Hydrogel Cross-Linked with Dynamic Covalent Bonding and

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Biological and Medical Applications of Materials and Interfaces

A Hydrogel Crosslinked with Dynamic Covalent Bonding and Micellization for Promoting Burn Wound Healing Ziyi Li, Fei Zhou, Zhiyong Li, Siyu Lin, Lei Chen, Lixin Liu, and Yongming Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08165 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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ACS Applied Materials & Interfaces

A Hydrogel Crosslinked with Dynamic Covalent Bonding and Micellization for Promoting Burn Wound Healing

Ziyi Lia, Fei Zhoub, Zhiyong Lia,*, Siyu Linc, Lei Chenb,*, Lixin Liua, Yongming Chena,*

a

School of Materials Science and Engineering, Center of Functional Biomaterials,

Key Laboratory of Polymeric Composite Materials and Functional Materials of Ministry of Education, GD Research Center for Functional Biomaterials Engineering and Technology, Sun Yat-sen University, Guangzhou 510275, China b

Department of Burns, First Affiliated Hospital of Sun Yat-sen University,

Guangzhou 510080, China c

School of Chemistry, Sun Yat-sen University, Guangzhou 510275, China

* Corresponding author. Tel.: 86 20 84113261 E-mail address: [email protected] (Y.M. Chen) [email protected] (L. Chen) [email protected] (Z.Y. Li)

Abstract A novel hydrogel (HA-az-F127 hydrogel) formed by reacting hydrazide modified 1

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hyaluronic acid (HAAD) and benzaldehyde terminated F127 triblock copolymers (BAF127) was developed in this work. The hydrogel with dynamic covalent chemically and micellar physically double-crosslinked networks exhibited rapid gelation and shear thinning property. Besides, the hydrogel possessed functions, such as adaptable mechanical strength, self-healability, liquid-absorption or drainage and tissue

adhesion,

which

are

important

for

wound

treatment.

Studies

on

cytocompatibility and histopathology by MTT tests, Live/Death staining and CCK-8 assay demonstrated excellent biocompatibility of the hydrogels. After applied in deep partial-thickness burn model, the hydrogel contributed effectively in promoting burn wound repair. Therefore, the HA-az-F127 hydrogel combined multiple functions in one system, demonstrating potential application in promoting burn wound healing.

Keywords: Burn wound healing; Dynamic covalent chemistry; Hydrogel; Self-healing; Wound dressing

1. Introduction Skin is a multilayer organ acting as a barrier that prevents body dehydration, defends body from pathogen invasion, and carries out biological functions.1-3 However, skin is extremely vulnerable to be damaged under burns such as heat, cold, electricity and chemicals.4 The burn wound is classified into different degrees dependent upon the thickness of burn injuries.5 When the injury extends into the dermis layer, it is a 2


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partial-thickness burn and blisters and crusts are often presented.6 During the medical treatments, wound dressings are often employed.7 The main purpose includes absorption and drainage of the exudation, protection and isolation of the injured burn sites from environment, and provision of moist plateau. The mostly used wound dressings include biological wound dressings such as allogenic amnion and synthetic tissue-engineering wound dressing8 such as Biobrane® and Transcyte®.9 However, the former has the risk of implantation rejection and virus infection, while the latter is often poor in biocompatibility and drainage, leading to risk of inflammation, infection, and scar formation.7-9 An ideal wound dressing should have adaptable mechanical strength and elasticity for recovering shape after external strain.10 Also, it should act as a barrier from invasion of the outside matters and, at the meantime, allow gaseous exchange.

