Oxidized dextrins as alternative crosslinking agents for polysaccharides: Application to hydrogels of agarose–chitosan

Acta Biomaterialia xxx (2013) xxx–xxx

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Acta Biomaterialia

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Oxidized dextrins as alternative crosslinking agents for polysaccharides:Application to hydrogels of agarose–chitosan

1742-7061/$ - see front matter � 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.actbio.2013.10.003

⇑ Corresponding author at: Institute of Polymer Science and Technology, CSIC,Juan de la Cierva 3, 28006 Madrid, Spain. Tel.: +34 915618806; fax: +34 915644853.

E-mail address: [email protected] (B. Vázquez).

Please cite this article in press as: Gómez-Mascaraque LG et al. Oxidized dextrins as alternative crosslinking agents for polysaccharides: Applicahydrogels of agarose–chitosan. Acta Biomater (2013), http://dx.doi.org/10.1016/j.actbio.2013.10.003

Laura G. Gómez-Mascaraque a,b, José Alberto Méndez c, Mar Fernández-Gutiérrez a,b, Blanca Vázquez a,b,⇑,Julio San Román a,b

a CIBER-BBN, Ebro River Campus, R&D Building, Block 5, Floor 1, Poeta Mariano Esquillor s/n, 50017 Zaragoza, Spainb Institute of Polymer Science and Technology, CSIC, Juan de la Cierva 3, 28006 Madrid, Spainc Escola Politècnica Superior, Edifici PI, Campus Montilivi, University of Girona, 17071 Girona, Spain

a r t i c l e i n f o

Article history:Received 29 May 2013Received in revised form 27 August 2013Accepted 3 October 2013Available online xxxx

Keywords:HydrogelsOxidized dextrinsAgaroseChitosanViscoelastic properties

a b s t r a c t

Hydrogel networks that combine suitable physical and biomechanical characteristics for tissue engineer-ing scaffolds are in demand. The aim of this work was the development of hydrogel networks based onagarose and chitosan using oxidized dextrins as low cytotoxicity crosslinking agents, paying specialattention to the study of the influence of the polysaccharide composition and oxidation degree of thedextrins in the final characteristics of the network. The results show that the formation of an interpen-etrating or a semi-interpenetrating polymer network was mainly dependent on a minimum agarose con-tent and degree of oxidation of dextrin. Spectroscopic, thermal and swelling analysis revealed goodcompatibility with an absence of phase separation of polysaccharides at agarose:chitosan proportionsof 50:50 and 25:75. The analysis of atomic force microscopy images showed the formation of a fibrillarmicrostructure whose distribution within the crosslinked chitosan depended mainly on the crosslinker.All materials exhibited the viscoelastic behaviour typical of gels, with a constant storage modulus inde-pendent of frequency for all compositions. The stiffness was strongly influenced by the degree of oxida-tion of the crosslinker. Cellular response to the hydrogels was studied with cells of different strains, andcell adhesion and proliferation was correlated with the homogeneity of the samples and their elasticproperties. Some hydrogel formulations seemed to be candidates for tissue engineering applications suchas wound healing or soft tissue regeneration.

� 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Hydrogels are hydrophilic polymer networks characterized bythe presence of physical or chemical crosslinks, entanglementsand/or rearrangements of hydrophobic and hydrophilic domains.Due to the properties derived from this specific structure these sys-tems have found applications as biomaterials in biomedicine [1].Both natural and synthetic polymers can be used for the synthesisof hydrogels, achieved by methods such as physical gelation,chemical crosslinking or self-assembly [2]. Polysaccharides areespecially important since they play a role in domains involvedin hydrophilicity, swelling, hydration, gelling, biodegradation, etc.Polysaccharides have a predominantly hydrophilic character andthe presence of hydroxyl groups in their macromolecules makespossible the formation of intra- and interchain hydrogen bonds,however they also have a small hydrophobic character arisingfrom–CH groups [3]. Due to their stereoregularity they often form

helical conformations in solution that frequently lead to the forma-tion of physical gels. In addition, many polysaccharides have recog-nized biological activities [4,5] that contribute to cell–materialinteractions. It is for those reasons and because polysaccharidesconstitute a large family with a variety of chemical structures thatthey play a significant role in the field of biomaterials for tissueengineering [6].

Advances in tissue engineering have focused on the develop-ment of three-dimensional hydrogel scaffolds that provide suitablephysical and biomechanical characteristics for cell culture [7].Extracellular matrix (ECM) can be considered a semi-interpene-trating network (IPN) of a crosslinked protein material interlacedwith high molecular weight molecules [8]. Hence, among the dif-ferent strategies to mimic ECM the preparation of semi-IPN [9]and IPN [10] hydrogels have received increasing attention. Semi-IPN and IPN hydrogels are an interesting class of systems basedon a combination of one or two polymer networks that are physi-cally interlocked on the molecular scale without covalent bondsbetween polymeric chains of different type [11]. This approach isopen to further research and, hence, the development of new

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2 L.G. Gómez-Mascaraque et al. / Acta Biomaterialia xxx (2013) xxx–xxx

scaffold platforms of this type with improved properties is stillappreciated.

Agarose is a neutral polysaccharide extracted from the cell wallsof certain Rhodophyceae algae, also known as agarophyte seaweeds.It consists of repeating units of alternating b-D-galactopyranosil and3,6-anhydro-a-L-galactopyranosil groups [12]. Its chemical struc-ture gives agarose the capacity to form very strong gels even atlow concentrations. Gels of agarose provide a moist environmentand enhance stability of the system when it is in combination withother polysaccharides. Due to these properties agarose has been em-ployed in the preparation of hydrogel scaffolds [13] for the regener-ation of cartilage [14,15], neural tissue [16] and wound healing [17].Agarose-containing hydrogels based on IPN [18] and semi-IPN [19]have also been proposed for tissue engineering applications.

Chitosan is a linear polysaccharide based on a binary co-poly-mer consisting of b-1,4 linked 2-acetamido-2-deoxy-b-D-glucopyr-anose (GlcNAc) units and 2-amino-2-deoxy-b-D-glucopyranose(GlcN) units whose proportions depend on the degree of acetyla-tion of chitin [20]. Chitosan has many useful properties for thepreparation of scaffolds for tissue engineering [21]. For example,due to its structural similarity to various glycosaminoglycans, scaf-folds for articular cartilage [22,23] and bioartificial liver [24] havebeen produced, and due to its biodegradation properties chitosanhas been demonstrated to be suitable for nerve regeneration[25]. In addition, its antimicrobial activity makes it a good candi-date for the development of engineering systems for wound heal-ing [26]. The presence of polar groups (–OH and –NH2 groups) inthe chemical structure means it is able to form secondary interac-tions via hydrogen bonds with other polymers. Using this chemis-try semi-IPN polymer network hydrogels based on chitosan haverecently been developed [27]. Chemical crosslinking of chitosanis the most direct method to obtain stable hydrogel networks bymeans of covalent bonds between macromolecular chains. Cross-linking with aldehyde groups, whereby Schiff bases are formed,has been one of the preferred choices, glutaraldehyde being theagent most widely used by far for this purpose [28]. However,due to the cytotoxicity of this compound high molecular weightpolyaldehydes obtained by partial oxidation of carbohydrates suchas starch [29] and dextran [30,31] have been formulated with goodresults. Using these approaches in situ gelable hydrogels [31] andIPN hydrogels [30] based on chitosan have recently been produced.

The objective of this research was the development of hydrogelsystems based on agarose and chitosan in which oxidized dextrinsare proposed as low cytotoxicity crosslinking agents for chitosan.These materials combine the desirable properties of chitosan interms of biodegradability, ECM mimicry and antimicrobial activitywith the enhanced mechanical stability of polymeric matrices pro-vided by agarose. The advantages of using oxidized oligosaccha-rides as crosslinking agents are improved biocompatibility andbetter control on the in vitro behaviour of the systems. The influ-ence of polysaccharide composition and degree of oxidation ofdextrins on the final properties of the hydrogels, as well as on theformation of semi-IPN or IPN structures, was also addressed. Forthis purpose the viscoelastic and swelling properties of the hydro-gels were evaluated, and their morphology was studied by atomicforce microscopy. The cytotoxicity of the systems was tested withvarious cell lines and preliminary studies on the capacity of the sys-tems for cell growth and proliferation were performed.

