Adjustable and ultrafast light-cured hyaluronic acid hydrogel: promoting biocompatibility and cell growth

Qianmin Zhang a, Xiaojuan Wei a, Yongli Ji b, Li Yin b, Zaizai Dong cd, Feng Chen *a, Mingqiang Zhong a, Jian Shen b, Zhenjie Liu *b and Lingqian Chang *cd
aCollege of Materials Science and Engineering, Zhejiang University of Technology, Hangzhou 310014, P. R. China. E-mail:
bSecond Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, P. R. China. E-mail:
cSchool of Biological Science and Medical Engineering, Beihang University, Beijing 100191, P. R. China. E-mail:
dInstitute of Nanotechnology for Single Cell Analysis (INSCA), Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing 100191, P. R. China

Received 10th December 2019 , Accepted 1st June 2020

First published on 4th June 2020

Bio-sourced hydrogels are attractive materials for diagnosing, repairing and improving the function of human tissues and organs. However, their mechanical strength decreases with an increase in water content. Furthermore, it is challenging to mold these hydrogels with high precision, which significantly limits their applications. Herein, we modified a biocompatible and biodegradable material, hyaluronic acid, with methacrylic anhydride and then cured it with a four-arm star structure cross-linking agent under ultraviolet light. The hyaluronic acid hydrogel was finally cured within 15 s with an adjustable cross-linking degree. The results demonstrated that the developed gel maintained good mechanical strength with a water content of 90%, while achieving micropatterns at a precision of 20 μm. The biological experiments showed that it could effectively promote the release of vascular endothelial growth factor (VEGF), which contributed to promoting cell growth, and has favorable biocompatibility. Overall, this hyaluronic acid hydrogel is a promising biomedical material with high forming accuracy, excellent mechanical properties, and favorable biocompatibility, which indicate its potential value in a variety of tissue engineering and biomedical applications.

1. Introduction

Biomass hydrogels have been widely applied in biological science, including wound healing,1–4 tissue scaffolds5–8 and drug release.9–11 Their noticeable benefits include degradability, green sources, and biocompatibility. Hyaluronic acid (HA) is a well-known biomaterial that widely exists in the extracellular matrix (ECM). It is a non-immunogenic, degradable and highly biocompatible natural linear polymer,12,13 which plays an important role in various biological processes such as tissue engineering,14,15 drug delivery16–18 and immune regulation.19,20 Furthermore, hyaluronic acid has an excellent water retention property and the hydrogels prepared using it have many advantages such as high water content, good transparency, and biocompatibility.

Nevertheless, the conventional gelling process requires the use of crosslinking agents, involving residues of small molecules, which are usually toxic to cells. Moreover, the low modification rate of HA limits the control of the molding precision, especially for molds on the micro-scale, which makes studies at the single-cell level difficult. For example, Gozde Eke et al.6 methacrylated gelatin and hyaluronic acid under similar conditions. The methacrylation degree of gelatin achieved was 63%, whereas that of hyaluronic acid was only 25%.

In the past decade, click chemistry21,22 has been widely used in the preparation of biological hydrogels because of its high yield, mild conditions and fast reaction. Biological hydrogels often need to meet the requirements of fast curing time and good mechanical properties. Therefore, photo-crosslinking is widely used in the rapid preparation of biological gels.23 The advantages of hyaluronic acid and alginate were combined with the efficiency and rapid nature of the thiol–yne click chemistry reaction to obtain biocompatible matrices with tailored properties.24 A hyaluronic acid hydrogel with good mechanical properties was also obtained by grafting the furan group onto the amino group of hyaluronic acid and crosslinking hyaluronic acid in the presence of light. The curing time was only 30 s and the compression modulus increased to 21 kPa.25

Excellent mechanical properties and rapid prototyping of high-precision biomass hydrogels26 are now the focus in the development of biomaterials.27 In this work, we report a new crosslinking agent by reacting N-acetyl-L-cysteine (NAC) with pentaerythritol (PE). We modified hyaluronic acid with methacrylic anhydride and then cured it with the prepared cross-linking agent under ultraviolet light to achieve the rapid formation and high-precision preparation of a hyaluronic acid hydrogel with an adjustable crosslinking degree. The hydrogel had little residual molecules and maintained excellent mechanical properties, even with a water content of 90%. The hydrogel was applied in biological experiments both in vitro and in vivo, which showed that our hyaluronic acid hydrogel could effectively accelerate the release of VEGF, contributing to promoting cell growth and differentiation in tissue engineering. The excellent biocompatibility observed in the in vivo experiment indicates that the hydrogel has excellent biocompatibility and its thiol groups do not hinder the biorthogonality of living cells.