Moreover,

injectability, self-healing,

tissue-adhesion,

and

exudation-absorption are also important for fitting the burn irregular wound and debridement of the necrotic tissue for promoting wound healing.10-12 Polymer hydrogel has a three-dimensional network in which water is captured and it is prepared by either chemical or physical crosslinking of polymer chains.13-15 It possesses soft tissue-like properties and acts as promising material for treatment of burns and other skin lesions.16, 17 However, conventional hydrogels often have severe limits which impair them from further application in wound dressing. For instance, many conventional biopolymer-based hydrogels are brittle, mechanically weak, and poor deformable.18-20 Additionally, the most of conventional hydrogels have no adequate tissue adhesion and, thus, it is difficult to integrate with surrounding tissue 3



after surgical operation.11,19, 21 Introduction of dynamic covalent bonding or physical associations is an effective strategy to improve the physicochemical properties of a hydrogel.22-25 Previously, we reported hydrogels with reversible/dynamic acylhydrazone bonding crosslinks,23 endowing the networks with exchanged crosslinking points for self-healability and adaptability of hydrogels. Recently, some studies have used dynamic covalent chemistry to design hydrogels for cell culture.26, 27 Micellization of amphiphilic block copolymers supplies a hydrogel with stretchability and compression performance attributed to energy consumption during hydrogel failure.25,28,29 Recently, we have reported the hydrogel combining dynamic covalent bonds with physical crosslinks, which showed excellent stretchability and toughness as well as self-healing property.25 Herein, we fabricated a novel hydrogel based on hyaluronic acid and biocompatible Pluronic F127 (PF127) utilizing dynamic acylhydrazone bonding and micellization crosslinking as an ideal material in promoting wound healing with adaptable physicochemical properties. Hyaluronic acid, a linear polysaccharide, is found in native extracellular matrix throughout the body and it also acts as a promoter of early inflammation, which is crucial in the whole skin wound healing process.30 Hyaluronic acid molecule, containing a large number of carboxyl and hydroxyl, plays an important role in water conservation for skin. F127, a nonionic triblock copolymer PEO−PPO−PEO, forms micellar solution by self-assembly at room temperature. As shown in Scheme 1, such hydrogel is formed by double-crosslinking based on acylhydrazone bonds and F127 micellization. The acylhydrazone bonds are formed by 4


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reacting hydrazine-modified hyaluronic acid (HAAD) and benzaldehyde modified F127 (BAF127) for micellization. The mixtures of HAAD and BAF127 formed hydrogels rapidly, and the obtained hydrogel showed shear thinning behavior, allowing the hydrogel to be injected and fitted to irregular burn wound. Moreover, the hydrogel dressing unites several features, like good self-healing and excellent deformability for maintaining the integrity of hydrogel, adaptable adhesion and efficient absorption and drainage of exudation for debridement of the necrotic tissue, which are important for wound dressing. We also evaluated their potential to promote the wound healing in deep partial-thickness burn model. Both macroscopic evaluation in vivo and histopathological examinations displayed that the hydrogels could be utilized as an advantageous dressing for treating burn wounds.

Scheme 1. Schematic presentation of double crosslinked HA-az-F127 hydrogel. (A) Polymer components including benzaldehyde modified F127 (BAF127) and hydrazine-modified hyaluronic acid (HAAD), (B) hydrogel architecture formed by dynamic acylhydrazone bonding and micellization crosslinking, and (C) burn site treatments involving multiple biological and physical 5



functions.

2. Materials and methods 2.1. Materials and modifications Hyaluronic acid (HA) from Bloomage Frida Biopharm Co. Ltd. (Shandong, China) and Pluronic F127 from Sigma-Aldrich (USA) were purchased and used without further purification. Adipic dihydrazide, carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were provided by Alladin (China). Cell Counting Kit -8 (CCK-8) was purchased from Keygen Biotech. Co. Ltd. (Nanjing, China). 3-(4, 5-Dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) was supplied by Sigma-Aldrich (USA). All the other reagents were used as received. HAAD and BAF127 were prepared according to the procedures described previously.23 The functionalization degree of HAAD and BAF127 was analyzed with 1H NMR (Bruker, AV400 FT-NMR). More details were given in the Supporting Information. 2.2. Preparation of HA-az-F127 hydrogels Lyophilized HAAD and BAF127 were dissolved in PBS (0.01M, pH 7.4) at a stock concentration of 10 wt% and 20 wt% (w/v), respectively. After mixing HAAD and BAF127 stock solutions at various functionality ratio (hydrazine to aldehyde) of 1/9, 3/7, 5/5, 7/3 and 9/1 at room temperature, HA-az-F127 hydrogels were formed and named as Gel1/9, Gel3/7, Gel5/5, Gel7/3 and Gel9/1, respectively. To evaluate the influence of solid content, the stock solutions were further adjusted to make the gels with different solid content. Then, 5 wt%, 7.5 wt%, and 10 wt% of HAAD solutions 6


