Multifunctional Hydrogels Prepared by Dual Ion Cross-Linking for

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Multi-functional hydrogels prepared by dualion crosslinking for chronic wound healing Yonghui Li, Yan Han, Xiaoya Wang, Jinliang Peng, Yuhong Xu, and Jiang Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 24 Apr 2017 Downloaded from http://pubs.acs.org on April 24, 2017

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ACS Applied Materials & Interfaces

Multi-functional hydrogels prepared by dual-ion crosslinking for chronic wound healing Yonghui Li1‡, Yan Han1‡, Xiaoya Wang1, Jinliang Peng2, Yuhong Xu2, Jiang Chang*1 1

Biomaterials and Tissue Engineering Research Center, Shanghai Institute of Ceramics,

Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, China. 2

School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240,

P.R. China Keywords: injectable, hardystonite, multi-functional, composite hydrogel, wound healing

ACS Paragon Plus Environment

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Abstract The creation of moist environment and promotion of blood vessel formation are critical for wound healing. Sodium alginate (SA) hydrogel, which has good biocompatibility and is able to provide moist environment, has been widely used as wound dressing. However, it lacks antibacterial and bio-activities, which would facilitate chronic wound healing. On the basis of gelation characteristics of SA and the bioactive hardystonite (HS) bioceramic, we designed a unique bioactive injectable composite hydrogel through double ion-crosslinking, in which divalent ions such as Ca2+ and Zn2+ function as crosslinkers, Zn2+ also functions as antibacterial component and nutrition for wound healing, and Si ions play the key role in determining the bioactivity of the hydrogel. With controlled release of divalent ions such as Ca2+ and Zn2+ from HS, the gelation process of the composite hydrogel could be efficiently controlled. In addition, in vitro results reveal that the composite hydrogel stimulated proliferation and migration of both human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs), and the in vivo results show that the wound healing process is obviously enhanced and the formation of epithelium and blood vessels are evidently advanced. This study indicates the potential of the SA/HS hydrogel as a multi-functional injectable wound dressing with the activity to inhibit bacterial growth and stimulate angiogenesis and wound healing.


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1. Introduction Severe wounds are a major health problem throughout the world1-2. Especially burdensome are chronic wounds, such as diabetic ulcers, which not only cause great suffering but also put patients at risk for severe infection and amputation3-5. There are two main ways to facilitate wound healing. On one hand, a clean and moist environment is beneficial6. On the other hand, the healing process can be accelerated by stimulating angiogenesis and addition of adequate nutrition elements7-9, since oxygen and nutrition supply are critical for chronic wound healing. In addition, avoiding infection is an important issue for wound care, in particular for chronic wound healing. Thus, it is necessary to develop bioactive materials with the ability to maintain moisture, stimulate angiogenesis, and inhibit bacterial growth to enhance the chronic wound healing process10-11. Because of their high-water content, hydrogels have been used as wound dressing materials to provide moist environment for wound healing1,

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. The sodium alginate (SA) hydrogel, a

naturally linear anionic polysaccharide extracted from brown algae 14, has good biocompatibility and is one of the most applied hydrogel materials for wound treatment13-14. Through the formation of ionic inter-chain bridges between adjacent alginate chains with multivalent cations, a SA solution can be turned to SA hydrogels15. Traditionally, a CaCl2 solution is used to crosslink SA to form SA hydrogels. However, due to the fast reaction between the Ca2+ and SA molecules it is almost impossible to create injectable SA hydrogels. Besides, the pure SA hydrogel does not have bioactivity to promote wound healing and inhibit bacterial growth16-17. Thus, the performance of this gel could be greatly improved through control of the crosslinking process to make it injectable, and the introduction of bioactive agents with the ability to stimulate wound healing and inhibit bacterial growth.


