Novel enzymatically crosslinked chitosan hydrogels with free-radicalscavenging property and promoted cellular behaviors under hyperglycemia

Dieu Linh Tran ,Phuong Le Thi ,Thai Thanh Hoang Thi ,Ki Dong Park

a Department of Molecular Science and Technology,Ajou University,Suwon,16499,Republic of Korea

b Biomaterials and Nanotechnology Research Group,Faculty of Applied Sciences,Ton DucThang University,Ho Chi Minh City,700000,Viet Nam

Keywords:

ABSTRACT

1.Introduction

The high blood-glucose level (hyperglycemia) associated with overproduction of reactive oxygen species (ROS) have promoted diabetic complication progression and development,such as delayed wound healing,cardiovascular complications,neuropathy,nephropathy,and retinopathy [1,2].It was indicated that ROS-induced oxidative stress triggered by hyperglycemia is the main reason for many pathways leading to cellular death,including pro-inflammation,procoagulation,apoptosis,and impaired nitric oxide release [3,4].In addition,cell migration and proliferation are also impaired under hyperglycemia-induced oxidative stress.Recent studies have demonstrated that the migration of fibroblast cells,the most important cells involved in producing and remodeling the extracellular matrix,were reduced 75% in hyperglycemia,compared with those in normal condition [5].Therefore,biomaterials that can both downregulate ROS production and improve cell migration under hyperglycemia play a pivotal role in mitigating the development of diabetic complications[6—8].

Chitosan (CS),a derivative of chitin,is the most abundant polysaccharide beside cellulose used in the biomaterial field [9].Because of its diverse biological activities,including antioxidant,biocompatible,biodegradable,antimicrobial,and hemostatic properties,chitosan has been widely used in various applications,ranging from the food industry to tissue engineering [10].Recently,chitosan-based hydrogels have been demonstrated to be ideal materials for treating diabetic wounds[11—14].The hydrogels can serve as both wound dressing to maintain a moist environment for the wound bed and as intracellular drug depot to deliver therapeutic agents (antibiotic drugs,growth factors,stem cells,etc.),therefore accelerating wound healing.Particularly,injectable chitosan hydrogels are advantageous because they are minimally invasive and precisely administered at the target site,with accurate filling of any irregular wounds.

To design the injectable hydrogels,the choice of crosslinking method is of utmost importance.Generally,chitosan-based hydrogels can be fabricated by physical or chemical crosslinking,and each method has its own advantages and limitations[9,10,15].For instance,physical crosslinking methods,such as hydrogen bonding,chain aggregation,ionic interactions,hydrophobic association,or crystallization,are simple and nontoxic,but require the specific reaction condition (temperature,pH,light,etc.).On the other hand,permanent and irreversible chemical bonding using chemical agents (glutaraldehyde,N,N-methylene-bisacrylamide,glyoxal,and genipin),or photopolymerization and radical polymerization can provide gels with high mechanical strength but impaired biocompatibility [16,17].Recently,enzymatic-triggered crosslinking reactions have been highly favorable because of their high mechanical stability without increasing the cytotoxicity of the resulting hydrogels [18,19].Among them,crosslinking reaction mediated by horseradish peroxidase (HRP)/hydrogen peroxidase (H2O2) has been described as an efficient way to prepare injectable hydrogels,given their mild reaction conditions,rapid hydrogelation,and ease of tuning hydrogels’ properties [20,21].The coupling of phenol-conjugated chitosan derivatives by means of an enzymatic crosslinking reaction of HRP/H2O2to obtain the injectable chitosan hydrogels has been reported previously [18,22,23].These hydrogels have been suggested to be ideal injectable biomaterials as drug-delivery carrier,tissue adhesives,wound dressings,or bone/cartilage engineering.However,the intrinsic ability of HRP-crosslinked injectable chitosan hydrogels for scavenging excessive ROS and promoting cellular activities under hyperglycemia has not been investigated.