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were mixed with 10 wt%, 15 wt%, and 20 wt% of BAF127 solutions at a fixed functionality ratio (hydrazine to aldehyde) of 5:5, respectively. The formed hydrogels were named as Gel1, Gel2, and Gel3. 2.3. Macroscopic self-healing experiments The HA-az-F127 hydrogel specimens, 10 mm in diameter and 5 mm in height, stained by Rhodamine B and Methylene Blue, respectively, were cut using a knife. Subsequently, two different pieces were placed together along their freshly cut surfaces in the original mold without external intervention. To avoid water evaporation, the mold was wrapped with a polyethylene film and then kept in a desiccator under moisture for 30 min at room temperature. At the set time, the self-healing behaviors of hydrogels were recorded with digital camera. 2.4. Rheology characterizations Rheological properties of all prepared hydrogels were studied by a rheometer with a cone and plate geometry (HAAK, D400-300CN). Viscoelastic properties of the hydrogels were measured at 1 % strain and 1 Hz. The thermo-sensitive sol-gel transition behavior was performed with a temperature cyclic step test between 4 °C and 60 °C, with a heating and cooling rate of 1 °C min-1, and the frequency (ω) and strain (γ) were held constant at 1 Hz and 1 %, respectively. The strain amplitude sweep was carried out at 37 °C and the frequency was kept as 1 Hz, the strain was changed from 10 % to 1000 % to obtain a strain failure. Finally, the continuous step change of oscillatory strain between 1 % and 300 % was conducted each two-minutes at 37 °C 7



and 1 Hz. 2.5. Swelling tests The freeze-dried hydrogel (W0) was accurately weighed and immersed in PBS solution, which was kept at 37 °C. At predetermined time intervals, the solutions were removed and the excess of water on the surface was absorbed with a filter paper. Then the remaining sample (Wt) was immediately weighed. The swelling ratio (SR) was given by (Wt − W0)/W0 × 100. The experiments were conducted triplicate. 2.6. Adhesive strength tests Bulk adhesive strength of the hydrogels was quantitatively tested by a lap shear testing.31 A testing-machine (GT-7001, Gotech, China) equipped with a 200 N load cell was used to perform the tensile tests, and the tests were carried out on different samples. On the one hand, a gelatin solution (30 wt%) was coated on a glass (25 mm × 50 mm) and dried at 37 °C for 24 h to imitate a skin tissue. Then, 200 µL of precursors of F127 hydrogel or Gel3 was applied onto the surface of the samples to form a hydrogel layer in 10 min, respectively. After that, the sample was pasted by another piece of gelatin coated glass with contacting area being 25 mm× 25 mm (Fig. 3A). On the other hand, the pieces of pig skins with an area of 25 mm × 50 mm were applied by replacing gelatin coated glass specimens. Between which, F127 or Gel3 was coated and the contact area of the two skin tissues was also kept to be of 25 mm × 25 mm. After holding for 30 min at room temperature, the samples were tested using the testing-machine, and the stretching rate was set as 10 mm min-1 at 37 °C. All 8