2. Experimental

2.1 Materials

Hyaluronic acid (Mw = 1[thin space (1/6-em)]000[thin space (1/6-em)]000) was purchased from Xi'an Reain Biotechnology Co., Ltd. Methacrylic anhydride (AMA), N,N-dimethylformamide (DMF), sodium chloride (NaCl), ethanol (99%), N-acetyl-L-cysteine (NAC), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and 4-dimethylaminopyridine (DMAP) were purchased from Aladdin Chemistry Co. Ltd, China. Photoinitiator (Irgacure 2959), pentaerythritol (PE), hyaluronidase from bovine testes (type I-S, 400–1000 units per mg) and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich Co. Ltd, China. All other reagents and solvents were commercially obtained at high purity grade and were used as received without further purification.

2.2 Synthesis of methacrylated hyaluronic acid (HAMA) and water-based tetrathiol crosslinker (PE(NAC)4)

Methacrylated hyaluronic acid (HAMA) was synthesized according to the reported method.28 Hyaluronic acid (HA) was dissolved in deionized water (2 wt%) to form a uniform and transparent solution. 1.5 volume of DMF was added with mechanical stirring and then the solution was cooled to 4 °C. 1.5 times the molar ratio of methacrylic anhydride (AMA) was slowly added dropwise (2 drops per second). The pH of the mixture was adjusted to about 8.5 with 0.5 M NaOH, which was maintained for 4 h. Subsequently, the mixture was maintained at 4 °C and mechanically stirred for 12 h, and then 0.5 M NaCl was added. Using ethanol as a precipitant, a white flocculent precipitate was obtained. The precipitate was washed with a mixture of water and ethanol (3/7, 1/4, and 1/9 volume ratio). Finally, the product was dissolved in deionized water (1 wt%), dialyzed for three days, and lyophilized to obtain methacrylated hyaluronic acid (HAMA). 0.01 mol L−1 NAC was dissolved in deionized water at room temperature, and 0.0001 mol L−1 EDC and 0.002 mol L−1 pentaerythritol (PE) were added. DMAP was added in portions and the mixture was left to react for 6 h. Then it was lyophilized to obtain the product PE(NAC)4.

2.3 Preparation of hyaluronic acid hydrogel (HAMA/PE(NAC)4) via UV-initiated thiol-click

HAMA was dissolved in deionized water, and then the crosslinker was added (PE(NAC)4). Then it was cured under UV light (405 nm, 5 cm) to obtain the hyaluronic acid hydrogel HAMA/PE(NAC)4. The raw materials of hydrogels of different cross-linking degrees are shown in Table 1. The curing time is defined as the time when the solution became completely gelated.
Table 1 Formulation of HAMA/PE(NAC)4 hydrogels with different degrees of crosslinking
Sample series HAMA (g) Water (mL) PE(NAC)4 (wt%) Curing time (s) Absorption ratio in normal saline (times)
HAMA/PE(NAC)4-0.5% 0.5 15 0.5 150 58.8
HAMA/PE(NAC)4-1% 0.5 15 1 60 23.1
HAMA/PE(NAC)4-5% 0.5 15 5 15 19.7

2.4 1H NMR spectra of HA and HAMA

The high-resolution proton nuclear magnetic resonance (1H NMR) spectra of HA and HAMA were obtained on a digital Fourier superconducting nuclear magnetic resonance spectrometer (Bruker AVANCE III 500 MHz) at room temperature at a 1H resonance frequency of 500 MHz with 32 scans. The methacrylation degree of HAMA was calculated via nuclear magnetic spectroscopy.

2.5 Scanning electron microscopy (SEM)

The pore structure of the methacrylated hyaluronic acid hydrogel (HAMA/PE(NAC)4) was analyzed via conventional scanning electron microscopy (SEM, VEGA3 TESCAN, Czech). The three samples (HAMA/PE(NAC)4-0.5%, 1%, and 5%) were swollen in 1× PBS buffer for 24 h, frozen in liquid nitrogen, and lyophilized. The dehydrated samples were used for the SEM test.

2.6 Swelling test

The three samples (HAMA/PE(NAC)4-0.5%, 1%, and 5%) were placed in different solutions. They were left to swell and then weighed to obtain their mass, M1. Subsequently, the samples were lyophilized and weighed to obtain mass M2. The swelling ratio was defined as follows:
Swelling ratio (%) = M2/M1 × 100%

2.7 Rheological property test analysis

The rheological properties of the three samples were measured on an advanced expansion rheometer (MCR 302, Anton Paar) equipped with a UV lamp. For time sweep assessment (constant frequency of 1 Hz and strain of 1%), 2 mL hydrogel was formed in situ with a gap of 5 mm. The UV lamp was turned on from the second minute for 5 min. The strain sweep test was performed immediately after the time sweep assessment from 0.1–1000% with a constant frequency of 1 Hz. The gelling point was defined as the point when the storage modulus (G′) increased rapidly.

2.8 Tensile and compression test

The tensile properties of the samples were measured on a high and low temperature double column test machine (Instron 5966, America). Dumbbell hydrogel samples were prepared, and the hydrogel samples were tested at a tensile rate of 5 mm min−1 until they were broken.