Herein,we created a new injectable chitosan hydrogel that can serve as a bioactive scaffold for regulating hyperglycemia-induced oxidative stress in diabetes-related diseases.Gallic acid (GA) is a powerful polyphenol with strong antioxidant activities because of its three hydroxyl groups,which play a key role in cleaving free-radical chain reactions [24,25].Moreover,under the hyperglycemia environment,GA can accelerate wound healing by promoting cell migration[26].It was also demonstrated previously that the pyrogallol moiety of polyphenols (e.g.,tannic acid,EGCG) can form hydrogels via intermolecular hydrogen bonds and coordination bonds,and the gelation was faster in the presence of HRP [27].We are therefore interested to see if HRP can crosslink the chitosan-gallic acid conjugates into hydrogels instead of the commonly used phenol-conjugated chitosan.To the best of our knowledge,this is the first study to use GA as both antioxidant and crosslinking moieties to fabricate HRP-crosslinkable chitosan hydrogels (Fig.1).We synthesized the chitosan-gallic acid conjugate (GGA) and investigated the physiological properties of the resulting hydrogels (e.g.gelation time,mechanical strength,and degradation time) by varying the concentrations of HRP and H2O2.To further explore the potential of GGA hydrogels for diabetic complication treatment,we evaluated the effect of CGA hydrogels on scavenging excessive ROS levels and on proliferation and migration of human dermal fibroblast cells under hyperglycemia.

2.Materials and methods

2.1.Materials

Chitosan (medium molecular weight,75—85% deacetylated),gallic acid(GA,3,4,5-Trihydroxybenzoic acid),horse radish peroxidase(HRP,type VI,250—330 units per mg solid),hydrogen peroxide (H2O2),1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC),and N-hydroxysuccinimide(NHS)were obtained from Sigma Aldrich(St.Louis,MO,USA).Penicillin-streptomycin (P/S),trypsin/ethylenediaminetetraacetic acid (EDTA),fetal bovine serum (FBS) and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Gibco BRL (Grand Island,NY,USA).All other chemicals and solvents were used such without further purification.

2.2.Synthesis and characterization of chitosan-gallic acid(CGA)conjugate

Chitosan-gallic acid (CGA) conjugate was synthesized by means of an EDC/NHS-mediated coupling reaction between the amine groups of chitosan and the carboxyl groups of gallic acid [28].Briefly,1 g of chitosan was dissolved in 100 mL of diluted hydrochloride acid (HCl,pH 3) and stirred overnight to allow complete dissolution.Afterwards,the pH was adjusted to 4.5 by using diluted HCl.EDC(3.8 g,20 mmol)and NHS(2.3 g,20 mmol)were added to GA(1.7 g,10 mmol)dissolved in the co-solvent of deionized water and DMF (volume ratio 3:2),followed by stirring for 1 h to activate the carboxyl groups of GA.The activated GA was then added to the CS solution and stirred for 24 h at room temperature.After the reaction,the resulting solution was dialyzed against deionized water for 3 days using the dialysis membrane(MWCO=3.5 kDa).Finally,the dialyzed solution was filtered and freeze-dried to obtain the sponge-like CGA conjugate.

CGA conjugates were characterized by UV—Vis spectra using a UV spectrophotometer (V-750 UV/vis/NIR,JACO,Japan) and1HNMR spectra using nuclear magnetic resonance spectroscopy (AS400,OXFORD instruments,UK).

Fig.1.In situ forming of CGA hydrogel through HRP/H2O2-mediated crosslinking for improving the antioxidation and cell migration under hyperglycemia.

The degree of substitution of GA groups (GA content) on the chitosan backbone was evaluated using the Folin-Ciocalteu method[29,30].Briefly,1 mL of Folin-Ciocalteu reagent solution was mixed with 1 mL of CGA solution(1 mg/mL)at 30°C in the dark.After 5 min,5 mL of Na2CO3solution (7.5 wt%) was added,and the mixture was incubated at room temperature for 2 h.Then,the absorbance of the mixture solution was measured at 760 nm using a microplate reader(Cytation™3 Cell Imaging Multi-Mode Reader,BioTek™,USA).The GA content was calculated referring to the calibration curve constructed from the known concentrations of GA (0.2—1 mg/mL) and the corresponding absorbance.

Thermogravimetric analysis(TGA)of CGA was done(Q50 machine,TA Instruments,USA)under nitrogen atmosphere(40 mL/min)from 30 to 700 °C with a ramp of 10 °C/min.