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Hardystonite (HS) is a Zn containing silicate ceramic (Ca2ZnSi2O7) that slowly releases Ca2+, Zn2+ and Si ions, of which Ca2+ and Zn2+ are known to be able to crosslink SA. In addition, Si ions have been demonstrated to stimulate angiogenesis. Furthermore, Zn2+ is well known to have antibacterial properties and is the most important part of the zinc finger protein, a key element for cell proliferation and differentiation. Previous studies have found that the zinc content of wound tissue is higher than that of the normal tissue8, 18. The lack of zinc delays wound healing, and additional supplemented zinc can promote the wound healing process8. So, our hypothesis is that if we combine SA with HS and control the ion release, we may be able to obtain an injectable multi-functional hydrogel able to stimulate wound healing and inhibit bacterial growth, which may enhance chronic wound healing. The remaining critical question is how we can effectively control the ion release from HS. In our previous experiments, we have found that silicate ceramics, including HS, create an alkaline environment when soaked in aqueous solution, and the behavior of ion release is affected by environmental pH. Therefore, a pH regulator may be able to control the ion release from silicate bioceramics. We know that acidic amino acids may affect the ion release of the silicate bioceramics by neutralizing the alkaline environment in silicate suspension. Therefore, in this study we propose the use of acidic amino acids such as aspartic acid (Asp) to regulate the ion release from HS, and investigate the formation and injectability of the SA/HS composite hydrogel, as well as its ability to stimulate angiogenesis, inhibit bacterial growth in vitro, and enhance wound healing in vivo. Figure 1 exhibits the design principal of the composite hydrogels.


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Figure 1. The design and principle of SA/HS composite hydrogel for wound healing. a) diagrammatic sketch of wound healing with hydrogel; b) HS particles release ions into solution; c) structure of SA/HS composite hydrogel. 2. Experimental section 2.1 Preparation of materials 2.1.1 Materials The main reagents used in this study included Asp (L-Aspartic acid, Sigma), Sodium alginate (low viscosity, Sigma) and CaCl2 (Sinopharm Chemical Reagent Co., Ltd). Hardystonite (Ca2ZnSi2O7, HS) powders were prepared by sol-gel method according to our previous report19. After being sieved, HS particles between 100 and 150 µm were obtained for further experiments.


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2.1.2 Preparation of the composite hydrogel The SA/HS composite hydrogels were prepared through the following process. Using a syringe, HS particles (1%, 2% and 5% of alginate aqueous weight) were dispersed into a stirred 1.5% (w/v) alginate aqueous solution. Then, Asp (0.25%, 0.5%, 0.75% and 1% of alginate aqueous weight) was added and dispersed into the alginate aqueous solution with HS in the same way. After dispersing HS particles in Asp into alginate aqueous solution, the solution was formed into a cylindrical gel using a cylindrical model. For the control experiments, pure alginate gels (without HS) were prepared by the addition of 0.1M CaCl2 solution into SA solution 20-21. 2.2 Physicochemical characterization of the composite hydrogels 2.2.1 Gelling time In order to evaluate the injectability of the composite hydrogel, the gelling time was determined as in our previous report22. Briefly, 5 mL of SA/HS gelling solution was injected into a 15-mL bottle at 37℃. The bottle was tilted ninety degrees and reset every 5 s until the composite hydrogel did not change its liquid level with tilting the bottle. The corresponding time was taken as the gelling time. 2.2.2 Mechanical properties In order to determine the mechanical strength of the SA/HS hydrogel, they were fabricated in cylindrical molds (1.0 cm in height and 1.5 cm in diameter), and tested after 12 h gelling using an Electronic Universal Testing Machine (T1-FR020 A50, Zwick, Japan) under the compressive speed of 1 mm min-1 without preload. The resulting maximum value of the stress-strain curve was recorded as the compressive strength. 2.2.3 Ion release behavior of Ca2ZnSi2O7 in Asp solution


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The ion release behavior of HS was investigated by adding 0.5% Asp and 2% HS ceramic in deionized water and incubated for 1 min, 5 min and 10 min, respectively. Then, the samples with ceramic and Asp were centrifuged twice at 1.2 ×104 g for 1 min, and the concentration of the zinc, calcium and silicon ions in supernatants were measured using inductively coupled plasma optical emission spectroscopy (ICP-OES, Varian 715-ES, USA). 2.2.5 The crosslinking ability of zinc ions and calcium ions In order to evaluate the crosslinking ability of Zn2+ and Ca2+ with SA, an experiment was conducted as follows. Briefly, 1.5% (w/v) SA solution was dropped into a Zn2+ solution with the initial concentration CZn0, a Ca2+ solution with the initial concentration CCa0, or a mixed solution containing both Zn2+(CZn0) and Ca2+ (CCa0), respectively. The volume of SA drops was V, and the volume of ionic solutions was 2V (V = 10 mL), so the ratio of SA solution/ionic solution is 1:2. Based on a prescreen experiment, we determined that the SA gelling reaction can reach equilibrium in 3 h, in which all the available crosslinking sites in SA have been reacted with ions. In order to the crosslinking degree of the gel, the gel microspheres formed in ionic solution were collected after 3 h, cut in half and photographed, and the concentrations of Ca2+ (CCaN) and Zn2+ (CZnN) in the solutions without SA gel were analyzed by ICP-OES. Finally, the amount of the ions participated in crosslinking with SA were calculated using the following equations: MZn=V·(10×CZn0-15×CZnN)/5