The compression characteristics of the samples were measured on a high and low temperature double column test machine (Instron 5966, America) using a compression fixture. Cylindrical hydrogel samples with a height of 15 mm and diameter of 20 mm were prepared using a 12-well plate as a mould. The sampling bandwidth was set to 50 Hz, and the test was continued until the sample was crushed.

2.9 Degradation in hyaluronidase

0.01 g swollen hydrogel sample was placed in 1× PBS buffer with 100 U mL−1 hyaluronidase. The degradation of the samples was carried out in a 37 °C shaker. The weight of the hydrogel was recorded at set intervals. Three replicates were performed for each sample.

2.10 Cell culture

Human umbilical vein endothelial cells (HUVECs) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Science. The cells were cultured in DMEM supplemented with 1.5 g L−1 sodium bicarbonate, 10% fetal bovine serum, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin at 37 °C in a humidified atmosphere of 5% CO2. Cells were refreshed with new medium every two days, and sub-cultured at 70–80% confluency.

2.11 Enzyme-linked immunosorbent assay (ELISA)

VEGF secretion in the cell culture supernatants was measured by ELISA, according to the manufacturer's standard protocols (SEA143Hu, CLOUD-CLONE CORP). Absorbance was read on a Multiskan FC plate reader and analyzed with SkanIt for Multiskan FC software (Thermo Fisher Scientific).

2.12 Western blot analysis

Proteins were extracted from the cultured cells using RIPA buffer and size-fractionated by denatured polyacrylamide gel electrophoresis. Membranes were incubated with antibodies against phosphoy-FAK (1[thin space (1/6-em)]:[thin space (1/6-em)]1000, #8556, Cell Signaling Technology, Danvers, MA, USA), FAK (1[thin space (1/6-em)]:[thin space (1/6-em)]1000, #71433, Cell Signaling Technology).

2.13 Immunofluorescence staining

Immunofluorescence staining was performed as previously described. HUVECs were seeded on HAMA(NAC)4 for 24 h, washed twice, and blocked with 0.5% (w/v) BSA in PBS for 15 min at 25 °C. Then, the cells were incubated with anti-F-actin (1[thin space (1/6-em)]:[thin space (1/6-em)]200, ab141420, Abcam) and anti-phosphoy-FAK (1[thin space (1/6-em)]:[thin space (1/6-em)]100, ab4792, Abcam) at 37 °C and for 1 h. The cells were washed three times and incubated with secondary antibody (1[thin space (1/6-em)]:[thin space (1/6-em)]1000, #33394, Invitrogen) together with Hoechst (1[thin space (1/6-em)]:[thin space (1/6-em)]5000, #33394, Invitrogen) at 37 °C and for another 1 h. Finally, the materials were mounted onto microscope slides. Microscope analyses were performed using a Leica TCS SP8 (Leica Microsystems Inc., IL, USA).

2.14 In vivo subendothelial transplantation

Evaluation of the biocompatibility in vivo of the HAMA/PE(NAC)4 hydrogel was performed by implanting the hydrogel into the dorsal subcutaneous connective tissue of C57BL/6 mice. All animal experiments were approved by the Animal Ethics Committee of Zhejiang University and performed according to the Animal Ethics Procedures. All mice were raised in a relative humidity of 50–60% and controlled temperature of 20–22 °C, under a 12 hour dark/light cycle and were allowed unlimited food and water. The dorsal hairs of the mice were removed by depilation so that a naked region was obtained for the operation.

The HAMA/PE(NAC)4 hydrogels were implanted in the dorsal subcutaneous tissue of the mice in the model group. The mice in the sham group received the surgery without implantation. After 1, 2 and 4 weeks, the mice were sacrificed, and the tissue surrounding the hydrogels was removed and immediately fixed in 4% paraformaldehyde for 24 h. Sections with a thickness of 5 μm were stained with hematoxylin–eosin. For immunofluorescent staining, the sections were stained with rat anti-mouse CD3 (Bio-Rad) and rat anti-mouse CD68 (Bio-Rad), respectively. The sections were further stained with Alexa Fluor 488 or 550 conjugated secondary antibodies (Invitrogen). After mounting with DAPI mounting medium, the samples were analyzed using a fluorescence microscope (Leica).

2.15 Data analysis

All data are presented as the mean ± SEM. The two-tailed Student's t-test was performed to compare the means of two groups. ANOVA followed by Bonferroni's multiple comparison was used to compare the means from three or more groups. A p-value of less than 0.05 was considered to be statistically significant.