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measurements were conducted triplicate. 2.7. Cytocompatibility evaluation To evaluate the cytocompatibility of each component in the hydrogel, 3T3 cells were seeded in 96-well microplates at a density of 1 × 104 cells well-1 in 100 µL well-1 of DMEM medium containing 10 wt% FBS and 1 wt% penicillin/streptomycin. After incubation for 24 h at 37 °C, the culture medium was replaced with 200 µL of culture medium containing different concentrations of BAF127, or HAAD solution and co-incubated for another 24, 48, and 72 h. Then, the medium was removed and cells were washed with PBS three times. Cell viability was evaluated using the MTT based in vitro toxicology assay with a microplate reader at 490 nm (BioTek Synergy2 Gen5). The Live/Dead assay and CCK-8 assay were further used to evaluate the cytocompatibility of Gel3. 3T3 cells were seeded onto a 24-well cell culture plate at a density of 1 × 104 cells well-1 in 500 uL of complete culture media and incubated at 37 °C and 5 % CO2 for 24 h. And then, the complete culture media was replaced with a precursor solution (300 µL well-1) of Gel3 and incubated at 37 °C for 30 mins. Afterwards, the hydrogels were formed and 500 µL of fresh culture media were added for another continuous culture. At the predetermined time, 500 µL of culture medium containing 50 µL of CCK-8 dye solution was added to each well and further incubated for 2 h at 37 °C. Absorbance at 450 nm was measured using a microplate reader (BioTek Synergy2 Gen5). Cultures in medium without any sample were used as control groups. Cell viability was expressed as absorbance relative to that of control. Then, the Live/Dead staining performed using the Live/Dead Kit and fluorescent 9



images were taken by Inverted Fluorescence Microscope (Leica, DM28, Germany) with an excitation wavelength of 488 nm and 514 nm excitation channels. Live cells were stained to green and dead cells to red. 2.8. In vivo wound healing in a deep partial-thickness skin burn model In this study, all experimental procedures were approved and performed in accordance with the guidelines of the Experimental Animal Administration Committee of Sun Yat-sen University. The Sprague Dawley (SD) rats, weighing 180-200 g, were anesthetized with inhaled gas anesthesia (O2, 2 L min-1; isoflurane, 2 %) prior to surgery, and the hair on their upper back was shaved. To induce a deep partial thickness burn, an aluminum cylinder with a diameter of 20 mm was preheated at ca. 100 °C for 5 min and then placed on the rat dorsum of the rat for 10 s. Then, the rats were divided into three groups randomly, with 18 rats in each group. Namely, the control group had no further treatment, the hydrogel group was covered with Gel3, and the Mepitel® group was covered with Mepitel® dressing. Then, all the groups were covered with elastic bandages. Additionally, the dressings were renewed every two days and the picture of the burn wounds were collected on 7, 14, and 21 days with a digital camera. To calculate wound closure rate, wound area was measured by gravitational planimetry and expressed as practical wound area. The wound closure rate was calculated by the equation of (Initial wound area-Practical wound area)/Initial wound area×100%. 2.9. Histopathologic examination

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Tissue samples were collected and fixed in 10 % PBS-buffered formaldehyde solution. After fixation, samples were embedded in paraffin following by cutting into 3 mm sections. After the samples were stained with H&E, Masson's trichrome and CD68, all images were recorded by a light microscope. 2.10. Statistical analysis SPSS (Version 13.0, Chicago, IL, USA) was used to analyze statistical significance. Significant difference was reported if the P value was less than 0.05. All data were presented as means ± standard deviations. 3. Results and discussions 3.1 In-situ gelation and injectability The preparation route of HAAD and BAF127 was presented in Figure S1. BAF127 was synthesized and the aldehyde functionality degree, 99.0%, was determined by 1HNMR (Figure S2). For comparison, benzaldehyde-terminated 4-arm star PEG (TPEG) was prepared and the aldehyde functionality degree was 94.7% (Figure S3). The structure of HAAD was characterized by 1H NMR (Figure S4A). The degree of hydrazine modification, 14.5%, was estimated by the Kaiser test (Figure S4B). Preparation of HA-az-F127 hydrogels was conducted by mixing HAAD and BAF127 solutions at physiological pH under room temperature (Figure S5A). The mixtures underwent gelling process and formed transparent hydrogel within a short timescale. And the hydrogels could be directly injected through a needle into a pH 7.4 solution or in air (Figure S5B and S5C). Then, the solid content and feed ratio were 11



