzle with a diameter D by applying an air pressure of P. Meantime, 
the nozzles move at a speed V to distribute the extruded fibers. As shown in Figure 2(B) and (C), 
the printed fiber diameter d can be effectively controlled by these printing settings. The effect of 
nozzle diameter D is self-evident. A larger nozzle diameter D means a larger printed fiber 
diameter d. Moreover, due to the die-swelling effect of viscoelastic ink, the extruded fiber 
diameter is generally larger than the nozzle diameter. Researches have showed that a larger 
extrusion force (air pressure) will cause a stronger die-swelling effect.10,41,42 Therefore, the 
printed fiber diameter d is larger at high air pressure. In addition, according to the volume 
conservation, fiber diameter d will decrease at higher printing speed V due to the traction effect. 
These results indicate that byadjusting these printing settings, the resolution of printed fibers can 
be effectively controlled, laying the foundation for high quality printing. Therefore, complex 
structures such as three-dimensional five-pointed star, crescent moon and hydrogel scaffold can 
be printed with high resolution and quality, as shown in Figure 2(D)-(E). Moreover, the SEM 
images further confirm the structures of printed scaffold (Figure 2(F)-(H)).
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Figure 2. (A) Typical setup for printing chitosan hydrogels. (Bi) Effect of air pressure P on 
printed fiber diameter d. (Bii) Effect of nozzle diameter D on fiber diameter d. (C) Optical image 
of a printed five-pointed star. Scale bar: 5 mm. (D) Optical image of a printed crescent moon. 
Scale bar: 2 mm. (E) Optical image of a printed scaffold (Scale bar: 2 mm) and its (F-G) 
scanning electron microscopy (SEM) images (Scale bar: 300 and 200 μm). (H) The SEM image 
of cross-sectional structures of printed chitosan hydrogel. Scale bar: 10 μm.
2.3. Mechanical properties of printed chitosan hydrogel
For in-site gelation printing, proper sol-gel gelation rate is crucial for printing strong 
structures. Because a slow gelation rate will cause printed filaments too weak to support the 
subsequent layers, while if the gelation rate is too fast, the subsequent parts cannot adhere with 
existing parts, resulting in a weaker and even separated structure. For this chitosan ink printed 
based on temperature-induced gelation, the material concentration and printing temperature are 
two key parameters that affect the gelation rate, and thereby affect the strength of printed 
structures. Thus we printed several tensile specimens under different conditions and evaluated 
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their mechanical properties to find the best material concentration and printing temperature. 
First, the effect of concentration of chitosan on the strength of printed structures was investigated 
as shown in Figure 3(A). It can be observed that the hydrogels with 4 wt% chitosan exhibit the 
best mechanical properties. A possible explanation is that at low concentration, the percentage of 
chitosan chain is less to construct a strong network.40 While when the concentration is too high, 
although the network is stronger, the gelation rate is too fast and then the adhesion between 
adjacent fibers and layers is too weak, thereby leading to a weak overall strength. Then the effect 
of printing temperature was evaluated as shown in Figure 3(B). The results show that 60 °C of 
coagulation bath is the best. This is because that at low temperature, the gelation rate is too slow 
for in-situ gelation printing, causing a weak printed structure. Whereas the gelation rate at higher 
temperature is too fast to form a robust adhesion between fibers. Overall, based on these results, 
the concentration of chitosan was determined as 4 wt% and the temperature of coagulation bath 
was set as 60 °C for further printing and characterization.
 
Figure 3. Mechanical properties of high strength chitosan hydrogels. (A) The mechanical 
properties of printed chitosan hydrogels with different concentrations, here the temperature of 
coagulation bath is 60 °C. (I) The tensile stress-strain curves and (II) the histogram for the 
calculated Young’s modulus and strength. (B) The mechanical properties of chitosan hydrogels 
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printed at different temperatures, here the concentration of chitosan is 4 wt.%. (I) The tensile 
stress-strain curves and (II) the histogram for the calculated Young’s modulus and strength. (C) 
Comparison of mechanical properties of printed sample and mold sample: (I) tensile properties 
and (II) compressive properties.