3. Results and discussion

3.1 Preparation of HAMA/PE(NAC)4 hydrogel

The methacrylated hyaluronic acid (HAMA) was prepared according to the literature,29 and the four-arm star structure cross-linking agent (PE(NAC)4) was prepared by esterification using N-acetyl L-cysteine, which is a type of raw biological material and pentaerythritol (the characterization of HAMA and PE(NAC)4 is shown in Fig. S1 and S2 (ESI), respectively). HAMA was dialyzed and lyophilized to remove unreacted small biological molecules. HAMA and the cross-linking agent were dissolved in deionized water at different ratios according to the required formula, as shown in Table 1. A rapid curing and transparent hyaluronic acid hydrogel was prepared via the high-efficiency click chemistry of the thiol and ene group30 under violet light (λ = 405 nm) within 10 s (Fig. 1C).
image file: c9tb02796c-f1.tif
Fig. 1 Preparation of methacrylated hyaluronic acid (HAMA), crosslinking agent, and UV-cured hyaluronic acid hydrogel. (A) Hyaluronic acid (HA) and methacrylic anhydride (AMA) were co-incubated to form methacrylated hyaluronic acid (HAMA). (B) N-Acetyl L-cysteine and pentaerythritol were dissolved to form a water-soluble tetrathiol crosslinker (PE(NAC)4). (C) Methacrylated hyaluronic acid (HAMA) was mixed with the crosslinker (PE(NAC)4) under 405 nm ultraviolet irradiation to form the hyaluronic acid hydrogel (HAMA/PE(NAC)4).

3.2 Morphology, rheological and mechanical properties

After swelling the hyaluronic acid hydrogel (HAMA/PE(NAC)4) of different cross-linking degrees in PBS buffered physiological saline, their water absorption rates were determined. As expected, the sample with a high crosslinking degree (HAMA/PE(NAC)4-5%) had a low water absorption rate (nearly 20 times), while the sample with a low crosslinking degree (HAMA/PE(NAC)4-0.5%) had a high water absorption rate (about 60 times) (Table 1). The water absorption ratio could be effectively adjusted by changing the proportion of the crosslinking agent of the hyaluronic acid hydrogel. To observe the pore structure of the three hydrogels, SEM images of the lyophilized hydrogels were obtained, and expectedly, their pore size decreased as the degree of crosslinking increased (Fig. 2a–c).
image file: c9tb02796c-f2.tif
Fig. 2 Morphology, rheological properties, swelling rate, and mechanical properties of the HAMA/PE(NAC)4 hydrogel. SEM images of the swollen (a) HAMA/PE(NAC)4-0.5%, (b) HAMA/PE(NAC)4-1%, and (c) HAMA/PE(NAC)4-5% hydrogels. (d) Time sweep assessment of the HAMA/PE(NAC)4 hydrogels (0.5%, 1%, and 5%) at a frequency of 1 Hz and a strain of 1%, where UV irradiation began at the second min and lasted until the maximum storage modulus. (e) Strain sweep assessment of the UV-crosslinked HAMA/PE(NAC)4 hydrogels (0.1–1000%) at a frequency of 1 Hz. (f) Swelling ratio of hyaluronic acid hydrogels with different crosslinking degrees in salt solution with different concentrations (n = 3). (g) Tensile performance of the HAMA/PE(NAC)4 hydrogel (0.5%, 1%, and 5%) at a tensile rate of 5 mm min−1. (h) Compression performance of the HAMA/PE(NAC)4 hydrogel (0.5%, 1%, and 5%) at a frequency of 50 Hz.

The gelation and mechanical properties of the HAMA/PE(NAC)4 hydrogels with different crosslinking degrees were also investigated. Initially, the UV curing behavior and mechanical properties of the HAMA/PE(NAC)4 (0.5%, 1%, and 5%) hydrogels were evaluated via a rheological time sweep assessment (Fig. 2d). After UV irradiation, the samples began to solidify. The gelling point was defined as the point that the storage modulus (G′) increased rapidly. The gelling points of HAMA/PE(NAC)4-5% and HAMA/PE(NAC)4-1% were around 50 s and 75 s, while that of HAMA/PE(NAC)4-0.5% was around 130 s. This indicates that the higher the content of crosslinker in the sample, the earlier the gelling point. As the concentration of the cross-linking agent increased, the chemically active sites in the thiol–ene click reaction increased. This led to a rapid response and an early gelling point. Comparing with the curing time in Table 1, the curing time was prolonged in the rheological test, which is because in the rheological test environment, ultraviolet light could not be fully irradiated to the sample. Further, the solidified samples were subjected to a strain sweep assessment (Fig. 2e). It was found that the viscoelastic region of the HAMA/PE(NAC)4-1% was wider than that of HAMA/PE(NAC)4-5%. The elastic modulus, G′, of HAMA/PE(NAC)4-5% was about 6 kPa, and that of HAMA/PE(NAC)4-1% and HAMA/PE(NAC)4-0.5% was about 4 kPa and 3.3 kPa. When deformation occurred, as the concentration of the crosslinking agent increased, the sample became harder, and the elastic modulus was small.