optimized. By time sweep tests, the gelation rate and mechanical strength of Gel1, Gel2 and Gel3 with different solid content but fixed functionality ratio of 5:5 (hydrazine to aldehyde) (Figure 1A). The Gel1 (7.5 wt%) with lowest solid content gelated at ca. 108 min (G′, G′′ crossover) (Figure S5D) and its storage modulus (G′) is close to the loss modulus (G′′) during the examined time. The G′ was as low as roughly 40.0 Pa. However, the gelation time of Gel2 (11.3 wt%) and Gel3 (15.0 wt%) decreased significantly to 10 min (Figure 1A) and 2 mins (Figure S5E). At the meantime, G′ increased remarkably to 900.0 Pa and ~3500.0 Pa (Figure 1A). Then, for Gel3, we studied the effect of ratio of functionalities (hydrazine to aldehyde) on the viscoelastic properties (Figure S6A and S6B). Herein, the Gel5/5 was the Gel3. We performed tests when the gels formed in humid environment for 24 h. The result indicated that G′, G″ and complex viscosity are the highest on ratio of functionalities being 5:5, at which the degree of crosslinking may reach maximum. Among the above hydrogels, the mechanical strength of Gel3 reached to that of natural skin tissue, whose elastic modulus G′ is in range of 1000.0 to 10000.0 Pa.32 Thus, we used Gel3 for further experiments. The present hydrogel has properties of temperature and shear induced sol-gel transformation. In order to elucidate the role of micellization, the hydrogel (HA-az-TPEG) made from 10.0 wt% HAAD and 20.0 wt% TPEG on ratio of functionalities being 5:5 was applied for comparison. Here, instead of BAF127, the four-armed PEG (TPEG) with benzaldehyde terminals was used a building block. The temperature sweep tests on Gel3 and HA-az-TPEG were conducted (Figure 1B). For 12


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Gel3, with increase of temperature, G′ jumps from ca. 300.0 Pa to 3000.0 Pa and then reached a steady value. When temperature was below 25 °C, the ratio of G″/G′ kept no change, whereas above 25 °C, the ratio G″/G′ became smaller, which suggests that the hydrogel became more elastic. In contrast, HA-az-TPEG hydrogel had much lower G′ and G″, and G″/G′ changed slightly throughout of testing temperature. Above comparison suggests that the mechanical strength of Gel3 is temperature sensitive and the transformation temperature matches the lower critical solution temperature (LCST) of F127.33 Thus, much higher strength at higher temperature was contributed by micellization of BAF127. Then, the static shear rate sweep was performed (Figure 1C), the complex viscosity of Gel3 decreased sharply with increase of shear rate. Therefore, the Gel3 had shear thinning property, attributing to the disassociation of self-assembly micelles in the gel network under shear.34,35 At the shearing rate of 20 1/s, the hydrogel showed a low viscosity, 18 Pa.s, which is low enough for injection.

Figure 1. Rheological results on (A) time sweep tests, (B) temperature sweep tests in temperature range of 4 to 60 °C in frequency of 1Hz, and (C) viscosity as a function of shear rate.

3.2 Self-healing and structure restoration We also conducted the strain sweep tests of Gel3 and HA-az-TPEG hydrogel 13



(Figure 2A). The results suggested that G′ and G″ of two samples versus strain curves similarly intersected at the strain ~115.0 %, indicating that the hydrogel transferred from solid to fluid state. With further increase of the strain to 1000.0 %, G′ dramatically decreased from ~3500.0 to ~40.0 Pa for Gel3 (400.0 to 0.5 Pa for HA-az-TPEG) due to the collapse of the hydrogel networks. Based on tests above, continuous step change of oscillatory strain between 300.0 % and 1.0 % under the same frequency (1 Hz) was performed to estimate the strain-induced damage and healing of the hydrogel (Figure 2B and 2C). We found that Gel3 and HA-az-TPEG hydrogel showed a similar behavior with a variation of strain. When the strain was of 300.0 % for 2.0 mins, G′ values of both hydrogels were lower than their G′′ value, which indicated a structure destruction. Once the strain returned to 1.0 %, G′ of two gels recovered to more than 95.0 % of the original G′ values. Therefore, both Gel3 and HA-az-TPEG hydrogel exhibited rapid recovery and showed excellent self-healing efficiency, indicating that the uncoupling and recoupling of the acylhydrazone linkages occurred dynamically in the hydrogel network. Also, we conducted direct visual experiments on self-healing. As shown in Figure 2D, two disks of Gel3 stained by Rhodamine B and Methylene Blue were cut into halves, respectively (Figure 2d1 and 2d2). Then, the two different colored samples were brought into contact along cut lines in the original mold at room temperature under moist environment for 30 min. Two hydrogel pieces merged into one hydrogel (Figure 2d3), which could withstand a tensile force from each direction without breakage and may return to its shape without strain (Figure 2d4-6). Additionally, the tensile 14