MCa=V·(10×CCa0-15×CCaN)/5

2.3 Antibacterial test of the composite hydrogel The antibacterial activity of composite hydrogels against Escherichia coli (E. coli) was determined by the plate-counting method. The 1 g hydrogel (composite hydrogel or pure SA


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hydrogel) was put into 10 mL solution of bacteria (4×104 CFU mL-1) in a tube with shaking (100 rpm) at 37℃ for 24 h. To determine bacteria viability, a 15 µL solution from each sample was diluted 10,000 times and plated onto a dish (diameter=10 cm) containing nutrient agar. After cultured at 37 ℃ for 12 h, the living bacteria in each dish were counted. Furthermore, a direct contact assay was conducted to evaluate antibacterial activity of the composite hydrogel, in which bacteria solution was directly seeded on the hydrogels. Briefly, 2 mL samples (SA/HS composite nutrient hydrogel, SA nutrient hydrogel or normal nutrient agar) were moved into a transwell. The hydrogel was prepared using the same nutrient solution same as for normal nutrient agar, including tryptone 10 g/L, yeast extract 5 g/L and NaCl 10 g/L. Then, 15 µL original bacterial solution (4 × 104 CFU mL-1) was moved in the transwell. Finally, after incubation for 12 h at 37 ℃, the living bacteria in each transwell were counted. Because composite hydrogels were milky, crystal violet staining of the samples was performed before the counting. C and X were the number of bacteria (CFU) in the control dish and the dish for hydrogel samples respectively. The antibacterial effect (R) was determined using the following equation: R%=100×(C-X)/C

In order to determine the cause of the antibacterial activity of the composite hydrogel, the pH of the extracts of the samples was also recorded using a pH meter (LeiCi, China). 2.4 In vitro cell culture experiments 2.4.1 Cell isolation and culture Human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) are two of the most important cells in the wound healing process. Therefore, these two types of cells


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were selected for evaluating the biological effect of the SA/HS hydrogels in this study. HDFs were isolated from the superficial layer of adult human skin as reported in the literature24, and cultured in DMEM (Gibco) supplemented with 10 vol.% FBS and 100 U mL-1 P/S. According to a method in a previous report23, HUVECs were isolated from the human umbilical cord vein, and cultured in ECM (Sciencell) supplemented with 5 vol.% FBS, 1vol.% ECGS (Sciencell) and 100 U mL-1 P/S. These two types of cells were cultured in a 5% CO2 atmosphere at 37℃.

2.4.2 Cell viability and proliferation determination In order to determine the cytocompatibility of the composite hydrogels, 100 µL extract of the composite hydrogel or pure SA hydrogel (without HS) was put into the 96-well plates, and HDFs and HUVECs were seeded at 96-well plates at 4×104 cells cm-2. Wells without hydrogels were used as the control groups. To investigate the cell viability and proliferation, cells cultured for 1, 3 and 7 days were detected by a utilizing Cell Counting Kit-8 (CCK-8, Beyotime) following the instruction of the manufacturer, and cell numbers were measured by utilizing an enzyme-linked immunoadsorbent assay plate reader (Synergy 2, Bio-TEK) at 450 nm. 2.4.3 In vitro scratch assay In order to determine the influence of the composite hydrogels on HDFs and HUVECs migration in vitro, the cells were seeded at 12-well transwell plates and cultured until confluency. Then, the cell monolayer was scrapped using a p200 pipette tip1, 25. After washing twice with fresh medium (the time was recorded as 0 h), cells were cultured for 24 h at 37℃ with low-serum medium (2 mL), which was the same as total medium except the FBS content is 1 vol.%. 100 µL composite hydrogels were put into the transwell, and the culture without hydrogel was used as the control group. At 0 h and 24 h, the cells were stained with crystal violet