The HAMA/PE(NAC)4 hydrogels were swollen with different salt solutions to investigate the effect of common cations on the water absorption ratio (Fig. 2f). The results showed that different cations have an effect on the water absorption rate but with no significant difference (Na+ > K+ > Ca2+). The swelling ratio of the hydrogel was mainly controlled by the crosslinking degree and the concentration of the liquid, where the degree of cross-linking was the dominant factor. The swelling ratio showed liquid concentration-dependent behavior only at a lower crosslinking degree (the HAMA/PE(NAC)4-0.5% gel swelled in normal saline up to about 60 times, and only about 20 times in 1 M sodium chloride solution). When it had a high degree of cross-linking, the influence of the liquid concentration was weakened (the swelling ratio of HAMA/PE(NAC)4-0.5% in different concentrations of liquid was almost the same).

To further characterize the mechanical properties of the HAMA/PE(NAC)4 hydrogels, tensile tests and compression tests were carried out (Fig. 2g and h). In the tensile test, HAMA/PE(NAC)4-1% exhibited a soft and tough property with a yield strength of 0.65 kPa, and elongation at break as high as 500%. The yield point of HAMA/PE(NAC)4-0.5% was only 0.4 kPa, and then the tensile stress decreased rapidly with fracture. HAMA/PE(NAC)4-5% and HAMA/PE(NAC)4-1% exhibited almost the same elastic modulus before the yield point due to the increase in crosslinking degree, which was about 200% that of HAMA/PE(NAC)4-0.5%. After the HAMA/PE(NAC)4-5% gel reached the yield point due to the high crosslinking degree, the tensile stress increased further after the yield point, but the necking process was significantly shorter than that of the HAMA/PE(NAC)4-1% gel (Fig. S3, ESI). As shown in Fig. S4 (ESI), the maximum compressive stress of HAMA/PE(NAC)4-0.5% and HAMA/PE(NAC)4-1% was 40.85 kPa and 44.96 kPa, respectively. The maximum compressive stress of HAMA/PE(NAC)4-5% was as high as 69.28 kPa, which was about 160% of that of the other two samples. Furthermore, the elastic modulus of the samples with a low crosslinking agent content was 119.5 kPa and 133.83 kPa, respectively. The elastic modulus HAMA/PE(NAC)4-5% reached 454.44 kPa, which increased by 260% compared to that of the other two samples. These comprehensive results indicate that the sample with a high degree of crosslinking had high pressure resistance. Most importantly, the rapid cure characteristics and storage modulus of the HAMA/PE(NAC)4 hydrogel meet the range of human soft tissue, which makes it particularly attractive for regenerative tissue medicine.

3.3 High-precision patterning

To obtain high-precision molded HAMA/PE(NAC)4 hydrogels, we used a micro-structured surface as a template. Different molding precisions were achieved by adjusting the degree of crosslinking of the hydrogel. After a few attempts, a 5% higher strength sample was applied to the surface of the micropattern template, and the precise pattern could be successfully reproduced by rapid initiation of curing via exposure to ultraviolet light. The surface of the sample after engraving the micropattern was observed using an inverted fluorescence microscope (Fig. 3a–f). The size of the sample was reproducible, and the flow path was structurally intact, ultimately retaining the template lines (Fig. 3a and b). More precise dot patterns (point diameters of about 25 μm) could also be successfully re-encoded (Fig. 3c). The magnified view showed that the engraved pattern was intact and the lines were smooth (Fig. 3d–f).
image file: c9tb02796c-f3.tif
Fig. 3 High-precision patterning of the HAMA/PE(NAC)4 hydrogel. (a) and (d) are replicated flow channel (width 50 μm) by HAMA/PE(NAC)4-5%; (b) and (e) are the replicated flow channel (width 20 μm) by HAMA/PE(NAC)4-5%; and (c) and (f) are the replicated periodic lattice (diameter 20 μm) by HAMA/PE(NAC)4-5%. (g) Channel fidelity and circularity of HAMA/PE(NAC)4-5% and HAMA/PE(NAC)4-1%.

The reprinting fidelity is defined as the ratio of the replica channel width to the original template width, and 1 represents 100% reprinting. The circle area of the lattice is divided by the square of the circumference of the circle as the roundness, and 1 represents the positive circle. Compared with HAMA/PE(NAC)4-1%, the channel with HAMA/PE(NAC)4-5% provided higher fidelity, and its roundness approached 1 (Fig. 3g). The mechanical properties of the samples increased with an increase in the crosslinking degree, which is beneficial for the replication of the path. The HAMA/PE(NAC)4-1% sample was distorted because its texture was relatively soft, resulting in tearing of hydrogel during the sampling process. HAMA/PE(NAC)4-0.5% had a low modulus and soft texture, which made it difficult to reproduce in a stable path. Thus, the fabricated hydrogel is expected to play an essential role in the application of high-precision hydrogel devices when its degree of crosslinking is appropriately high.