2. Experimental section

2.1. Materials

Water-soluble chitosan chloride (Ch) for medical applications(Protasan UP CL 213, Mw > 150 kDa, Novamatrix), agarose (Aga)

Please cite this article in press as: Gómez-Mascaraque LG et al. Oxidized dexthydrogels of agarose–chitosan. Acta Biomater (2013), http://dx.doi.org/10.1016

(Agarose D1-LE, Hispanagar), dextrin (Dex) from corn starch (Sig-ma–Aldrich), sodium periodate (NaIO4) (Sigma–Aldrich), glutaral-dehyde (GTA) (Fluka), glacial acetic acid (Scharlab), t-butylcarbazate (tBC) (Sigma–Aldrich), 1 M trinitrobenzene sulfonic acid(TNBS) aqueous solution (Sigma–Aldrich), trichloroacetic acid(TCA) (Sigma–Aldrich), formaldehyde (Fisher Scientific), phos-phate-buffered saline, pH 7.4 (PBS) (Sigma–Aldrich), pH 6 and pH2 buffer solutions (Titrisol�, Merck), amphotericin B from Strepto-myces spp. (Fluka), 37% hydrochloric acid (HCl) (VWR Interna-tional), sodium hydroxide (NaOH) (Panreac), acid potassiumphthalate (KHP) (Riedel-de-Haën), potassium bromide FTIR grade(KBr) (Sigma–Aldrich), sodium nitrate (NaNO3) (Sigma–Aldrich),sodium dihydrogen phosphate (NaH2PO4) (Sigma–Aldrich)and recombinant human lysozyme (expressed in rice,P100,000 U mg protein�1) (Sigma Aldrich) were used as received.For biological assays Thermanox� (TMX) control discs were sup-plied by Labclinics S.L. and polypropylene tubes were purchasedfrom Sarstedt. Foetal bovine serum (FBS) was supplied by Gibco.Tissue culture media, additives, trypsin, dimethyl sulfoxide(DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazoliumbromide (MTT) were purchased form Sigma–Aldrich and the Ala-mar Blue reagent from Serotec.

2.2. Preparation of oxidized dextrins (DexOx)

19 g of dextrin were dissolved in 133 ml of PBS overnight atroom temperature. 0.234 and 0.467 M NaIO4 aqueous solutionswere prepared. Required volumes of the corresponding NaIO4 solu-tions were added to the dextrin solution at room temperature. Themolar ratios of sodium periodate to dextrin were 0.76:10 and1.51:10, respectively. The reaction was stopped after 4 h by addi-tion of pure ethanol. The resulting solution was dialysed againstdistilled water for 3 days using a cellulose membrane (Spectra/Por Dialysis Membrane, molecular weight cut-off 1000 Da) andlyophilized (Virtis lyophilizer). The as-obtained dextrins weretermed Dex1 and Dex2, respectively. Commercial dextrin (Dex)was also submitted to the dialysis process for comparisonpurposes.

2.3. Preparation of Aga/Ch hydrogels

A 1 wt.% aqueous agarose solution was prepared at 85 �C. Aftercomplete dissolution the temperature was lowered to 40 �C. Sepa-rately, a 1 wt.% aqueous chitosan chloride solution was prepared atroom temperature and then heated to 40 �C. The masses of Aga andCh in the respective solutions were adjusted to obtain hydrogelswith Aga/Ch mass ratios of 10:90, 25:75 and 50:50. Stock solutionscontaining the crosslinking agent (1 wt.% aqueous DexOx solution)were prepared at room temperature and then heated to 40 �C. Analiquot of this solution (10 wt.% DexOx with respect to chitosan)was added to the agarose solution and respective amounts of themixture were finally added to the Ch solution at 40 �C. The mixturewas vigorously stirred for 5 min, ultrasonicated, placed into a Tef-lon mould, transferred to an oven at 37 �C and kept there untilcomplete evaporation of the solvent. Likewise, samples of Aga/Chhydrogels were prepared in the absence of DexOx and Aga geland Ch/DexOx samples were prepared separately under the sameconditions. The compositions of the different formulations are dis-played in Table 1. Finally, chitosan was treated with commercialDex using the protocol described (termed Ch/Dex).

2.4. Physical and chemical characterization

2.4.1. Determination of relative average molecular weightsNumber and weight average molecular weights were deter-

mined by size exclusion chromatography (SEC) in a Shimadzu

rins as alternative crosslinking agents for polysaccharides: Application to/j.actbio.2013.10.003


Table 1Composition of the Aga/Ch formulations used for the preparation of hydrogels.

Sample Aga(wt.%)

Ch(wt.%)

DexOx (wt.% with respectto Ch)

Aga gel 100 0 0Ch/Dex1 0 100 10Ch/Dex2 0 100 10Aga/Ch 50:50 no Dex 50 50 0Aga/Ch 50:50 Dex1 50 50 10Aga/Ch 50:50 Dex2 50 50 10Aga/Ch 25:75 no Dex 25 75 0Aga/Ch 25:75 Dex1 25 75 10Aga/Ch 25:75 Dex2 25 75 10Aga/Ch 10:90 no Dex 10 90 0Aga/Ch 10:90 Dex1 10 90 10Aga/Ch 10:90 Dex2 10 90 10

Table 2Yields of the oxidation reactions, molecular weight parameters and degree ofpolymerization (DPn) of dextrins from corn starch and the oxidized dextrin samples.

Sample Yield (%) Mn (kDa) Mw/Mn DPn

Dex – 1.7 4.1 10.5Dex after dialysis 60 5.2 2.0 32.1Dex1 59 5.3 1.9 32.7Dex2 58 4.5 1.9 27.8

DPn was calculated as Mn/162.

L.G. Gómez-Mascaraque et al. / Acta Biomaterialia xxx (2013) xxx–xxx 3

SIL-20A HT with a LC-20AD isocratic pump connected to a RID-10Arefractive index detector. Three columns of PL aquagel-OH 30, 40and 50 nm nominal porosity (Varian) mixed bed were used to elutethe samples (1 mg ml�1 concentration) at 1 ml min�1 flow rate.Calibration of SEC was carried out with monodisperse standardpullulan samples in the range 180–708 � 103 Da obtained fromVarian. The eluent was an aqueous solution of 0.2 M NaNO3 and0.01 M NaH2PO4 adjusted to pH 7 using a 10 wt.% NaOH solutionfor the dextrin samples. For Aga the pH was adjusted to pH 3 usinga 10 wt.% glacial acetic acid solution. The SEC was conducted atroom temperature for Dex samples, whereas the columns wereconditioned at 40 �C for the Aga sample.

2.4.2. Chemical analysis by spectroscopic techniques1H NMR spectra were recorded in a Mercury 400BB spectropho-

tometer, operating at 400 MHz. The spectra for the initial and oxi-dized dextrins were recorded at 80 �C in deuterated water (D2O),with a relaxation delay of 4 s and 128 scans. The spectra of theAga/Ch hydrogel extracts were recorded in D2O at 25 �C. Attenuatedtotal reflectance Fourier transform infrared (ATR-FTIR) spectra wererecorded on a Perkin-Elmer Spectrum One spectrophotometer.