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(CV) for 30min, optical images of each well were taken using an optical microscope, and the proportion of initial scratch (A0) and healing scratch (A1) were calculated using an Image J software program (NIH). The migration ratio (A) was obtained by the following equation: A=(A0-A1)/A0×100%

2.5 In vivo wound healing In this study, the Shanghai Jiao Tong University School of Medicine approved the animal experimental protocols. Balb/c nude mice, purchased from Shanghai Laboratory Animal Center, CAS (SLACCAS), were 6 weeks old at the beginning of experiments. A total of 32 mice were used for this study, and all mice were housed in Laboratory Animal Center of the Shanghai Jiao Tong University School of Medicine in a pathogen-free environment. The mice were injected with 10 µL Methylprednisolone Sodium Succinate and wounds were created by cutting off one piece of circular skin (diameter=1 cm) on dorsa region. Then, the composite gelling solution (SA/HS), pure SA hydrogel, or Recombined Human Epidermal Growth Factor Derivative (positive control) was placed on the wound area. For the negative control experiments, one group of mice had no hydrogel but were injected with 10 µL Methylprednisolone Sodium Succinate as all the others groups. Each nude mouse was placed with only one kind of sample on two wounds, and each experiment was carried out in quadruplicate. At day 3, day 7, day 14 and day 21, mice were sacrificed and the skin samples at the wound area were collected for further analysis. 2.6 Histological and immunohistochemical analysis The samples were formalin-fixed for 24 h, paraffin-embedded and cut into 7 µm sections. First, the samples were stained by hematoxylin and eosin (H ＆ E, Sigma-Aldrich) by the manufacturer instructions. The number of blood vessels was determined by counting three


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randomly selected areas. In addition, immunostaining was performed. Briefly, the sections were deparaffinized by immersing xylene, and soaked in 0.3 vol.% H2O2 in methanol for half an hour and washed twice by PBS. Then, the samples were immersed in 0.1M sodium citrate solution at 99 °C for half hour. After cooling to 25 °C, the samples were washed with PBS twice. After being blocked with 5% goat serum in PBS at 25 °C for 1 h, the sections were incubated with primary antibody at 4 °C for 12 h. In this study, the primary antibodies included CD31 (ARG52748, Arigo) and K14 (ab181595, Abcam). Under the instruction of manufacturer, the samples were washed twice by PBS, then reacted with DAB kit (Gene Tech). At last, the samples were stained by hematoxylin, dehydrated, and covered with coverslips. Images were captured by a digital camera (DMI 4000, Leica). 2.7 Statistical analysis The data shown in this study was expressed as mean ± standard deviation (SD) and analyzed using a one-way analysis of variance with a Post Hoc test. The level of a p value < 0.05 was considered statistically significant. 3. Results 3.1 Physicochemical characterization of hydrogels In order to determine the appropriate amount of HS and Asp, gelling time and compressive strength of the hydrogels with different concentrations of HS and Asp were examined, and the results are shown in Figure 2. Results show that the gelling process became faster with the increase of HS and Asp (Figure 2a), and the gelling time is controllable from 30 s to 15 min with the control of these contents, which may meet different requirements of the hydrogel for different applications. In addition, there is no difference between the gelling time of 2% HS group and 5% HS group with the change of Asp contents. Generally speaking, a gelling time


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between 3 min and 5 min might be the most suitable considering practical applications of injectable hydrogels, for which 0.25% or 0.5% Asp and 2% or 5% of HS are needed.

Figure 2. Physicochemical characterizations of SA/HS composite hydrogels. a) gelling time; b) compressive strength. Data represent means ± SD (n=5) Figure 2b shows that the compressive strength of the SA/HS hydrogels increased with the increase of HS and Asp contents. In addition, there is no significant difference between the compressive strength of 2% HS group and 5% HS group with the change of Asp contents. From the perspective of practical applications, taking into account various factors included the gelling time, compressive strength, and the amount of the additives, 0.5% Asp and 2% HS group was selected for further experiments. Ion release is one of the most critical issues in the formation and functionality of the composite hydrogels designed in this study. Therefore, the concentrations of different ions released form HS in 0.5% Asp aqueous solution were determined and the results are shown in Table 1.