3.4 In vitro loading and release of VEGF

Subsequently, we investigated the effects of the fabricated hydrogel on cell growth. To explore the biocompatibility of the HAMA/PE(NAC)4 hydrogels interacting with cells, human umbilical vein endothelial cells (HUVECs) were cultivated on their surface. As shown in Fig. 4a, compared with the control group, the HUVECs cultured on HAMA/PE(NAC)4-0.5% and HAMA/PE(NAC)4-1% showed the typical morphology, while the surface cells of HAMA/PE(NAC)4-5% showed a poor morphology, indicating that the growth of cells was hindered with a high degree of crosslinking.
image file: c9tb02796c-f4.tif
Fig. 4 Biocompatibility of HAMA/PE(NAC)4 hydrogels. (a) Immunofluorescence staining of p-FAK in human umbilical vein endothelial cells (HUVECs) cultured on HAMA/PE(NAC)4 hydrogels (0.5%, 1%, and 5%) at 37 °C (n = 3). (b) Western blotting of p-FAK and FAK in HUVECs cultured on the HAMA/PE(NAC)4 hydrogels (0.5%, 1%, and 5%). (c) VEGF stably released from the HAMA/PE(NAC)4 hydrogels (0.5%, 1%, and 5%)-cultured HUVECs at 37 °C (n = 3). (d) Degradation curves of the HAMA/PE(NAC)4 hydrogels (0.5%, 1%, and 5%) at 37 °C (n = 3).

Besides, we examined whether the surface of the HAMA/PE(NAC)4 hydrogel altered the Y397 phosphorylation level of focal adhesion kinase (FAK), a major phosphorylation-activated cell surface protein, which regulates VEGF-A synthesis and release. We observed the down-regulation of FAK pY397 in the HUVECs seeded on the HAMA/PE(NAC)4-0.5% and HAMA/PE(NAC)4-1% group compared with the polypropylene dish and HAMA(NAC)4-5% group (Fig. 4b). The tunable-EM HAMA/PE(NAC)4-0.5% and HAMA/PE(NAC)4-1% created a “soft” extracellular matrix cytoskeleton to mimic the physiological microenvironment to culture HUVECs, while HAMA/PE(NAC)4-5% represented a stiffness condition which prevented cell attachment.

Based on our previous study,31 the production of VEGF can be modulated by the elastic modulus (EM) of the cell-adhered material, and thus the soft material showed the ability to inhibit the over-expression of VEGF-A in vitro. Fig. 4c shows that the HUVECs cultured on the three samples could effectively release VEGF. The VEGF released in HAMA/PE(NAC)4-0.5% was slightly lower than that in HAMA/PE(NAC)4-5%, while that in HAMA/PE(NAC)4-1% was the highest. This is due to the excessive cross-linking of HAMA/PE(NAC)4-5%, leading to the high modulus of the sample, which affected cell growth. Besides, the UV crosslinked HAMA/PE(NAC)4 hydrogel exhibited degree of crosslinking-dependent degradation behavior in 1× PBS buffer. For the three samples, the higher crosslinking degree, the lower the degradation rate (Fig. 4d). All three samples showed good degradation stability and required at least two weeks to degrade completely.

The sample with a high crosslinking degree completely degraded after three weeks. Thus, the results indicate the HAMA/PE(NAC)4-1% is a promising wound dressing material in the fields of tissue engineering and reconstructive medicine.

3.5 In vivo subendothelial transplantation

We finally conducted an in vivo test on animal models. The histological response of the HAMA/PE(NAC)4 hydrogel implanted into the subcutaneous tissue was analyzed by H&E staining and immunofluorescent staining (Fig. 5). One week after implantation of the HAMA/PE(NAC)4 hydrogel, the inflammatory reaction was mild compared with the sham group. However, the severity of inflammation decreased after two weeks, and almost no inflammatory cells were observed at four weeks. Two weeks after implantation, the hydrogels degraded, and the inflammatory reaction was alleviated. The immunofluorescent analysis indicated the changes in the quantity and distribution of the cells due the inflammatory reaction of subcutaneous tissue (Fig. 5B and C). Macrophages and lymphocytes are immune cells that participate in the immune response after activation. In the first week, a large number of lymphocytes (marked by CD3) and macrophages (marked by CD68) appeared in the subcutaneous tissue. In the second week, the number of inflammatory cells decreased significantly. It is reported in the literature that the thiol toxicity of modified bio-macromolecules will affect the bio-orthogonality of bio-hydrogels in living tissues.32 However, after four weeks, the presence of rare inflammatory cells indicates that the HAMA/PE(NAC)4 hydrogel has no adverse effect on living organisms after implantation, which shows its excellent biological potential. Thus, combined with the high precision forming of the hydrogel, the application of this hyaluronic acid hydrogel can be extended to the field of biochips and high precision 3D printing of biological tissue model.
image file: c9tb02796c-f5.tif
Fig. 5 In vivo biological experiments of implanting the hydrogels in mice. (A) Photomicrographs of H&E staining in subcutaneous tissue around the hydrogels after 1, 2 and 4 weeks of implantation, where the scale bar is 100 μm. (B and C) Immunofluorescent staining of the subcutaneous tissue stained by CD3 (B) and CD68 (C) after 1, 2 and 4 weeks of implantation, where the scale bar is 50 μm.