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stress-strain curve was tested, and the elongations at break of Gel3 was 2400.0 % (Figure S7A). After Gel3 experienced three continuously cyclic compression at a strain of 90.0 %, the transient recovery rate of deformation was 85.2 % (Figure S7B), indicating excellent compression-resistant property. All the results are attributed to energy dissipation from the micelle dissociation.28, 29

Figure 2. Evaluation of self-healing properties of Gel3. (A) G′ and G″ of HA-az-F127 (Gel3) and HA-az-TPEG from strain amplitude sweep (γ=1%-1000%), at a fixed frequency (1HZ). (B) and (C) G′ and G″ of Gel3 and HA-az-TPEG from the continuous step strain measurements (1 % → 300 % → 1 %). (D) Photographs of self-healing process: (d1) the as-formed hydrogel; (d2) two halves of the hydrogel in PTFE mold; (d3-d4) the self-healed hydrogel after 30 min; (d5) stretching with forceps; and (d6) recovered the shape.

3.3 Bio-adhesiveness The adhesion with surrounding materials is an important feature of hydrogels.11,19,21 The shear adhesive strength was evaluated according to the lap-shear approach suggested by ASTM F2255-05, which uses tension loading parameters to get the lap shear strength.36 The glass substrate, gelatin film or porcine skin, and the 15



hydrogel specimen were fabricated in parallel to the direction of extension as shown in Figure 3A, converting a tensile load to a shear load. The lap shear strength was calculated by equation as the tensile force divided by the overlap area. To study the effect of dynamic acylhydrazone bonding on bio-adhesiveness properties of the hydrogel, the micellar crosslinked F127 hydrogel was used for comparison. Figure 3B gives the curves of lap shear strength versus movement for Gel3 compared with the F127 hydrogel by using porcine skins as substrates. It clearly shows that the lap shear strength firstly increases quickly until a plateau and then keeps a long movement until an upturn appears. The growth regime in initial movement is attributed to the loading of tension, the upturn at late regime is attributed to the infinite area of overlaps, while the plateau value gives the lap shear strength value quantitatively. A higher plateau value suggests a stronger adhesion. Then the lap shear strength, i.e. adhesion strength, of Gel3 and F127 hydrogels on both gelatin film and porcine skin were collected in Figure 3C. It was observed that the strength of Gel3 on either gelatin film or porcine skin were all much higher than that of F127 hydrogel. Better adhesion properties of Gel3 was attributed to those free and pendant acylhydrazone bonds and aldehyde that may form hydrogen interaction and chemical bonding with the protein enriched substrates, respectively. It is noteworthy that the strength of Gel3 on gelatin is higher than that on porcine film. This is understandable since the porcine film may contain fat that may retard the interaction.

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Figure 3. (A) Schematic presentation of the adhesion tests suggested by the ASTM F2255-05. (B) Lap shear strength versus movement for Gel3 and F127 hydrogel. The substrate was porcine skin. (C) The lap shear strength for F127 hydrogel adhering to gelatin film, F127 hydrogel to porcine skin, Gel3 to gelatin film and Gel3 to porcine skin (n=3).

3.4 Liquid absorption Swelling ratio is another important parameter in burn wound dressing, which requires capacity of exudate absorption and drainage.10,37 The swelling ratio of the Gel1, Gel2, and Gel3 with time was examined as shown in Figure 4A. The Gel3 swelled up to ~2600.0 % in 5 min and remained integrate for more than ~50 h. Though swelled rapidly to ratio of 3300.0 to 4500.0 % in 3.0 min, the Gel1 and Gel2 disrupted gradually. It is explained that the Gel1 and Gel2 with relatively low solid content, i.e.