Furthermore, we compared the mechanical properties of printed samples with those made by 
conventional method for further verification, as shown in Figure 3(C). Although for in-situ 
gelation printing, the decrease in structural strength is almost unavoidable, after systematic study 
to choose the best printing conditions, this decrease is modest and acceptable. More importantly, 
as shown in Table 1, compared with existing reports, the mechanical properties of chitosan 
hydrogels printed in this report is great. Even compared with those utilizing nanofillers or 
blended with other polymers to reinforce43–45, the results are also comparable. While these 
reinforcements sometimes partly sacrificed the intrinsic properties of chitosan. 
Table 1. Comparison of the Mechanical Properties of Chitosan Hydrogel in this Report with 
Existing Reports
Tensile CompressiveSolvent 
System
Fabrication 
Method σ(MPa) ε (%) σ(MPa) ε(%)
This work Alkali/Urea DIW Method 0.229 79 2.31 81
Chitosan Scaffolds27 Distilled Water 3D Printing 0.16 - - -
Chitin/Chitosan43 Acidic Solvent Cast - - 0.135 -
Chitosan Actuators46 Acidic 3D Printing 0.15 15 - -
Chitosan/LiOH/Urea47 Alkali/Urea Solvent Cast - - 1.78 74
Chitosan/ t-Acon acid45 Acidic Solvent Cast 1.3 -
Chitosan Scaffolds48 Acidic Solvent Cast - - 0.52 60
β-TCP/chitosan 
scaffolds44
Acidic Solvent Cast - - 1.73 -
2.4. Biocompatibility of chitosan hydrogel scaffolds
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Further, the biocompatibility of chitosan scaffolds printed with the method proposed here was 
studied. The chitosan scaffolds were seeded with HUVECs to characterize the interaction with 
cells and their capacity to allow cell growth for investigation of biocompatibility. Figure 4 
shows the fluorescent images of the HUVECs seeded on the scaffolds within a 3-day culture. 
Figure 4(Ai) demonstrates the cells could cover and attach to and the surface of the scaffold in 1-
day cell culture. The 3D image of the scaffold seeded cells is presented in the Figure 4(Aii), 
demonstrating that cells could grow along the sides of the pores. Figure 4(B) shows that the cell 
viability values on the printed scaffolds were above 90%, indicating no cytotoxicity. The Live-
Dead staining results after 3-days culture, which are imaged with the confocal fluorescence 
microscopy, are shown in Figure 4(C). Figure 4(D) shows the F-actin and nucleus morphology of 
the seeded HUVECs on the scaffold after 3-day culturing. The cells were found well attaching 
and gradually proliferating and spreading on the scaffold before forming a uniform and complete 
HUVEC monolayer. Consequently, the presented results showed that the chitosan hydrogel 
scaffold had favorable biocompatibility for cell survival, attachment and growth, which verified 
its great potential in tissue engineering. 
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Figure 4. Biocompatibilityof printed scaffolds. (A) Merge images of chitosan hydrogel scaffold 
seeded with cells. (Green: live cells, red: dead cells) (B) The cell viability values on the printed 
scaffolds. (C) The Live-Dead staining results of the chitosan hydrogel scaffold. (D) The F-actin 
and nucleus morphology of the seeded HUVECs on the scaffold (Red: F-actin, blue: nucleus).
2.5. Wound healing tests of chitosan hydrogel scaffolds
To further investigate the biocompatibility of printed high strength chitosan scaffolds in vivo, a 
typical wound healing model was constructed and investigated (Figure S1). Specifically, gauzes 
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and printed chitosan scaffolds were used to compare the wound healing effect. With 3D printing 
techniques, a porous 3D strutures can be easily fabricated, which could support a sufficient 
nutritional supply for new cells and tissues. As shown in Figure 5, it can be observed that a 
significant inflammation occurred in gauze-treated wounds on Day 3. Whereas on chitosan-
treated wounds, the formation of scabs was observed. Moreover, the wound size measurements 
throughout the treatment period shows that wounds treated with chitosan scaffolds showed a 
faster contraction (Figure 5(B)). Then, hemotoxylin and eosin (H&E) staining and Masson’s 
trichrome staining were used for histomorphological examination of the wound healing (Figure 
S2 and S3). And the quantitative analysis of the re-epithelialization and collagen production is 
shown in Figure S4. The H&E staining showed that the epithelialization process in the chitosan-
treated wounds was faster and better than gauze-treated wounds. The Masson’s trichrome 
staining showed that more newly produced collagen was deposited in chitosan-treated wounds 
than in gauze-treated wounds. These results indicate the wounds repair much faster and better in 
chitosan-treated group. Further, the immunohistochemical method was used to detect the 
expression of vascular (α-SMA 、 CD31) and pro-inflammatory factors (IL-6 、 TNF-α) in 
wounds (Figure S5 and 6). On the Day 7 and Day 14, the expression levels of neonatal blood 
officers (CD31) and mature blood vessels (α-SMA) in wounds treated with chitosan scaffolds 
were significantly higher than those treated with gauze. At the same time, the expression levels 
of pro-inflammatory factors (TNF-a, IL-6, and IL-1β) in hydrogel-treated wounds were 
significantly reduced, and anti-inflammatory factor (TGF-β1) expression was significantly 
increased. These results show that hydrogel can reduce the expression of proinflammatory 
factors and increase the expression of vascular factors and anti-inflammatory factors in rats’ 
wounds, thereby playing an important role in the wound healing process. Western blot analysis 
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of CD31, α-SMA, TGF-β1, TNF-a, IL-6, and IL-1β and mRNA level analysis further support 
these results. Because compared to the gauze-treated wounds, there were less pro-inflammatory 
factors and more anti-inflammatory and vascular factors in hydrogel-treated wounds.