2.4.3. Determination of aldehyde groups in DexOxThe content of aldehyde groups in DexOx was determined using

tBC. An aqueous acid solution (1% w/v trichloroacetic acid) of thecorresponding DexOx (500 ll, 10�2 M) was mixed with an aqueoussolution of tBC (500 ll, 2.5 � 10�3 M) and the mixture allowed toreact for 24 h at room temperature. An aliquot of 250 ll was addedto an aqueous solution of TNBS (2.5 ml, 4 � 10�3 M) and allowed toreact for 30 min at room temperature. Once this time had elapsed250 ll of the mixture were diluted with 4 ml of an acid aqueoussolution (0.5 vol.% HCl). The absorbance of this solution was mea-sured with a Schimadzu UV-160A spectrophotometer at 334 nm,to determine the concentration of the trinitrophenyl complex ob-tained with the unreacted of tBC. To normalize the absorbance/aldehyde concentration values a calibration curve was determined.Different aqueous acid solutions (1% w/v trichloroacetic acid) ofknown aldehyde concentration (formaldehyde) were reacted withtBC solution and the excess of reactant was determined as men-tioned above. Finally, a calibration curve (R = 0.994) of the absor-bance (334 nm) as a function of the aldehyde concentration wasobtained using formaldehyde solutions of known concentration(4.20, 5.04, 6.30 and 7.56 mM) which was used to determine thecontent of free aldehyde groups in the DexOx samples. The degreeof oxidation (DO) was calculated as the percentage moles of alde-hydes as a function of moles of anhydroglucose units (AGUs).

2.4.4. Determination of free amino groups in the hydrogelsThe number of amino groups was measured for Ch samples

crosslinked with DexOx in the absence of agarose by pH titration


[32]. In this method 25 ml of a 0.1 N HCl solution were added in ex-cess to approximately 0.2 g of the corresponding sample, allowingenough time (24 h) to charge all proton binding groups. Subse-quently the solution was titrated with 0.1 N NaOH, previously nor-malized using acid potassium phthalate. Pure chitosan chloride(0.2 g) was also dissolved in the HCl solution and titrated with0.1 N NaOH solution. Two different equivalence points could berecognized. The percentage amino groups (% NH2) was calculatedusing Eq. (1):

% NH2 ¼ ½MNaOHðV2 � V1Þ � 197:5=W� � 100 ð1Þ

in which MNaOH is the molarity of the NaOH solution, V1 and V2 arethe volumes required to neutralize the excess HCl and protonatedamino groups, respectively, 197.5 is the molecular weight of thedeacetylated monomeric units of chitosan chloride and W is themass of chitosan chloride in the dry state before titration.

2.4.5. Thermal propertiesThermogravimetric analysis (TGA) was performed in a TGA

Q500 (TA instruments) thermogravimetric analyser, under a nitro-gen flow at a heating rate of 10 �C min�1 in a range of 30–600 �C.

2.4.6. Morphological characterizationAtomic force microscopy (AFM) experiments were performed in

tapping mode using a Multimode AFM (Veeco Instruments, SantaBarbara, CA) equipped with a Nanoscope IVa control system (soft-ware version 6.14r1). Silicon tapping probes (RTESP, Veeco) wereused with a resonance frequency of �300 kHz and a scan rate of0.5 Hz. 10 � 10, 5 � 5 and 2 � 2 lm2 AFM images were taken foreach sample. Topography was examined by topographical AFM,whereas composition was explored using phase imaging AFM.

2.4.7. Swelling and degradation propertiesFor swelling experiments Aga/Ch xerogel discs (14 mm diame-

ter and 29–43 lm thickness) were dried at 60 �C and accuratelyweighed (Wo). Afterwards each disc was immersed in 5 ml of thecorresponding pH buffered solution (pH 7.4 or pH 2) at 37 �C andkept under static conditions. The buffered solution was previouslytreated with an antifungal agent (1% amphotericin B). At differenttime intervals the sample was removed from the solution, dried,and weighed (Wt). Then it was returned to the same container.Water uptake (WU) was calculated using Eq. (2) in which the influ-ence of the salt weights was not accounted for in order to simplifythe analysis:

WU ð%Þ ¼ ½ðW t �WoÞ=Wo� � 100 ð2Þ

Once the swelling reached equilibrium, when three consecutiveWU values showed no significant differences, the samples werewashed with distilled water and dried in an oven at 60 �C untilthey reached a constant weight (Wd). Equilibrium water uptake(EWU) and equilibrium weight loss (EWL) were calculated fromEqs. (3) and (4), respectively:

EWU ð%Þ ¼ ½ðW t �WdÞ=Wo� � 100 ð3ÞEWL ð%Þ ¼ ½ðWo �WdÞ=Wo� � 100 ð4Þ



Table 3Characteristic ATR-FTIR absorption bands of agarose D1-LE, chitosan chloride Protasan UP CL213, Ch/Dex2 and Aga/Ch Dex2 xerogels of different compositions.

Assignment Wavenumber (cm�1)

Aga Ch Ch/Dex2 Aga/Ch/Dex2

50:50 25:75 10:90

m bonded O–H and N–H⁄ 3367 3234 3265 3283 3280 3256m bonded C@O acetamide (amide I) – 1624 1636 1632 1632 1629m NHþ3 – 1509 1524 1521 1522 1520m C–O glycosidic linkages 1042 1066 1080 1035 1037 1028

Scheme 1. (Left) Representation of the procedure applied for the preparation of hydrogels and (right) the chemical structure of chitosan network obtained by crosslinkingwith DexOx.


Enzymatic biodegradation of a specific Aga/Ch hydrogel, Aga/Ch25:75 Dex2, was studied in the presence of recombinant humanlysozyme at 37 �C. The discs were immersed in lysozyme solution(1.5 lg ml�1) in PBS at pH 7.4 and 37 �C. After determined timeintervals the sample was removed from the lysozyme solutionand dried in an oven at 60 �C until it reached a constant weight(Wdt). The extent of in vitro degradation was expressed as a per-centage of the weight loss applying Eq. (5).

WL ð%Þ ¼ ½ðWo �WdtÞ=Wo� � 100 ð5Þ

In all the experiments a minimum of three samples were mea-sured and averaged. The data obtained from swelling experimentswere analysed using one-way analysis of variance (ANOVA) at asignificance level of p < 0.05.

Table 4

2.4.8. Viscoelastic propertiesRheological experiments were carried out with a TA-ARG2

rheometer, using the parallel plate geometry to measure the stor-age modulus (G0), the loss modulus (G00) and the loss tangent (tand). The parallel plate diameter was 20 mm. Three samples of each

Fig. 1. pH titration of chitosan chloride Protasan UP CL213 alone and after reactionwith Dex1 and Dex2 separately.


formulation were allowed to equilibrate in PBS (pH 7.4). The testswere performed at a controlled temperature of 37 ± 0.1 �C. Smallamplitude oscillatory shear experiments were performed to evalu-ate the viscoelastic properties. Preliminary strain sweep tests at afixed oscillation frequency (0.5 Hz) (consisting of monitoring theviscoelastic properties while logarithmically varying the strainamplitude c0) were performed on the materials to determine thestrain amplitude range in which linear viscoelasticity is valid. Fre-quency sweeps between 0.05 and 100 Hz were then conducted at37 �C and 0.5% strain to evaluate the mechanical properties ofthe hydrogels. The evolution of G0 with temperature was also fol-lowed by applying temperature sweeps between 10 and 100 �C,and then decreasing it back from 100 to 10 �C, both cycles at a scanrate of 10 �C min�1. The oscillation frequency was 1 Hz and strainwas 0.5%.

2.5. Biological characterization

The biological response to the materials was tested with humanembryonic skin fibroblasts (HFB, Innoprot), human corneal

TGA results for the ingredient materials and Aga/Ch xerogels in an N2 atmosphere.