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Table 1. Concentration (mM) of calcium ions, zinc ions and silicon released from HS. (n=3) Time/min

Ca

Si

Zn

1

13.61±0.22

13.37±0.13

6.42±0.18

5

21.19±0.44

20.41±0.24

10.01±0.34

10

21.88±0.25

20.84±0.32

10.24±0.29

Considering the content of each element in the ceramic (Ca2ZnSi2O7), it is clear to see from Table 1 that the release of Ca and Zn ions is in the same ratio as the molecular ratio in the ceramic. Figure 3 shows the amount of ions crosslinked with SA. It is clear that the total amount of ions crosslinked with SA increased as the initial ion concentration increased in the case of single ion crosslinking (Fig 3a). When comparing the crosslinking of different ions, there was no significant difference between Zn and Ca ions crosslinked with SA at lower initial ionic concentration (10 and 20 mM) of the crosslinking solution, while at higher initial concentration (40 mM) Ca2+ ions seem to participate more in the crosslinking than Zn2+ ions (Fig 3a). In contrast, in the case of dual ion crosslinking, the amount of ions crosslinked with SA increased when the initial concentration of ions increased from 10 mM to 20 mM, but then decreased when the initial ion concentration further increased to 40mM. Because the maximal amount of ionscrosslinking was limited by volume of SA, the amount of ions-crosslinking did not increase proportionally when initial concentration of ions in solution increased. Specially, the amount of ion-crosslinking deceased when initial concentration of ions in solution increased in third group of Figure 3b, because the initial ions concentration was so high that a layer of dense hydrogel


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covered the SA drop and stopped the reaction as seen in Figure S1b. So, the crosslinking ability of Ca2+ is slightly stronger than Zn2+.

Figure 3. The amount of ions crosslinked with SA. a) single ion crosslinking (Zn2+ or Ca2+); b) dual ion crosslinking with both Zn2+ and Ca2+, Zn2+ or Ca2+ had the same concentration. Data represent means ± SD (n=3) 3.2 Antibacterial performance of the composite hydrogel One of the important expected functions of the composite hydrogel is the antibacterial activity resulting from Zn2+ ions released from HS. The results confirmed the assumption and showed that the antibacterial rate of the SA/HS composite hydrogel was 100% in both extract assay and direct contact assay. In contrast, the pure SA hydrogel did not show any inhibitory effect on E. coli growth.


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Figure 4. Antibacterial effects of the SA/HS composite hydrogel against Escherichia coli. normal plate-counting method: a) control group; b) pure SA hydrogel group; c) SA/HS composite hydrogel group. Contact type plate-counting method: d) control group; e) SA nutrient hydrogel group; f) SA/HS composite nutrient hydrogel group. As table 2 shows, SA/HS composite hydrogel was almost neutral. So, the antibacterial property of the composite hydrogel was derived entirely from Zn2+. Table 2. pH of the pure SA hydrogel and SA/HS composite hydrogel. (n=3)

pH

SA hydrogel

Composite hydrogel

7.2±0.1

7.3±0.1

3.3 Bioactivity of SA/HS hydrogels on HDFs and HUVECs in vitro 3.3.1 Cell viability assessment of HDFs and HUVECs cultured with the hydrogels For evaluation of cytocompatibility and proliferation of cells, both HDFs and HUVECs were incubated with the extracts of hydrogels for 1 day, 3 days or 7 days. From Figure 5 it is clear to see that the composite hydrogels are biocompatible with both HDFs and HUVECs. Furthermore, the extracts of the composite hydrogels at certain concentration range can also stimulate


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proliferation of both HDFs and HUVECs, while the active concentrations are different for different type of cells. Figure 5a shows that the extracts from composite hydrogels diluted from 1/2 to 1/128 significantly stimulated HDFs proliferation at day 3 and day 7 compared to the blank group, while at day 7, even the original extract without dilution also promoted HDFs proliferation. For HUVECs, the extracts of composite hydrogels only showed activity to stimulate cell proliferation in the dilution range from 1/4 to 1/64 at day 7 (Fig 5b).