Figure 5. (A) Representative photographs of the healing processes of gauze-treated group and 
chitosan-treated group. (B) The wound area as a functional of treatment days.
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Figure 6. (A) Western blot analysis of CD31, α-SMA, TGF-β1, TNF-a, IL-6 and IL-1β on Day 7 
and Day 14. (B) Quantitative analysis of CD31, α-SMA, TGF-β1, TNF-a, IL-6 and IL-1β on Day 
7 and Day 14. And the mRNA levels of (C) α-SMA, (D) CD31, (E) TGF-β1, (F) TNF-a, (G) IL-
6 and (H) IL-1β.
3. Conclusion
In this study, we demonstrated a new chitosan ink and corresponding DIW printing method for 
fabricating high strength chitosan hydrogels. The chitosan ink is obtained by dissolving chitosan 
into aqueous alkali solution and possesses unique temperature-induced gelation property. Based 
on this, a DIW printing method was developed to achieve the in-situ gelation printing of chitosan 
hydrogels. More specifically, the chitosan ink was extruded into hot water to achieve the in-situ 
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gelation. The rheological behaviors of chitosan ink were tested to study the sol-gel change of 
chitosan hydrogel during printing. To print structures with high quality, the printability and 
printing parameters was systematically investigated. In addition, the mechanical properties of 
printed chitosan hydrogel under different printing conditions were evaluated to get the best 
chitosan concentration and printing temperature. And under optimal conditions, the mechanical 
properties of printed chitosan hydrogels are great compared with existing work. Moreover, the 
results of cell culture show that the scaffolds printed with this proposed method have good 
biocompatibility. And the wound healing model further shows that the printed chitosan scaffolds 
are suitable for tissue engineering We expect that this work would promote the applications of 
chitosan hydrogels especially in tissue engineering.
ASSOCIATED CONTENT
Supporting Information. Materials and methods. Figure S1 to S4. (docx)
AUTHOR INFORMATION
Corresponding Author
*E-mail: yongqin@zju.edu.cn (Yong He).
Author Contributions
#H. R. and L. Z. contributed equally to this work.
Funding Sources
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This paper was sponsored by the National Nature Science Foundation of China (U1609207), the 
National Key Research and Development Program of China (2018YFA0703000), the Science 
Fund for Creative Research Groups of the National Natural Science Foundation of China (No. 
51821093).
Notes
The authors declare no competing financial interest. 
ACKNOWLEDGMENT
The authors thank Prof. Jun Yin and his student Qian Wu for granting the access to the 
rheometers and their help in rheological test. This paper was sponsored by the National Nature 
Science Foundation of China (U1609207), the National Key Research and Development 
Program of China (2018YFA0703000), the Science Fund for Creative Research Groups of the 
National Natural Science Foundation of China (No. 51821093). The wound-healing model of 
printed chitosan scaffolds was evaluated in male Sprague–Dawley rats (Center for Disease 
Control of Hubei Province, China) weighing 250–300 g. All animal experimental procedures 
were performed in obedience to guidelines and protocols of the Animal Experimental Ethics 
Committee of Zhejiang University.
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TOC
TOC Text:
Here, a novel direct ink printing method was created to print high strength chitosan hydrogel 
scaffolds without any organic solvents.
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