Sample Tmaxa (�C) Residue (%)

Aga/Ch 50:50 no Dex 209 ± 9 36.7Aga/Ch 50:50 Dex1 197 ± 6 36.4Aga/Ch 50:50 Dex2 197 ± 6 35.9Aga/Ch 25:75 no Dex 206 ± 6 36.5Aga/Ch 25:75 Dex1 219 ± 6 36.7Aga/Ch 25:75 Dex2 222 ± 9 34.0Aga/Ch 10:90 no Dex 227 ± 9 35.3Aga/Ch 10:90 Dex1 213 ± 6 35.6Aga/Ch 10:90 Dex2 205 ± 8 33.9Aga 347 0.0Ch 226 33.3Dex1 328 19.5Dex2 322 23.5

a Tmax refers to the temperature at which the maximum degradation rate occurs.



Fig. 2. Topographical (A) AFM image, (B) phase image and (C) amplitude image of the 50:50, 25:75 and 10:90 Aga/Ch samples prepared with Dex2 along with those of Agaand Ch.


epithelial cells and human epidermal keratinocytes (HEK, Inno-prot). The culture medium was Dulbecco’s modified Eagle’s med-ium enriched with 4500 mg ml�1 glucose (DMEM) (Sigma)supplemented with 10% FBS, 200 mM L-glutamine, 100 units ml�1


penicillin and 100 lg ml�1 streptomycin and modified with HEPES(complete medium). The culture medium was changed at selectedtimes with care taken to cause little disturbance to the culture con-ditions. Fibrin gel and TMX discs were used as negative controls.



Table 5Equilibrium water uptake values of Aga and Aga/Ch hydrogels conditioned in bufferedmedia at different pH values at 37 �C.

Sample EWU (%) EWL (%)

pH 7.4 pH 2 pH 7.4 pH 2

Aga 452 ± 26 485 ± 18 3 ± 1 18 ± 2Aga/Ch 50:50 no Dex 403 ± 17 3604 ± 250 13 ± 6 48 ± 3Aga/Ch 50:50 Dex1 696 ± 72 1829 ± 129 12 ± 2 51 ± 2Aga/Ch 50:50 Dex2 487 ± 75 1701 ± 260 11 ± 4 34 ± 9Aga/Ch 25:75 no Dex 2124 ± 646 ns 49 ± 8Aga/Ch 25:75 Dex1 729 ± 51 ns 31 ± 3Aga/Ch 25:75 Dex2 593 ± 81 ns 19 ± 3Aga/Ch 10:90 Dex1 1652 ± 584 ns 20 ± 5Aga/Ch 10:90 Dex2 502 ± 49 ns 12 ± 2

ns, non-stability of the sample in the medium.


All tested materials were sterilized with a UV lamp (HNS Osram,263 nm, 3.6 UVC/W) at a power of 11 W for 4 h.

2.5.1. Cytotoxicity experimentsThe cytotoxicity of oxidized dextrins was assessed by a modifi-

cation of the standard MTT assay [33–35] using Alamar Blue as a

Fig. 3. Storage and loss moduli and loss tangent as a function of oscillation frequency


dye due to the chemical interaction of MTT dye with dextrins.The cytotoxicity was determined for a series of dilutions madefrom a stock solution (50 mg ml�1) in FBS-free supplementedDMEM to calculate the median inhibitory concentration (IC50).Cells were seeded at a density of 8 � 104 cells ml�1 in completemedium in a sterile 96-well culture plate and incubated to conflu-ence. After 24 h incubation the medium was replaced with the cor-responding dilution and incubated at 37 �C in humidified air with5% CO2 for 24 h. Then 0.1 ml of a solution of Alamar Blue (10%inphenol red-free DMEM) was added and the plates were incubatedat 37 �C for 3–4 h. Afterwards the absorbance was measured with aBiotek Synergy HT detector using a test wavelength of 460 nm anda reference wavelength of 630 nm. The percentage cell viability(CV) was calculated from Eq. (6):

CV ð%Þ ¼ 100� ðODS � ODBÞ=ðODC � ODBÞ ð6Þ

where ODS, ODB and ODC are the optical density for the sample (S),blank (B) and control (C), respectively. A dose–response curve of rel-ative cell viability was plotted to delineate the concentrations oftest material that depressed CV by 50% (IC50 value).

The cytotoxicity of Aga/Ch xerogels discs (14 mm diameter,29–43 lm thickness) was determined using the standard MTT test

for the Aga gel and Aga/Ch hydrogels of different compositions obtained at 37 �C.



Fig. 4. Storage modulus as a function of temperature during heating cycles for Aga/Ch hydrogels prepared with different blend compositions and dextrins. The bottom graphshows both heating and cooling cycles for the Aga/Ch hydrogels prepared with Dex2.


Please cite this article in press as: Gómez-Mascaraque LG et al. Oxidized dextrins as alternative crosslinking agents for polysaccharides: Application tohydrogels of agarose–chitosan. Acta Biomater (2013), http://dx.doi.org/10.1016/j.actbio.2013.10.003


Table 6IC50 values of DexOx samples using different cell lines.

Cell line IC50 (mg ml�1)

Dex1 Dex2

Human dermal fibroblasts 5.2 ± 0.11 4.4 ± 0.21Human epidermal keratinocytes 5.2 ± 0.43 3.4 ± 0.21Human corneal epithelial cells 8.9 ± 0.14 7.9 ± 0.17


and TMX discs as negative controls. Samples were set up in 5 ml ofFBS-free supplemented DMEM and placed on a shaker at 37 �C. Themedium was removed at different time periods and replaced with5 ml of fresh medium. All the extracts were obtained under sterileconditions. Cells were seeded at a density of 8 � 104 cells ml�1 incomplete medium in a sterile 96-well culture plate and incubatedto confluence. After 24 h incubation the medium was replaced withthe corresponding extracts and incubated at 37 �C in humidified airwith 5% CO2 for 24 h. A solution of MTT (0.5 mg ml�1) was pre-pared in warm FBS-free supplemented DMEM and the plates wereincubated at 37 �C for 3–4 h. Excess medium and MTT were re-moved and DMSO was added to all wells in order to dissolve theMTT taken up by the cells. This was mixed for 10 min and theabsorbance was measured with a Biotek Synergy HT detector usinga test wavelength of 570 nm and a reference wavelength of630 nm. CV was calculated from Eq. (6). Analysis of variance (ANO-VA) of the results for Aga/Ch hydrogels was performed comparinghydrogels with TMX (p < 0.05) and comparing hydrogels amongthemselves (p < 0.05).

Fig. 6. Alamar Blue results obtained from fluorescence emission measurements at590 nm for Aga/Ch 25:75 DexOx and for the control TMX. All the results are shownas means ± SD (n = 16). ⁄Significant difference between the Aga/Ch hydrogel and thecontrol (⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001); +significant difference comparing bothAga/Ch formulations between themselves (p < 0.05).

2.5.2. Cellular adhesion and proliferationQuantitative analysis of cell adhesion and proliferation on the

Aga/Ch hydrogels was carried out by means of the Alamar Blue test[36]. Cells were seeded at a density of 14 � 104 cell ml�1 over thetest specimens (previously swollen in complete medium) in a 24-well culture plate for 24 h. After that 2 ml of Alamar Blue dye(10% Alamar Blue solution in phenol red-free DMEM) was addedto each specimen. After 4 h incubation 100 ml (n = 4) of culturemedium from each test sample was transferred to a 96-well plateand the fluorescence emission measured at 590 nm in a BiotekSynergy HT. The specimens were washed with PBS twice to removethe remaining reagent and 1 ml of culture medium was added tomonitor the cells over the materials. This step was done at differenttimes. Analysis of variance (ANOVA) of the results for the testedhydrogels was performed comparing hydrogels with TMX andamong themselves, at the p < 0.05 significance level in both cases.

Fig. 5. SEM images of the colonization by different types of cells of Aga/Ch 25


2.5.3. Cellular morphologyTo determine the cell morphology of attached cells Aga/Ch discs

were placed in a 24-well plate (in duplicate) and complete mediumwas added in order to swell the samples to equilibrium. Swollensamples and fibrin gel samples used as controls were seeded at a

:75 Dex2 hydrogels compared with a control fibrin gel 24 h after seeding.