Figure 5. Cell viability assessment of HDFs and HUVECs cultured with hydrogel extracts for 7 days. a) HDFs; b) HUVECs. Data represent means ± SD (n=5). (* P < 0.05) 3.3.2 Migration of HDFs and HUVECs cultured with the hydrogels Cell migration is a critical step in the wound healing process. Figure 6 shows the results of the cell migration assay of HDFs and HUVECs in the presence of the hydrogel extracts, and it is clear to see that the composite hydrogel stimulated migration of both cell types compared with the extracts of pure SA hydrogel (Figure 6c).


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Figure 6. Migration behavior of HDFs and HUVECs with hydrogels. a) HUVECs and b) HDFs at 0 h and 24 h after scratched, BF, bright field), CV, crystal violet); c) migration ratio of HDFs and HUVECs. Scale bar=200 µm. Data represent means ± SD (n=5) (* P < 0.05) 3.4 Effect of the composite hydrogel on wounding healing in vivo Figure 7 shows wound closure at different time points. It is clear that the wound treated with the SA/HS composite hydrogel already closed with a thin layer of tissue 3 days after the treatment, while wounds in all the other groups still had a clear defect. After two weeks, although new tissue formation is obvious, the wounds of the control groups and the pure SA hydrogel group still showed incomplete healing. In contrast, the wounds treated with the SA/HS composite hydrogel showed the formation of new skin tissue that fills the whole defect,


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indicating a fast healing stimulated by the composite hydrogel. Furthermore, it is clear to see the extensive the new blood vessel formation in the newly formed skin tissues in the composite hydrogel and SA hydrogel groups at day 7 and 14 compared to control groups (Fig 7).

Figure 7. Wound closure at day 3, day 7, day 14 and day 21 after treatment with the composite hydrogel dressing.


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Figure 8. a) H＆E staining of tissue sections at day 3, day 7, day 14 and day 21. The dotted line is the interface between tissue and hydrogel. White arrows indicate blood vessels; H, hydrogel;


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NE, neo-epidermis. Scale bar=200 μm. b) Number of blood vessels at day 7, day 14 and day 21. Data represent means ± SD (n=3) (* P < 0.05) The histological results confirmed the observation of optical images of the wound samples. As seen in Figure 8a, the H&E staining showed that a neo-epidermis layer already formed at day 7 in the composite hydrogel group, while no epidermis formation could be seen in the control groups. In addition, Figure 8b revealed a significantly increased neovascularization in the regenerated tissue of SA/HS composite hydrogel treated wound than the other groups, especially at day 7 and day 14. The number of blood vessels in SA/HS composite hydrogel group decreased to the level of the other groups by day 21, indicating the remodeling stage of the healing process.


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Figure 9．Immunohistochemical staining of CD31 and K14 at day 7 and 14. The black dotted line is the interface between tissues and hydrogels. The neo-epidermis is above the white dotted line. Black arrows indicate blood vessels. H, hydrogel; WE, wound edge; W, wound. The left side of images is new organization, and the right side is center of the wound. Scale bar=200 μm. Figure 9 shows the immunohistochemical staining of CD31 and K14, which are markers for endothelia cells and keratinocytes, respectively. The results showed clearly that an intensive staining of CD 31 in the composite group indicating the stimulation of angiogenesis and enhanced blood vessel formation in the period from day 7 to day 14. In addition, the intense staining of K14 in the newly formed epithelia layer suggests the stimulation of keratinocyte migration and proliferation in the wound area. 4.Discussion Moisture maintenance is required for wound healing, and hydrogel dressings have the ability to provide a moist environment. However, most hydrogels do not have the ability to stimulate blood vessel formation and inhibit bacteria growth, which are critical for chronic wound healing. In the present study, we proposed a novel approach by utilizing bioactive ions released from a Zn-Ca-Si bioactive ceramic to crosslink SA, which results in a bioactive hydrogel with the activity to enhance blood vessel formation and inhibiting bacteria growth. Our results proved our concept and demonstrated that the SA/HS composite hydrogel indeed has the ability to not only inhibit bacteria growth, but also to stimulate angiogenesis of HUVECs in vitro and blood vessel formation in vivo during the wound healing. SA is a well-known biomaterial that can form a hydrogel through divalent ion crosslinking. The traditional way to prepare alginate hydrogel is to add alginate into CaCl2 solution20-21, but