2.3.Preparation and characterization of CGA hydrogel

To prepare CGA hydrogels,100 μL of CGA polymer solution (1 wt%) dissolved in DIW were separately mixed with either HRP(0.005—0.1 mg/mL) or H2O2(0.025—0.1 wt %) in two vials.Then,the solutions in the two vials were mixed together (volume ratio=1:1)with gentle shaking to form a hydrogel.The gelation time was measured using vial tilting [31—33],which is defined as the time at which no flow was observed.

The elastic modulus(G’)of the CGA hydrogels was measured using a Rheometer (Advanced Rheometer GEM-150-050,Bohlin Instruments,Cranbury,NJ,USA) at 37 °C in oscillation mode,with a frequency of 0.1 Hz and a strain of 0.01% (strain control) using parallel-plate geometry (diameter=25 mm,gap=0.5 mm).To do the measurement,150 μL of the CGA polymer solution containing either HRP (0.05 mg/mL) or H2O2(0.025—0.1 wt%) were mixed on the bottom plate of the rheometer.Then,the upper plate was immediately lowered down to the desired gap.The elastic modulus of CGA hydrogels was recorded for 30 min at 37 °C.

The microporous structure of the CGA hydrogel was investigated by Field Emission Scanning Electron Microscopy (FE-SEM).For the analysis,600 μL of hydrogel was prepared using different concentrations of HRP and H2O2,followed by the lyophilization.The dried sponge-type hydrogels were cross-sectioned and sputter-coated with gold before the observation by SEM.

2.4.In vitro ROS scavenging activities of CGA hydrogels

The ROS-scavenging effect of CGA hydrogels was evaluated by means of free-radical-scavenging assays,including 1,1-diphenyl-2-picrylhydrazyl,hydroxyl radical,and a ferric-reducing antioxidant power assay.In these experiments,the ROS-scavenging activities of GA(4 μmol/mL),CS (1 wt%),CGA polymer (1 wt%),and CGA hydrogels(CGA_0.025,CGA_0.05,and CGA_0.1,presented in Table 1)were tested and denoted as test samples.

Table 1 Hydrogel samples used in ROS scavenging test and further in vitro studies.

2.4.1.1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity

The DPPH radical-scavenging activities of test samples were measured according to the reference with some modification [25,34].Briefly,we incubated 300 μL of the test samples in 1 mL of DPPH solution (0.5 mM in absolute ethanol) for 100 min in the dark.In the control group,300 mL of DIW was substituted for the sample.Afterwards,the absorbance of the incubated mixture was measured at 517 nm,using a microplate reader (Cytation™3 Cell Imaging Multi-Mode Reader,BioTek™,USA).The DPPH scavenging effect (%) was calculated by the following formula:

where ODtestand ODcontrolrepresent the optical densities of DPPH solution after incubation with test samples and DIW,respectively.

2.4.2.Hydroxyl radical (OH·) scavenging activity

The hydroxyl radical-scavenging activity was done as described before [35].We immersed 300 μL of test samples,including 600 μL of FeSO4(2 mM) and 500 μL of Safranin O (360 μg/mL),in the reaction mixture for 10 min at room temperature.Instead of test samples,300 μL of DIW was used as the control sample in this step.After that,800 μL of H2O2(6 wt%) or DIW was added to the reaction and incubated for 60 min at 55 °C.Then,the absorbance of the reacted mixtures was measured at 492 nm using a microplate reader.The scavenging effect of test samples on the hydroxyl radicals was obtained by the equation:

where ODtestand ODcontrolrepresent the optical densities of test samples and DIW,respectively.

2.4.3.Ferric reducing antioxidant power assay (FRAP)

The metal chelating activity of CGA hydrogel,which can reduce potassium ferricyanide (Fe3+) to potassium ferrocyanide (Fe2+) and then reacts with ferric chloride to form ferric—ferrous(strong absorption at 700 nm),was evaluated based on the previous report [36].We immersed 500 μL of test samples in 2.5 mL of phosphate buffer saline(PBS),followed by adding 2.5 mL potassium ferricyanide (1 wt%) and incubated it for 20 min at 50 °C.Then,we added 2.5 mL of trichloroacetic acid (10 wt%) and the mixtures were centrifuged at 3000 rpm for 10 min.Afterwards,2.5 mL of the supernatant was collected and mixed with 2.5 mL of DIW and 2.5 mL of ferric chloride solution(0.1 wt%).After 10 min,the absorbance of the reacted mixture was measured at 700 nm.Instead of a test sample,300 μl of DIW was used as the control.The total reducing power of the test samples was calculated as below:

where ODtestand ODcontrolrepresent the optical densities of test samples and DIW,respectively.