Fig. 7. MTT test results for Aga/Ch 25:75 DexOx formulations crosslinked withDex1 and Dex2 against (A) human epidermal keratinocytes and (B) human cornealepithelial cells. All the results are shown as means ± S.D (n = 16). ⁄Significantdifference comparing the results for the Aga/Ch formulation with the control(p < 0.05); +significant difference comparing both Aga/Ch formulations betweenthemselves (p < 0.05).


density of 14 � 104 cells ml�1 and incubated at 37 �C. After 24 hculture the cells were fixed with glutaraldehyde (2.5% solution indistilled water) and the samples sputter-coated with gold forexamination by scanning electron microscopy (SEM) using a Phi-lips XL 30 apparatus at an accelerating voltage of 25 kV.

3. Results and discussion

3.1. Preparation of oxidized dextrins

Dextrins are oligosaccharides traditionally obtained by partialhydrolysis of starch. Dextrins from corn starch (Dex) with a num-ber average molecular weight (Mn) of 1.7 kDa, determined by SECin this work, was oxidized to different extents with sodium perio-date. In this reaction it is assumed that the periodate ion attacksone of the hydroxyl groups of the vicinal C2–C3 diol of the anhydro-glucose unit, breaking the C–C bond and forming aldehyde groups,which take the place of C–OH groups with increasing aldehydecontent [37]. Reaction yields were around 60% (Table 2) in bothoxidation reactions. Molecular weight distributions of DexOx sam-ples gave Mn values of �5 kDa and polydispersity index (Mw/Mn) of�2 for both DexOx samples (Table 2), indicating that the oxidizedoligosaccharides had been enriched in high molecular weight spe-cies. This phenomenon could be related to cleavage of the dextrinbackbone during oxidation and subsequent removal of the lowmolecular weight species during dialysis. Thus, to shed light onthis possible event, the initial dextrins were dialysed under thesame conditions and subsequently characterized by SEC (Table 2).It was found that commercial Dex underwent a weight loss in thedialysis process (�60 wt.%) comparable with the oxidation reactionyields. Moreover, the Mn and Mw/Mn values of the dialysed Dex ap-proached those of the treated samples. This finding supports the


hypothesis that the same fraction of low molecular weight speciesis removed from both commercial Dex and the DexOx samples dur-ing dialysis and, therefore, cleavage of the oligosaccharide duringoxidation, as has been described for oxidized dextrans [38–40],can be disregarded.

Structurally, DexOx were characterized by 1H NMR spectros-copy. The signals corresponding to the hydroxyl protons C2–OHand C3–OH in the range 5.7–5.8 p.p.m. and those of C6–OH at4.8 p.p.m. [41,42] were found in the spectra of all samples. The sig-nal due to resonance of the a-1,4 protons appeared at 5.4 p.p.m.,whereas a very small signal due to the a-1,6 protons appeared at5.0 p.p.m., showing that a-1,4 linkages were the major form inthe native oligosaccharide sample [43]. The rest of the signalsdue to protons of the anhydroglucose ring appeared in the range3.7–4.5 p.p.m. [44]. However, the signals assigned to the aldehydeprotons were not clearly discerned [38]. DexOx were also charac-terized by ATR-FTIR spectroscopy. The most remarkable bandswere the strong band centred at 3317 cm�1 assigned to the –OHstretching vibration of hydroxyl groups and the characteristic bandat 1013 cm�1 due to C–O bond stretching of AGU [41]. The alde-hyde group band was not observed by FTIR spectroscopy(1730 cm�1), as has been reported for polysaccharide samples witha low level of oxidation [38].

The content of aldehyde groups was quantified using a modifi-cation of the tBC method described elsewhere [45]. In that previouswork the method used a calibration curve made with tBC solutionsof known concentration, whereas in the present work the calibra-tion curve was built using formaldehyde solutions of known con-centration. The rationale of using the present method foraldehyde determination is that it takes into account the overlapbetween the UV–vis signals of the tBC/TNBS complex and semi-carbazones derived from reaction between tBC and aldehydegroups, as reported in the literature [18,46,47]. Thus tBC was addedto each aldehyde solution and the excess reacted with TNBS. Thenthe absorbance of each solution was determined and a calibrationcurve of absorbance vs. aldehyde concentration was plotted. Byapplying this calibration curve the degrees of oxidation of Dex1and Dex2 were determined to be 15.8% and 17.5%, respectively.In addition, the content of aldehyde groups in the dialysed originaldextrins was calculated using the same methodology, giving a va-lue of 15.3%, which can be explained by the contribution of reduc-ing end groups generated during the hydrolysis of starch, whichare directly related to the degree of hydrolysis [48,49].

3.2. Preparation and characterization of Aga/Ch hydrogels

The systems developed in this work were based on polysaccha-rides, the major components being Aga and a water-soluble Ch[50]. Our hydrogel systems were obtained by a simple methodwhich basically consists of mixing and briefly stirring two aqueoussolutions, one of them containing Aga and the crosslinking agent(DexOx) and the other Ch. The mixed solutions were transferredto a mould which was settled at 37 �C until evaporation of waterwas complete. The key point of this procedure is the temperatureof the mixture, which is governed by the solubility of Aga. Thistemperature was 40 �C in order to avoid early agarose gelation.Upon mixing the two solutions DexOx comes into contact withthe chitosan and it is presumed that the aldehyde groups of DexOxreact with the amine groups of Ch to form Schiff bases [51], leadingto the formation of a chemical network of Ch. This reaction mech-anism has been widely demonstrated in the literature, for examplein recent works reporting the formation of IPN hydrogels based onCh and oxidized dextran [30] or self-crosslinking of a Ch function-alized with aldehyde groups [52]. Scheme 1 shows a simplifiedrepresentation of the procedure applied and the chemical structureof the network produced by the crosslinking reaction. By applying




this procedure Aga/Ch hydrogels with the compositions displayedin Table 1 were prepared. The xerogels were characterized byATR-FTIR spectroscopy along with those of the initial Aga and Chmaterials. The main characteristic absorption bands of the differentsamples are summarized in Table 3.

The spectrum of commercial Aga showed a broad band centredat 3367 cm�1 attributed to stretching vibrations of OH groups andan intense band at 1042 cm�1 attributed to the deformation modeof the C–O groups in the sugar molecules [53]. The band at892 cm�1 is mainly associated with C–H bending at the anomericcarbon in b-galactose residues [54] and the band at 930 cm�1 isattributed to vibration of the C–O–C bridge of 3,6-anhydro-D-gal-actose [54,55]. The ATR-FTIR spectrum of chitosan chloride exhib-ited a strong and broad band with a maximum at 3234 cm�1

attributed to the –O–H stretching vibration and –N–H stretchingvibration [32,56,57], a band at 1624 cm�1 ascribed to amide I(C@O stretching) [56,58], a band at 1509 cm�1 attributed to N–Hof NHþ3 groups [52] and a band at 1066 cm�1 assigned to the C–Ostretching mode in sugar rings [56]. In the spectrum of theCh/Dex2 sample a displacement in the band attributed to NHþ3 tohigher wavenumber (1524 cm�1) with respect to pure Ch wasobserved. A similar trend was seen in the spectra of the Aga/ChDexOx xerogels (1520–1522 cm�1). This shift to higher wavenum-ber can be related to the formation of the Schiff base as a conse-quence of the crosslinking reaction, as has been reported for thecrosslinking of chitosan with glutaraldehyde [54,58] and for aSchiff base modified Ch treated with p-dimethylaminobenzalde-hyde [59]. On the other hand, in the latter spectra the absorptionband due to m C–O glycosidic linkages shifted to a lower wavenum-ber (1028–1035 cm�1). This shift may suggest the existence ofinteractions between polysaccharides, possibly due to the forma-tion of hydrogen bonds. These interactions will favour interpene-tration between both polysaccharides and, consequently, thecompatibility between the two components [53].