4. Conclusion

In this work, we reported a fully bio-sourced rapid curing hyaluronic acid hydrogel (HAMA/PE(NAC)4). This hydrogel was initiated by ultraviolet light through the rapid click-chemistry of thiol and ene. The hydrogel could be rapidly cured within 15 s. By adjusting the concentration of the four-arm star structure tetrathiol crosslinking agent, HAMA/PE(NAC)4 hydrogels with different degrees of crosslinking and different water absorption ratios were obtained. We further studied the effect of different ions on the water absorption rate of the hydrogels. The species and concentration of cations both affected the water absorption rate of the hydrogels, but these effects were not apparent. Rheology tests were performed to confirm the sensitivity of the HAMA/PE(NAC)4 hydrogel to ultraviolet light, and we discussed its mechanical properties initially. The compression test further verified that the compressive stress of the hydrogel could reach 70 kPa, which is similar to the vessel strength. Degradation under hyaluronidase conditions revealed that the hydrogel was degraded entirely in about 3 weeks. The degradation time could be easily controlled by adjusting the degree of crosslinking. Also, the hydrogel was successfully reproduced in a micro-scale flow path structure and lattice structure, indicating it can be molded on the micro-scale. The in vitro biological experiments using the hyaluronic acid hydrogels showed that these hydrogels could promote the release of VEGF, and the cells maintained a good morphology and growth on the surface of the hydrogels. Meanwhile, the in vitro biological experiments also proved that the hydrogels have excellent biocompatibility in animals. Ultimately, the hyaluronic acid hydrogel is expected to show potential value in a variety of tissue engineering and biomedical applications.

Conflicts of interest

There are no conflicts to declare.


This study was funded by the Natural Science Foundation of China (No. 81670433 and No. 81970398), the Natural Science Foundation of Zhejiang Province (No. LY18E030009 and LY19E030007), the Medical Scientific Research Foundation of Zhejiang Province (No. 2016KYA097). L. C. acknowledges the funding from the Institute of Nanotechnology for Single Cell Analysis (INSCA), Beijing Advanced Innovation Center for Biomedical Engineering.