2.5.In vitro viability and migration of human dermal fibroblasts (hDFBs)

The viability and migration of hDFBs incubated with CGA hydrogel in both normal and hyperglycemia conditions were investigated using the insert systems [37].We cultured hDFBs at a density of 5×104cells/mL in 24-well plates with DMEM supplemented with 10%of FBS and 1%of PS,and incubated them at 37°C for 24 h to allow cell adhesion.Before the test,polymer (1 wt%),HRP (0.05 mg/mL),and H2O2(0.025—0.1 wt%) were dissolved in sterilized DIW and filtered by a syringe filter (pore size=0.2 μm) for sterilization.Then,300 μL of CGA hydrogels were prepared in the insert system (Falcon® Permeable Supports,Corning,USA) as described above.

Cell viability was assessed according to the ISO 10993 standard using WST-1 assay [38].The insert system containing hydrogel was placed in the cell pre-cultured 24-well plate and incubated with 1 mL of culture media.The culture media was supplemented with normal(5.5 mM) or high (55 mM) glucose to mimic the hyperglycemia environment.After 24 h,100 μL of WST-1 reagent was added to each well and incubated for another h at 37 °C.The absorbance of the incubated media was measured at 450 nm using a microplate reader(Cytation™3 Cell Imaging Multi-Mode Reader,BioTek™,USA).Tissue culture plates(TCPs) were used as control samples.The percentage viability of cells was calculated according to the following equation:

where ODtestand ODcontrolrepresent the optical densities of hDFBs cultured with hydrogel and DMEM,respectively,and ODblankis the optical density of the pure DMEM medium.

The in vitro migration rate of hDFBs was examined using the scratch assay [39].We cultured hDFBs (1 × 104cells/mL) in 24-well plate at 37 °C until a confluent monolayer was formed.Then,the P200 pipet tips were used to create straight scratches on the cell layer of a similar size.The debris was washed out by DPBS,and the fresh media supplemented with normal (5.5 mM) or high (55 mM) glucose was added,followed by the placement of the insert system containing hydrogel.The change in size of scratches was captured at pre-determined time points by a microscopy (Leica Microsystems,Korea).GA (10 μM) was used as a positive control in this experiment.The percentage of wound closure was calculated according to the following equation:

whereA0andAtrepresent the wound area recorded at h 0 and h t,respectively.

2.6.Statistical analysis

All experiments were repeated at least three times and each condition was analyzed in triplicate.The data was analyzed using one-way ANOVA followed by t-test and was presented as means±standard deviations.Significant levels were considered as P ＜0.05.

3.Results and discussion

3.1.Synthesis and characterization of chitosan-gallic acid (CGA)conjugates

The CS backbone contains a high density of the free amine groups,providing the reactive sites for conjugation with carboxyl groups of GA by means of a carbodiimide coupling method (Fig.2a) [40].In this study,GA was activated by EDC/NHS and reacted with CS to form CGA conjugate via amide and ester linkages.Interestingly,the interruption of the intermolecular H-bond and crystalline structure of chitosan by the hydrophilic functional groups of GA significantly improved the solubility of CS at neutral pH,up to 4 wt% (inset of Fig.2b) [41].

The chemical structure of CGA conjugations were then examined by UV—Vis and1HNMR spectra.As shown in UV—Vis spectra (Fig.2b),GA exhibited an absorbance peak at 265 nm,corresponding to the aromatic hydrocarbons.After we conjugated onto CS,this peak was shifted from 265 nm to 260 nm,because less energy is required for the n—π＊ and π—π＊transition[42].To measure the GA content in CGA,we used Folin-Ciocalteu,and calculated GA content to be 434.08 μmol/g.