The content of free amino groups in the hydrogels after thereaction of Ch with the DexOx in the absence of Aga was deter-mined by pH titration. The results are shown in Fig. 1. Analysisof the curves provided a reduction of free amino groups of 1.2%for Ch treated with Dex1 and 5.7% for the sample treated withDex2, indicating the consumption of free amino groups after thecrosslinking reaction in both cases.

3.3. Thermal properties of Aga/Ch hydrogels

Thermal degradation analysis of Aga/Ch xerogels of different ra-tios was performed by TGA under N2 atmosphere. Aga, Ch and Dex-Ox were also analysed. Table 4 summarizes the values of the mainTGA results.

Due to the hydrophilic character of polysaccharides an initialloss of free water (�5%) was observed in the TG curves of all sam-ples. For Aga the major weight loss occurred between 150 and400 �C as a result of decomposition of the polysaccharide [60].The Ch TG curve showed a main degradation event in the temper-ature range 200–400 �C, attributed to deacetylation of the mainchain [61], cleavage of glycosidic linkages [62] and, finally, com-plete decomposition involving pyrolitic processes [61]. The TGcurves of DexOx presented the main weight loss at the tempera-tures at which the maximum degradation rates occurred, 328and 322 �C for Dex1 and Dex2, respectively, which has been relatedto dehydration and depolymerization processes [63]. Although thepristine polysaccharides exhibited different degradation profiles,the Aga/Ch xerogel samples underwent thermal degradation inonly one stage, with temperatures at which maximum degradationrates occurred in the range 205–227 �C. In general the degradationbehaviour of Aga/Ch xerogels approached that of Ch, even for high


Aga contents. This reflects a good interconnection of both types ofpolysaccharide macromolecular chains in the hydrogels.

3.4. Morphology of Aga/Ch xerogels

Morphology of the Aga/Ch networks was examined by AFM.AFM images of different Aga/Ch Dex2 xerogels are shown inFig. 2, with Supplementary Fig. S1 showing those for xerogels ob-tained with Dex1. The topographical image of pure Aga revealeda rough surface with a dense fibrillar structure characteristic ofthe formation of a gel, as has been reported in AFM [64] andUFM [65] studies for this Aga. However, the topographical imageof Ch was characterized by a smooth surface without any indica-tion of the presence of orientated polymer chains or aggregatesor organized systems of crystalline domains (see Fig. 2).

The surface characteristics of Aga/Ch DexOx xerogels were com-position dependent. The fibrillar topography characteristic of Agawas clearly observed in the Aga/Ch 50:50 Dex2 xerogels, even witha relative orientation of fibrils on the surface. For the Aga/Ch 25:75Dex2 sample the image shows the formation of microfibrillarstructures of Aga or Aga–Ch that can be stabilized by crosslinkingof the Ch. For the Aga/Ch 10:90 Dex2 xerogel the topographical im-age shows only a slight tendency towards the formation of fibrillarstructures although they were not as well defined as in the previ-ous compositions (Fig. 2). With respect to the samples preparedwith less DexOx (Dex1), formation of fibrillar structures could alsobe observed in the AFM topographical image of Aga/Ch 50:50 Dex1.However, for the composition Aga/Ch 25:75 no fibrillar structurecold be discerned and there even appeared a trend for the forma-tion of microdomains. A similar but more marked pattern was seenfor the composition Aga/Ch 10:90, in fact, the surface of this xero-gel resembled that of pure Ch (Supplementary Fig. S1). Roughnesswas higher for Aga/Ch Dex2 xerogels than for those obtained withDex1, and in both cases, roughness decreased with decreasing Agaconcentration in the blend. Thus we can say that both the Aga fi-brils and the crosslinking network contribute to the roughness ofthe surface. Analysis of phase images revealed an absence of phaseseparation in the microdomains of Aga/Ch hydrogels crosslinkedwith Dex2 at all compositions and of Aga/Ch 50:50 Dex1 hydrogels,what can be attributed to the presence of DexOx that contribute tostabilization of the system, but also to the presence of Aga. The restof the hydrogels tended to form microdomains. In general homoge-neity of the samples was better for the Aga/Ch Dex2 xerogels.

Overall, the analysis of AFM images suggests that the gelling ofAga took place during curing of the systems at 37 �C for Aga/Chsamples containing 50% or 75% Ch in the presence of Dex2. The for-mation of agarose gels at around 38 �C has already been demon-strated [66]. When agarose solutions are cooled Aga chains formhydrogen bonds and hydrophobic interactions, leading to the cre-ation of double helices. It is the generation of these double helices[16] followed by their aggregation to form microcrystalline junc-tions that are the precursors of the gel structure [66], which is as-sumed to give rise to a solid Aga hydrogel. In this sense, gelling ofAga in the presence of gellan [18] or k-carrageenan [10] has beendemonstrated, giving physical interpenetrating networks withoutphase separation at the molecular level. The molecular weight ofAga is another factor that can influence the formation of a gel.The molecular weight of our agarose was relatively low(Mn = 17.5 kDa, determined by SEC in this work) and this couldcontribute to the formation of double helices in the presence ofCh and a crosslinker at specific Aga/Ch ratios.

3.5. In vitro behaviour: swelling and degradation properties

The stability of Ch/DexOx and Aga gel samples was studied inPBS, pH 7.4. Ch/DexOx samples swelled rapidly in the first




5–10 min and afterwards remained insoluble, whereas Ch/Dex2samples maintained their dimensional stability, achieving anEWU of 792 ± 191%. The Ch/Dex1 samples folded and/or frag-mented, becoming unmanageable, which complicated the mea-surement of their EWU. This confirms that the higher degree ofoxidation of Dex2 leads to the formation of a more compact andstronger network which can bear less water and maintain its struc-ture. This is due to the higher number of aldehyde groups gener-ated during the oxidation of Dex2. In contrast the Ch sampletreated with the commercial Dex (Ch/Dex) dissolved rapidly andcompletely in the same buffer, indicating that aldehyde groups atthe reducing ends of commercial Dex are unable to crosslink Ch.This confirms that the aldehyde groups generated during the oxi-dation reaction are essential for the formation of the chemical net-work. The physical Aga gel was stable at neutral pH and presenteda EWU value of 452 ± 26%. It has been reported that Aga hydrogelsswell little in pure water [19,60]. This is probably due to its un-charged nature and to the low polarity resulting from its transitionto a helical form during gelation. This transition must have de-creased the number of sites for interaction with water.

Aga/Ch hydrogels prepared in absence of DexOx were stable atpH 7.4 for the 50:50 and 25:75. However, the composition 10:90dissolved completely with time. These results indicate that eventhough chemical network of Ch is not formed, the Aga gel andinterpenetration of the two polysaccharides due to entanglementof the macromolecules and interactions between functional groupsprovides enough dimensional stability and hindered the dissolu-tion of Ch whenever agarose is present at a certain concentration.The EWU of stable Aga/Ch hydrogels increased with Ch content,and was 5-fold higher for hydrogels containing 75 wt.% Ch. The val-ues are displayed in Table 5. Swelling of the same order of magni-tude was reported for Aga/Ch membranes obtained withoutchemical crosslinking [19].

All Aga/Ch DexOx hydrogels were stable in PBS, 7.4. EWU oscil-lated in the range 500–1600% and was lower for the systems pre-pared with Dex2, independent of the Aga content, what can beattributed to the formation of the most compact chemical networkwith the most DexOx, as was observed for the Ch/DexOx samples.

It is worth noting that Aga/Ch hydrogels of all compositions ob-tained with or without DexOx, were transparent after reachingequilibrium in PBS, what is considered a signal of good compatibil-ity between the two polysaccharides and absence of phase separa-tion at the macroscopic level.