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low crosslinking density, had a higher swelling ratio. But at the higher water content, the reversible dynamic bonding may be disassociated and thus the network got disrupted. Therefore, one has to make a compromise between swelling capacity and material intactness. The Gel3 having capacity of absorption was also showed by Figure 4B. When 0.2 mL of 2.0 wt% sodium alginate (SA) solution was added to 0.1 mL of Gel3, the liquid was observed into hydrogel in 1 h. This property may benefit wound dressing hydrogels with absorption of wound exudate.

Figure 4. (A) Influence of the solid content on the swelling properties (n=3). (B) Liquid absorbable ability of Gel3: (b1) Gel3 was prepared, (b2) 2 % SA solution was added above the Gel3, and (b3) the SA solution was absorbed after 1 h later.

3.5 In vitro cytocompatibility We quantified the viability of 3T3 fibroblasts incubated with HAAD and BAF127 of different concentration by MTT assay respectively with culture media as control group. As shown in Figure 5A and 5B, the relative cell viability values of the cells

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cultured with the polymers for 24, 48 and 72 h were all higher than 90 %, indicating that both HAAD and BAF127 nearly had no cytotoxicity. To estimate the cytocompatibility of the Gel3, the quantification of cell viability was performed by CCK-8 assay. The cells cultured with the Gel3 exhibited the same proliferation with the incubation time as that only with DMEM (Figure 5C). The relative cell viability ratio after cultured with Gel3 for 24, 48 and 72 h remained higher than 85 %, relatively to the one cultured only in DMEM (Figure 5D). We further employed the Live/Dead assay for an intuitive observation of cell status shown in Figure 5E and 5F. The cells were proliferated healthily as that cultured in DMEM only. Therefore, the Gel3 displayed good cytocompatibility in vitro.

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Figure 5. In vitro biocompatibility of polymer materials. (A) HAAD and (B) BAF127. (C-F) In vitro biocompatibility of Gel3. The corresponding quantification of (C) cell proliferation and (D) cell viability performed by CCK-8 assay. Representative Live/Dead fluorescence images of 3T3 cells cultured with (E) Gel3 or (F) cultured in control group (DMEM without hydrogel).

3.6 Wound healing in a deep partial-thickness burn model Wound healing process is very complex.38 For deep partial-thickness burn, skin has some self-healing ability because there is still some healthy dermis. Therefore, the wound healing is associated with load of the crusts on burn site. Delay of the decrustation always leads to increasing local inflammatory response and prolonging wound healing.39 Tangential excision is a routine treatment for deep partial thickness 20


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burn, but the side effects are obvious, such as bleeding, surgical stress and removing extra normal tissue.40 It urgently needs an ideal wound dressing with multifunctional properties for maintaining a moist wound environment, offering protection from continuous injury and infections, clearing away wound eschar and promoting tissue healing.41 Therefore, a deep partial-thickness burn model was established for evaluation the present hydrogel dressing (Figure 6A). Mepitel®, a standard-of-care dressing, was served as a positive control.42 The representative photographs of burn wounds on day 0, 7, 14, 21 are shown in Figure 6A. As contrast to that in control group, the crust or scab in Gel3 group was not obvious, similar with that in Mepitel® group, during early healing stage. Thus, Gel3 provided better performance of autolytic debridement, which is important to aid wound closure.43 As shown in Figure 6B, wound close rate in three groups was increased with the time. At the same timeline, the Gel3 group showed relatively better performance than either Mepitel® or the control group. For example, at the 7th day, wound closure rate for Gel3 was 31.3±7.9%, while that for the Mepitel® and control groups were 18.6±4.4 % and 17.1±0.6 %, respectively.

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Figure 6. (A) Evaluation on the effect of treatments at day 0, 7, 14 or 21. (B) Wound closure rate (%) *

at day 7, 14 or 21 of treatment ( P

Hydrogel Cross-Linked with Dynamic Covalent Bonding and

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