1HNMR spectra of GA,CS and CGA conjugate are shown in Fig.2c.The1HNMR spectrum of CS showed multiple peaks at 4.3 ppm,3.3—3.8 ppm,and 2.9 ppm,corresponding to H-1,H-3 to H-6,and H-2 of CS,respectively.Compared to the peaks of the CS spectrum,there is a new peak at around 7.0 ppm in the CGA spectrum,correlating with the phenyl protons of GA.

TGA measurement was used to find the thermal stability and the fraction of volatile components of compounds by percent weight loss(Fig.2d).GA graph showed the initial weight loss of 8.8% (at 80—100°C)caused by the removal of physical absorbed water,followed by other stages of weight-loss of 41.0% (at 264 °C) and 22.5% (at 325°C)caused by the dehydroxylation of GA[43].Meanwhile,the TGA graph of CS is a smooth curve with the first stage of weight loss of 10%(at 30—100 °C) resulting from the loss of bound water,and another weight-loss stage of 40% (at 280—380 °C),associated with the dehydration of the saccharide rings,and depolymerization and decomposition of the chitosan units [44].The remaining weight of the CGA conjugate was higher than that of GA,indicating the increased thermal stability of GA in the CGA conjugates.Taken all together,these results confirmed the successful conjugation of GA to the CS backbone and improved water solubility of chitosan derivatives.

3.2.In situ formation and characterization of CGA hydrogel

The CGA hydrogels were formed in situ by means of HRP-catalyzed oxidative crosslinking of pyrogallol moieties on CGA conjugates(Fig.3a).It was reported that the autoxidation of galloyl moieties in aerobic condition over a period of time resulted in the formation of pyrogallol dimers [45].In the presence of HRP and H2O2,pyrogallols were oxidized into semiquinone radicals,and the coupling was triggered [46].Herein,CGA hydrogels were rapidly formed after simply mixing the solutions of CGA polymer containing either HRP or H2O2.The controllable gelation time of a hydrogel is an important parameter that affects its successful application.Depending on different applications,the gelation time is required to be fast or slow.The hydrogel with fast gelation,which can prevent the burst diffusion of bioactive molecules,can be used as a drug delivery system.Meanwhile,a hydrogel with adequate gelation time is needed to fulfill the irregular-shaped defects in tissue-engineering applications.We found that a higher concentration of HRP induced a faster hydrogel formation,ranging from 180 to around 30 s.CGA hydrogels were formed within 60 s when 0.05 mg/mL of HRP and 0.05 wt%of H2O2was used(Fig.3b).Based on this result,HRP with concentration of 0.05 mg/mL was selected for further studies (Table 1).

The viscosity (G″) and elastic modulus (G′) profiles of hydrogels during gelation were investigated by rheological studies(Fig.3c and d).As shown in Fig.3c,G′values gradually increased and crossed-over the G″values in all samples,indicating the phase transition from sol to gel.The crossover points of G′ and G”,which characterized the gelation time of CGA hydrogels;and were in agreement with the results obtained from vial titling test.Fig.3d showed that the elastic modulus of CGA hydrogel could be well controlled by varying the H2O2.G’ values measured in 10 min of CGA hydrogels were increased from 190 Pa to 260 Pa when increasing the H2O2concentrations from 0.025 wt% to 0.1 wt%.After 30 min,these values were increased to over 300 Pa because of the continuous but slow autooxidation in an aerobic condition and crosslinking between semiquinone moieties.Moreover,FESEM images (Fig.3d) demonstrated the microporous cellular structure of hydrogel with a diameter ranging from 5 to 25 μm.These results confirmed that a higher concentration of H2O2produced stiffer hydrogels with a smaller pore size by increasing the crosslinking density.In addition,in vitro degradation study also showed that CGA hydrogels were completely degraded after 7 days of incubation with lysozyme in PBS solution (pH 7.4,37 °C) (Fig.S1).This enzymatic degradation behavior of CGA hydrogels is favorable for wound healing treatment to prevent the secondary injury after fulfilling their function [9,13,23].

These results indicated that the conjugation of GA not only increase the water solubility of CS derivatives but also provides the functional groups for rapid hydrogel formation via an HRP-mediated crosslinking reaction.