  1. Y. Hong, F. F. Zhou, Y. J. Hua, X. Z. Zhang, C. Y. Ni, D. H. Pan, Y. Q. Zhang, D. M. Jiang, L. Yang, Q. N. Lin, Y. W. Zou, D. S. Yu, D. E. Arnot, X. H. Zou, L. Y. Zhu, S. F. Zhang and H. W. Ouyang, Nat. Commun., 2019, 10, 2060–2070 CrossRef PubMed .
  2. N. K. Dehkordi, M. Minaiyan, A. Talebi, V. Akbari and A. Taheri, Biomed. Mater., 2019, 14, 035003 CrossRef CAS PubMed .
  3. B. Kocaaga, O. Kurkcuoglu, M. Tatlier, S. Batirel and F. S. Guner, J. Appl. Polym. Sci., 2019, 136, 47640 CrossRef .
  4. A. Ehterami, M. Salehi, S. Farzamfar, H. Samadian, A. Vaez, S. Ghorbani, J. Ai and H. Sahrapeyma, Chitosan/Alginate Hydrogels Containing Alpha-Tocopherol for Wound Healing in Rat Model, J. Drug Delivery Sci. Technol., 2019, 51, 204–213 CrossRef CAS .
  5. A. P. Haring, E. G. Thompson, Y. X. Tong, S. Laheri, E. Cesewski, H. Sontheimer and B. N. Johnson, Biofabrication, 2019, 11, 025009 CrossRef CAS PubMed .
  6. G. Eke, N. Mangir, N. Hasirci, S. MacNeil and V. Hasirci, Biomaterials, 2017, 129, 188–198 CrossRef CAS PubMed .
  7. Y. Zou, L. Zhang, L. Yang, F. Zhu, M. M. Ding, F. Lin, Z. Wang and Y. W. Li, J. Controlled Release, 2018, 273, 160–179 CrossRef CAS PubMed .
  8. P. T. Kuhn, R. T. Rozenbaum, E. Perrels, P. K. Sharma and P. van Rijn, Polymers, 2017, 9, 149–157 CrossRef PubMed .
  9. Z. Wang, Y. Duan and Y. W. Duan, J. Controlled Release, 2018, 290, 56–74 CrossRef CAS PubMed .
  10. Z. G. Huang, F. M. Lv, J. Wang, S. J. Ca, Z. P. Liu, Y. Liu and W. Y. Lu, Int. J. Pharm., 2019, 556, 217–225 CrossRef CAS PubMed .
  11. Y. Wang, W. Wang, R. Xu, M. F. Zhu and D. Yu, Flexible, Chem. Eng. J., 2019, 360, 817–828 CrossRef CAS .
  12. H. Ying, J. Zhou, M. Wang, D. Su, Q. Ma, G. Lv and J. Chen, Mater. Sci. Eng., C, 2019, 101, 487–498 CrossRef CAS PubMed .
  13. M. Taz, P. Makkar, K. M. Imran, D. W. Jang, Y. S. Kim and B. T. Lee, Mater. Sci. Eng., C, 2019, 99, 1058–1066 CrossRef CAS PubMed .
  14. W. B. Jia, M. Li, L. Z. Kang, G. F. Gu, Z. W. Guo and Z. G. Chen, J. Mater. Sci., 2019, 54, 10871–10883 CrossRef CAS .
  15. H. Kenar, C. Y. Ozdogan, C. Dumlu, E. Doger, G. T. Kose and V. Hasirci, Mater. Sci. Eng., C, 2019, 97, 31–44 CrossRef CAS PubMed .
  16. R. Yegappan, V. Selvaprithiviraj, A. Mohandas and R. Jayakumar, Colloids Surf., B, 2019, 177, 41–49 CrossRef CAS PubMed .
  17. N. Sahiner, S. S. Suner and R. S. Ayyala, Colloids Surf., B, 2019, 177, 284–293 CrossRef CAS PubMed .
  18. T. J. Yin, J. Y. Liu, Z. K. Zhao, Y. Y. Zhao, L. H. Dong, M. Yang, J. P. Zhou and M. R. Huo, Adv. Funct. Mater., 2017, 27, 1604620 CrossRef .
  19. C. X. Li, Y. Zhang, X. Dong, L. Zhang, M. D. Liu, B. Li, M. K. Zhang, J. Feng and X. Z. Zhang, Adv. Mater., 2019, 31, 1807211 CrossRef PubMed .
  20. S. Yu, Y. Y. Duan, X. G. Zuo, X. Y. Chen, Z. W. Mao and C. Y. Gao, Biomaterials, 2018, 180, 193–205 CrossRef CAS PubMed .
  21. H. Wang, G. Y. Zha, H. Du, L. L. Gao, X. D. Li, Z. Q. Shen and W. P. Zhu, Polym. Chem., 2014, 22, 6489–6494 RSC .
  22. Z. Zhang, J. Du, Y. L. Li, J. C. Wu, F. Yu and Y. Chen, J. Mater. Chem. B, 2017, 30, 5974–5982 RSC .
  23. Y. B. Zhang, S. Y. Liu, T. Y. Li, L. Q. Zhang, U. Azhar, J. C. Ma, C. C. Zhai, C. Y. Zong and S. X. Zhang, Carbohydr. Polym., 2020, 236, 116021 CrossRef CAS PubMed .
  24. M. M. Perezmadrigal, J. E. Shaw, M. C. Arno, J. A. Hoyland, S. M. Richardson and A. P. Dove, Biomater. Sci., 2020, 1, 405–412 RSC .
  25. G. Wang, X. D. Cao, H. Dong, L. Zeng, C. X. Yu and X. F. Chen, Polymers, 2018, 10, 949 CrossRef PubMed .
  26. J. H. Guo, J. J. Duan, S. Q. Wu, J. M. Guo, C. Huang and L. N. Zhang, J. Mater. Chem. B, 2017, 5, 8446–8450 RSC .
  27. Y. Z. Zhu, J. Shen, L. Yin, X. J. Wei, F. Chen, M. Q. Zhong, Z. Gu, Y. Xie, W. Jin, Z. J. Liu, C. Chitrakar and L. Q. Chang, Chem. Eng. J., 2019, 366, 112–122 CrossRef CAS .
  28. J. L. Guo, Y. S. Kim and A. G. Mikos, Biomacromolecules, 2019, 20, 2904–2912 CrossRef CAS PubMed .
  29. L. Messager, N. Portecop, E. Hachet, V. Lapeyre, I. Pignot-Paintrand, B. Catargi, R. Auzely-Velty and V. Ravaine, J. Mater. Chem. B, 2013, 1, 3369–3379 RSC .
  30. X. J. Wei, J. Shen, Z. Gu, Y. Z. Zhu, F. Chen, M. Q. Zhong, L. Yin, Y. Xie, Z. J. Liu, W. Jin, M. Nouri and L. Q. Chang, ACS Appl. Bio Mater., 2018, 1, 2167–2175 CrossRef CAS .
  31. J. Shen, Y. Xie, Z. J. Liu, S. N. Zhang, Y. P. Wang, L. L. Jia, Y. D. Wang, Z. J. Cai, H. Ma and M. X. Xiang, J. Mol. Cell. Cardiol., 2018, 122, 140–151 CrossRef CAS PubMed .


Electronic supplementary information (ESI) available. See DOI: 10.1039/c9tb02796c
Qianmin Zhang, Xiaojuan Wei, Yongli Ji contributed equally to this work.

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