In parallel, the weight loss of Aga/Ch hydrogels at pH 7.4 wasevaluated at different times. Weight loss of the Aga/Ch hydrogelstook place mainly in the first 5–10 min after immersion. EWL val-ues are shown in Table 5. For samples obtained without DexOxEWL increased with decreasing Aga content. For the rest of thesamples EWL was higher for the hydrogels prepared with less Dex-Ox, as expected. The soluble fraction of each sample was isolatedand analysed by 1H NMR spectroscopy. In all cases the characteris-tic resonance signals of Ch were observed, indicating that therewere uncrosslinked chains of Ch in the samples that dissolved inthe medium upon immersion. Nevertheless, the resonance signalsof the crosslinker were not detected. As this component is alsowater soluble this finding confirms that the DexOx had been cova-lently linked to the Ch macromolecules.

The stability of Aga/Ch hydrogels was also analysed at acidicpH. In this medium only the Aga gel and the Aga/Ch 50:50 hydro-gels remained stable over time and maintained their dimensionalstability. Hydrogels of other compositions disintegrated withtime. The EWU values of stable samples at acidic pH (Table 5)were much higher than in neutral pH and they hardly changedwith increasing degree of oxidation of dextrins in the blend. How-ever, the EWU value of the Aga/Ch 50:50 no Dex sample wastwice as high at �3600%. This behaviour was reported for Ch


cryogels crosslinked with different amounts of glutaraldehyde[58] and can be interpreted in the light of ionization of the aminogroups that are responsible for the ionic nature of Ch. The EWLvalues of the Aga/Ch 50:50 samples at acidic pH were also signif-icantly higher than at neutral pH. Considering that the EWL ofagarose was around 18% in this medium, weight loss of theAga/Ch hydrogels at acidic pH can be attributed to contributionby both Ch and Aga.

Regarding degradability of the systems, although Ch is not nat-urally present in the mammalian body it is degraded by the actionof the proteolytic enzyme lysozyme and other enzymes such aspepsin and pancreatin. The degradation of Ch in the presence oflysozyme has been widely studied. It is known that degradationdepends on the degree of deacetylation in the sense that totallydeacetylated Ch is not sensitive to the proteolytic enzyme. In fact,it seems that three consecutive acetylated units are required forthe substrate to be recognized by the enzyme. Thus highly deacet-ylated Ch is degraded more slowly than less deacetylated forms.The degradation mechanism has not been completely elucidated,but it is generally admitted that the crystallinity and the accessibil-ity of the enzyme are important. Other parameters such as molec-ular weight and pH also have an influence [67].

In order to gain an insight into enzymatic biodegradation of theprepared Aga/Ch hydrogels specific samples (i.e. Aga/Ch 25:75Dex2) were subjected to recombinant human lysozyme solutions(1.5 lg ml�1) in PBS at 37 �C. We observed an initial weight lossof 19 ± 2% in the first day, which is consistent with the value ofEWL obtained for these samples in the swelling assays. After thisinitial loss a slow weight loss of about 0.02% day�1 is observed,achieving a total weight loss of 27 ± 2% after 1 month.

3.6. In vitro behaviour: viscoelastic properties

The viscoelastic behaviour of Aga/Ch hydrogels was studied forsamples equilibrated in PBS, pH 7.4) at 37 �C using a rotational rhe-ometer with a parallel plate configuration. In order to determinethe rheological parameters it was necessary to establish the rangeof validity of linear viscoelasticity. To this end preliminary strainsweep tests at a fixed oscillation frequency were performed onthe hydrogels. The viscoelasticity range extended to 1% strain, soa strain of 0.5% was fixed for further tests.

The viscoelastic properties of the different Aga/Ch hydrogelcompositions were studied by measuring the storage and lossmoduli (G0 and G00) by performing frequency sweeps. The viscoelas-tic properties of the Aga gel were also analysed. The results areshown in Fig. 3. For all samples G0 was higher than G00 and G0 wasrelatively independent of frequency, indicating behaviour typicalof gels. Interpenetrating hydrogels based on agarose and cross-linked polyacrylamide showed similar behaviour [64]. However,this was not the case for hydrogels composed of Ch, dextran andmethylcellulose, for which G0 was higher than G00 at all frequencies,but both exhibited parallel slopes over the whole frequency range[68]. In this case the material exhibited a typical viscoelasticbehaviour to that observed, for example, for branched systemswith a low degree of crosslinking [69,70].

The Aga gel exhibited a G0 of 72.5 ± 8.9 kPa at 0.5 Hz, notablyhigher than those obtained for any Aga/Ch hydrogel. The Aga/Ch50:50 samples obtained with no Dex exhibited a G0 value of8.9 ± 1.2 kPa at 0.5 Hz, one order of magnitude higher than thatof Aga/Ch 25:75 no Dex (0.95 ± 0.1 kPa at 0.5 Hz), which canmainly be attributed to both a higher Aga content and a lower de-gree of swelling (by approximately a fifth). Values of G0 reported inthe literature for Ch–Aga hydrogels prepared by gelation of Aga at4 �C were much lower and oscillated between 20 and 70 Pa [71].

For systems obtained with DexOx the viscoelastic propertieswere determined by both the Aga content and the degree of Dex




oxidation. When Dex1 was used the Aga content was the dominantinfluence. The G0 value was 5.26 ± 0.9 kPa at 0.5 Hz for an Aga con-tent of 25%, and 13.9 ± 2.1 kPa at 0.5 Hz for an Aga content of 50%in the hydrogel. However, when Dex2 was used as the crosslinkerthe Ch chemical network played an important role in the viscoelas-tic properties, giving non-significantly different values of G0 of20.0 ± 2.5 and 20.2 ± 3.9 kPa at 0.5 Hz for hydrogels with Aga con-tents of 50% and 25%, respectively, only decreasing somewhat foran Aga content of 10% (11.7 ± 1.7 kPa at 0.5 Hz), provided thatthe degree of swelling of these hydrogels was not significantly dif-ferent. The degree of oxidation of polysaccharides has also been re-lated with a higher stiffness in the literature [32].

Fig. 4 shows the variation of G0 with temperature for Aga/Chhydrogels of different compositions. The behaviour of Aga gelswith temperature is also included in the upper graph of theFig. 4. For the Aga gel G0 was constant in the temperature interval10–60 �C, but started to drop above 60 �C due to gel–sol transitionof Aga. The evolution of G0 with temperature for hydrogels of allcompositions obtained using DexOx was intermediate betweenthat of the Aga gel and that of the corresponding Ag/Ch hydrogelprepared with no Dex, i.e. at low temperatures G0 was nearly inde-pendent of temperature, but at higher temperatures G0 dropped. Inall cases the drop was more pronounced for Dex1 systems.

Before the transition G0 was higher for samples crosslinked withDex2 compared with those crosslinked with Dex1 or prepared withno Dex. The transition of hydrogels containing Dex 2 and Aga/Chratios of 50:50 and 25:75 started at a temperature above 70 �C,whereas for Aga/Ch 10:90 it approached that of the Aga gel. Thebeginning of the transition of Dex1 or no Dex containing sampleswas in the interval 50–55 �C, slightly lower than that of the Agagel (�60–65 �C). Above the transition temperature G0 remainedhigher for Dex2-containing samples, but it rapidly decreased forsamples containing Dex1 or no Dex. This can be attributed to thereduced impediment to the ‘‘melting’’ of Aga in the presence of aless chemically crosslinked network, but also to the higher watercontent of these hydrogels, which may contribute to the mobilityof Aga molecules and favour their dissolution.

The overall results seem to indicate the importance of Aga con-tent in the hydrogels prepared with Dex1 or no Dex, which makes agreater contribution to the viscoelastic properties before transitionthan do the characteristics of the chemical network. However, forAga/Ch hydrogels obtained with Dex2 both the chemical networkand a thermodynamically stable gel of Aga result in an increasein G0 below and above the gel–sol transition [14], especially forthe Aga/Ch compositions 50:50 and 25:75.