3.3.In vitro ROS scavenging activities of CGA hydrogel

Fig.2.Synthesis and characterization of CGA.(a)Synthesis route of CGA,in which GA was conjugated onto the CS backbone via EDC/NHS-mediated coupling,(b)UV—vis absorbance spectra and the increased solubility of CGA conjugate(inset),(c) 1HNMR spectra of CS,GA and CGA conjugate in D2O,and(d)TGA graph of GA,CS and CGA.

Oxidative stress induced by overproduction and accumulation of reactive oxygen species (ROS) contributes to the impairment of tissue repair and regeneration from diabetes-related disease.Especially,under hyperglycemia,oxidative stress can damage enzymes and cellular machinery and increase insulin resistance.Therefore,the injectable chitosan hydrogel with ROS-scavenging activities will benefit the treatment of diabetic disease by preventing these oxidative stress injuries.The antioxidant property of GA has been well documented in the literature [24,47].The excellent radical-scavenging effect of GA is mainly dependent on the ortho hydroxyl groups,which not only provide the antioxidant properties by formation of intramolecular hydrogen bonding but also stabilize the formed antioxidant radical (Fig.4a)[24—26,47].In this study,we investigated the antioxidant activities of GGA hydrogels by assessing the scavenging effect on free radicals (2,2-diphenyl-1-picrylhydrazyl (DPPH),hydroxyl (OH·)),and total reducing power.

DPPH is a free radical with an unpaired valence electron at one atom of the nitrogen bridge,which shows a hydrogen receptor towards the antioxidants.In the DPPH assay,the antioxidant activities of CGA hydrogels were displayed by the decolorization of the DPPH radical solution,from the purple solution (strong absorption at 517 nm) to the colorless one [48].As shown in Fig.4b,after conjugation onto the CS backbone,the antioxidant property of GA was preserved,leading to the significant increase in the greater DPPH scavenging effect of CGA than that of pure CS.It was found that the DPPH scavenging effect of CGA hydrogels decreased with increases of the H2O2 concentration.For instance,CGA_0.025 hydrogel exhibited a DPPH scavenging effect (almost 90%) comparable with that of GA and CGA,whereas the scavenging effect of CGA_0.05 and CGA_0.1 hydrogels decreased to around 70% and 54%,respectively,perhaps because the higher H2O2concentration decreased the pore size of the hydrogel matrices,which decreased the diffusion rate of DPPH into hydrogels to react with remaining GA moieties.

We evaluated the capacity of CGA hydrogels to scavenge the OH·generated from the Fenton reaction between Fe2+and H2O2.Fig.4c shows that the CGA polymer and CGA_0.025 and CGA_0.05 hydrogels can effectively scavenge more than 90% of OH·,comparable to the scavenging effect of free GA [49].Meanwhile,the scavenging effect of CGA_0.1 was lower,around 65%.

Then,we also evaluated the total reducing power of CGA hydrogels by a ferric-reducing antioxidant power assay (FRAP).Similar to the results of DPPH and OH·scavenging studies,the CGA exhibited a reducing power comparable to that of GA(95%and 100%,respectively),which was significantly higher than that of pure CS(29%)(Fig.4d).The reducing power of CGA was preserved after hydrogel formation.Almost 85% of the Fe3+was reduced by CGA_0.025,whereas around 70% of Fe3+was reduced by CGA0.05 and CGA_0.1 hydrogels.

Overall,the conjugation of GA on CS significantly increased the antioxidant activities of the CS derivative.After hydrogel formation,the antioxidant activities of the CGA were preserved,depending on the used H2O2concentrations.These results can be explained by the denser crosslinking and higher hydrogel stiffness when a higher concentration of H2O2was used,which reduced the amount of free hydroxyl groups on the aromatic ring of GA and also eliminated the interaction of these groups with free radicals.

3.4.In vitro viability and migration of human dermal fibroblasts (hDFBs)

Fibroblasts play an important role in the remodeling of impaired tissues,especially during wound healing[50,51].Therefore,we investigated the proliferation and migration of hDFBs under the high-glucose conditions that mimic the hyperglycemia in diabetes [39].The viability of hDFBs incubated with CGA hydrogel was evaluated using a WST-1 assay and live/dead staining.As presented in Fig.5a and b,the cell viabilities of CGA hydrogels were the same as in the control group (TCPs) in normal condition (≥100%),except for CGA_0.1 hydrogel group (20%),which can be explained by the toxicity of residual H2O2released from the hydrogel(Fig.S2).Notably,under hyperglycemia,the viabilities of the cells without hydrogel treatment decreased dramatically(3.2%),indicating the high cytotoxicity of the glucose-rich environment.These results suggested the good cytocompatibility and protection by CGA hydrogels against ROSinduced oxidative stress triggered by hyperglycemia,supported by the ROS-scavenging activities of hydrogels.