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the gelling process is too fast to get homogeneous gelation, and the SA-CaCl2 hydrogel system is almost non-injectable. Many other divalent cations have been found to be able to crosslink alginate, including zinc. However, there is no report about the effect of the crosslinking with the combination of Ca and Zn ions, in particular in the case of a controlled release of these two ions from HS simultaneously by using acidic amino acids such as Asp. Here we demonstrated that Asp can control the ion release from HS in a dose dependent manner. The dual-ion crosslinking of SA using the combination of Ca and Zn is effective, which makes the SA/HS composite hydrogel injectable, and the gelling time and mechanical strength of the SA/HS hydrogel can be controlled by adjusting the amount of Asp, which regulates the ion release from the HS ceramics. The analysis of ion release (Table 1) also indicates that both Ca2+ and Zn2+ contributed to the gelation of the SA/HS composite hydrogel, and the proportion of the Ca2+ and Zn2+ involved in chelation is about to 2:1. One of the key points of our approaches is to endow the hydrogel with multiple functions required for enhancing wound healing by incorporation of multiple bioactive ions which not only function as crosslinkers but also play a role in wound healing stimulation. It is known that blood vessel formation and bacteria inhibition are important for wound healing. Our main hypothesis of the present study is that the bioactive ions released from HS bioceramics are not only functioning as crosslinkers, but also have the activity to stimulate angiogenesis and inhibit bacteria growth. Zinc is known as an important element in wound healing and common antibacterial agent26, so one of our assumptions is that the incorporation of Zn in the hydrogel will endow it with antibacterial activity. Our results demonstrated that, even the most of Zn2+ released by HS participated in the crosslinking with SA, the composite hydrogel indeed had excellent antibacterial activity, and this antibacterial effect is not due to the pH change as seen in


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some silicate bioceramics27, since the pH of the composite hydrogel is in the neutral range. Additionally, Si ions have been found to enhance wound healing by stimulating cell proliferation, migration and blood vessel formation28. In the composite hydrogel, we have shown that not only Ca and Zn ions, but also Si ions are released under the regulation of Asp. We also confirmed that the bioactive ions released from the HS stimulated proliferation and migration of both fibroblasts and endothelial cells, which are critical for wound healing. Furthermore, our in vivo results revealed that the composite hydrogel significantly enhanced wound healing. Histological and immunohistochemical results clearly revealed that the bioactive composite hydrogels stimulated blood vessel formation and epithelia formation, which are closely related to the activated cell proliferation and migration. 5.Conclusions Bioactive composite hydrogels were formed through dual-ion crosslinking SA with Zn2+ and Ca2+ released from HS incorporated in SA. The injectability of the hydrogel could be controlled by the addition of Asp, which regulated the ion release of HS bioceramics. The ions released from HS bioceramics not only function as crosslinkers, but also as bioactive agents, capable of not only bacterial growth inhibition, but also stimulation of cell proliferation and migration in vitro, and blood vessel formation and epithelia formation during wound healing in vivo, significantly enhancing chronic wound healing. Our results suggest that the application of bioactive ceramics with the ability to release multiple bioactive ions is an effective way to design multi-functional biomaterials, and the SA/HS composite hydrogel has great application potential for chronic wound healing.

Author information


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Corresponding Author *E-mail: [email protected] (J.C.), Tel./Fax: 86-21-52412804. Jiang Chang: orcid.org/0000-0003-1462-6541

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡The two authors contributed to the work equally.

Notes The authors declare no competing financial interest.

Supporting Information. The supporting information are available free of charge. Structure of gel microspheres, Figure S1 (PDF)

Acknowledgements Authors thank Dr. Daniel B. Shropshire from University of Texas Health Science Center, for help to improve the language of the whole manuscript. This work was supported by grants from the National Key Research Program of China (2016YFC1100201), the National Natural Science Foundation of China (Nos. 81190132, 31200714 and 31470918), the Natural Science Foundation of Shanghai Municipal (No. 12ZR1413900), Shanghai Pujiang Talent Program (No.


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13PJ1404100), and the Innovation Program of Shanghai Municipal Education Commission (No. 14ZZ032).


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TOC Graphic


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Multifunctional Hydrogels Prepared by Dual Ion Cross-Linking for

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