Fig.3.Preparation and characterization of CGA hydrogel via HRP/H2O2-catalyzed reaction.(a) Schematic presentation for CGA hydrogel formation under catalytic reaction of HRP/H2O2;(b) Effect of HRP concentration on the gelation time of hydrogel (0.025 wt% H2O2 and 1 wt% polymer were used for hydrogel formation);(c) The elastic modulus (G′,solid lines) and the loss modulus (G″,dashed lines),(d) Mechanical strength,and (e) SEM images of CGA hydrogels depending on the H2O2 concentration.Data presented as the mean ± SD (n≥3) and analyzed using one-way ANOVA,＊＊p ＜0.01.

Next,we looked at the migration of cells incubated with/without hydrogels under both normal and high-glucose conditions.As mentioned above,CGA_0.1 caused cytotoxicity because of the high H2O2residue;so,we used only CGA_0.025 and CGA_0.05 hydrogels in the cell migration assay.We found that under the normal condition,the untreated cells migrate slowly and cover just 70%of the wound after 48 h(Fig.5c and d).In contrast,the high-glucose condition induced cell death and no migration of cells toward the center.Most interestingly,the migration rate was accelerated for cells treated with hydrogels in both normal and high-glucose concentration,comparable to the positive control group(GA).In detail,under normal conditions,cells treated with CGA hydrogels covered around 60% of the wound after only 24 h and healed the wound after 48 h (Fig.5c and d).However,the cell migration rate was attenuated in the high-glucose condition.After 48 h,almost 80% of the wound was closed by treating cells with CGA_0.025 and CGA_0.05,which was twice as fast as the control samples did(TCPs).The improved migration rate of hDFBs treated with CGA hydrogel resulting from the migratory effect of GA moieties on fibroblasts in both normal and hyperglycemic conditions,which was previously reported [26].

4.Conclusions

In the present study,a new chitosan-based hydrogel was developed that can scavenge excessive ROS and promote cell proliferation and migration under a hyperglycemia-mimicking environment.GA was conjugated onto a chitosan backbone by means of EDC/NHS chemistry,which not only improved the solubility of chitosan derivatives but also functioned as active sites for hydrogel crosslinking and antioxidant activities.We found that the CGA hydrogel was rapidly formed in situ by means of an HRP/H2O2-mediative oxidative crosslinking reaction with controllable physicochemical properties.The resulting CGA hydrogels presented an excellent scavenging effect toward free radicals,which promoted the proliferation and migration of fibroblasts in both normal and hyperglycemic conditions.These positive features suggested the potential applications of CGA hydrogels as a bioactive injectable matrix for treatment of diabetes-related complications.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This study was supported by a grant from Priority Research Centers Program (2019R1A6A1A11051471)and Korea government(2018R1A2B2004529) funded by the National Research Foundation of Korea (NRF).

Fig.4.Free-radical-scavenging activities of GA,CGA polymer,and CGA hydrogels formed by different H2O2 concentrations.(a) Ortho-hydroxyl groups responsible for scavenging activity of gallic acid groups in CGA.Scavenging effect of CGA hydrogels with different concentrations of H2O2 on (b) DPPH and (c) OH· radical,and (d) total reducing power.Data presented as the mean ± SD (n≥3) and analyzed using one-way ANOVA,＊＊p ＜0.01.

Fig.5.Effect of CGA hydrogels on fibroblast (hDFBs) activities under hyperglycemia.(a) Quantitative analysis of cell viability by WST-1 assay and (b) Live/dead staining image of cells cultured with CGA hydrogels after 1 day.(c)Optical microscope images of the cell migration and(d)migration rate of cells calculated by Image J after 2 days of incubation.Data presented as the mean ± SD (n≥3) and analyzed using one-way ANOVA,＊＊p ＜0.01 versus control.

Appendix A.Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.pnsc.2020.08.004.