Additionally, after heating the samples to 100 �C a cooling cyclewas carried out by decreasing the temperature back to 10 �C. Theheating–cooling cycles for Aga/Ch hydrogels crosslinked withDex2 are given at the bottom of Fig. 3. All samples showed hyster-esis loops, with lower G0 values in the cooling cycle. G0 increasedwith decreasing temperature, showing an important increase ataround 40–30 �C, corresponding to Aga gelation. The final valueof G0 at 10 �C for the Aga/Ch 50:50 sample was superior to thatmeasured in the heating cycle. A similar behaviour was seen inthe hysteresis loop for pure Aga gel, what may be attributed to dif-ferent gelation temperatures [72]. However, for the Aga/Ch 25:75ratio a value two orders of magnitude lower was achieved in thecooling cycle. When Dex1 was used the final G0 value after coolingwas significantly lower for all Aga/Ch blends (data not shown),indicating poor chemical network attainment due to the lower de-gree of oxidation of Dex1. For hydrogels obtained with no Dex theG0 vs. temperature curve in the cooling cycle was unstable for theAga/Ch blend of 50:50 in the initial temperature interval, but gela-tion of agarose was observed. However, a proper curve was not ob-tained in the cooling cycle for hydrogels containing only 25%agarose.


3.7. Cellular behaviour

The cytotoxicity of the DexOx was evaluated using different celllines. In all cases the compound exhibited a dose-dependent effectand the IC50 values could be calculated. The results are shown inTable 6. GTA is the most widely used crosslinker of Ch and for thatreason it is frequently used for comparison purposes in the study ofnovel crosslinkers [28]. The IC50 value of GTA obtained in this workusing human dermal fibroblasts was 0.276 ± 0.016 mg ml�1, whichcorrelates well with results reported in the literature using of hu-man embryonic lung fibroblast [73], indicating lower cytototxicityof the crosslinker.

The cytotoxicity of all Aga/Ch hydrogels was analysed in humanfibroblast cultures. CV was nearly 100% in the presence of extractsfrom every sample at any time, independent of the presence or notof DexOx and its degree of oxidation. Thus we can say that the sys-tems do not compromise in vitro viability of this type of cells.

To study the potential of these hydrogels for tissue engineeringapplications cells of different strains were seeded on hydratedAga/Ch hydrogels with varying Aga contents and observed bySEM 24 h after seeding. The response to the Aga/Ch 25:75hydrogels was the most positive, independent of DexOx used inthe formulation. A detail of this cellular adhesion is shown inFig. 5 for the samples crosslinked with Dex2. The cells in thesesamples showed a spread morphology indicative of good materialrecognition. It has been speculated that both the surface chargeand stiffness of the gel have an influence on cell adhesion to poly-saccharide samples. For example, incorporation of Ch and dextranto Aga–methylcellulose gels produced softer hydrogels withimproved adhesion of dorsal root ganglion cells. In particular, thebeneficial effects of including Ch were mainly attributed to thelower storage modulus and increasing surface charge of thesehydrogels [68]. Other works based on simple Aga–Ch gels supportthe above results, indicating that Ch–Aga hydrogels show bettersupport of neuron adhesion than plain Aga hydrogels [71].Recently Vandar et al. [19] studied the in vitro response to semi-IPN hydrogels of Aga–Ch vs. Aga–alginate using L929 fibroblastsand found that fibroblast adhesion, proliferation and penetrationwere higher on positively charged Ch-containing hydrogels. Look-ing at our results, the Aga/Ch 50:50 and 25:75 hydrogels preparedwith Dex2 had comparable G0 values, so the best cellular adhesionto Aga/Ch 25:75 hydrogels should be attributed to an increasedamount of positive charges on the surface of the hydrogel, whichallows electrostatic interactions with the negative charges on thesurface of the cell membranes [74].

Cellular proliferation on the surface of Aga/Ch 25:75 hydrogelsproduced with either Dex1 or Dex2 was quantified over a period of3 weeks using the Alamar Blue test. The results are shown in Fig. 6.The pattern of proliferation of cells on the Aga/Ch hydrogels dif-fered depending on the strain, but in all cases the pattern resem-bled that of the control. Fibroblast growth increased with timeand cell proliferation was significantly lower on the hydrogels atnearly all times, except for the Aga/Ch 25:75 Dex2 sample, onwhich cell proliferation was not significantly different from thecontrol after 3 weeks. Comparing the two hydrogels with respectto the type of DexOx, no significant differences were observed.

The population of human keratinocytes was also significantlylower on the hydrogels, but comparing the two compositionsshowed a significantly higher proliferation on days 14 and 21 inthe sample containing Dex2.

The proliferation of human corneal epithelial cells was charac-terized by a decrease in the population of cells throughout thewhole period, with the behaviour of the sample prepared withDex2 not being significantly different to the control at all times.Cell growth on this sample was significantly higher than on thesample containing Dex1.




To disregard the possible influence of cytotoxic extracts on theproliferation of keratinocytes and epithelial cells a typical MTT as-say was conducted. The results are shown in Fig. 7. No significantdifferences were found in CV for keratinocytes comparing hydro-gels. However, for human epithelial cells a significantly lower CVon days 4 and 7 and a significantly higher CV on days 14 and 21was measured for hydrogels prepared with Dex1. These resultsdo not correlate with higher cell proliferation on Dex2 formulatedhydrogels at any time. CV was around or higher than 80% in allcases and according to assessment of the standard ISO 10993-5:2009 [75] biocompatibility was not compromised. Thus we canconclude that cytotoxicity does not have an influence on cell pro-liferation of keratinocytes or epithelial cells.

Taking the cellular results overall, we can say that the Aga/Ch25:75 hydrogel formulated with Dex2 provided the most positiveresponse of cells of different strains, particularly significant inhuman corneal epithelial cells. Considering that this hydrogel pos-sesses a comparable surface charge to that formulated with Dex1,this response can be correlated with (a) a slightly higher roughnessand homogeneity of the Dex2-containing sample (as observed byAFM images), (b) better elastic properties (G0 = 19.1 ± 3.5 kPa forDex2 vs. 5.3 ± 0.9 kPa for Dex1) and (c) a lower swelling(ECW = 593 ± 81% for Dex2 vs. 729 ± 51% for Dex1), factors that,altogether, could favour cell attachment and subsequent prolifera-tion. Thus it is clear that the composition and physico-chemical andelastic properties must be analysed when selecting a material as ascaffold for tissue engineering experiments.

4. Conclusions

Hydrogels based on Aga and Ch were obtained using a simplemethod with low toxicity oxidized dextrins as crosslinkers. Spec-troscopic, thermal and swelling analyses revealed good compati-bility of the components of the hydrogels, with an absence ofphase separation of the two polysaccharides at Aga/Ch proportionsof 50:50 and 25:75. The analysis of AFM images showed the forma-tion of Aga fibrils, more or less defined and homogeneously distrib-uted depending on the hydrogel composition. Viscoelastic analysisshowed a constant storage modulus independent of frequency,typical of gels, for all materials, and indicated that their stiffnesswas strongly influenced by the degree of oxidation of the cross-linker. The overall results showed that by adjusting the Aga con-tent and varying the degree of oxidation of the crosslinker it waspossible to obtain IPN polymer networks of physical gels of Agaand chemical gels of crosslinked Ch, or that semi-IPN networksformed between the crosslinked Ch and Aga polysaccharides. Final-ly, biological assays showed that cell adhesion and proliferationwas dependent on an array of properties of the hydrogels thatshould be analysed in order to choose a particular material for tis-sue engineering purposes. As a main conclusion of this work wehave to consider that the formulation of Aga/Ch 25:75 crosslinkedwith DexOx has shown the best behaviour for potential applica-tions in wound healing or soft tissue regeneration.

Acknowledgement

We thank the Ministry of Economy and Competitivity (projectMAT2010-18155) for financial support. The support of Gema Rod-ríguez Crespo in the recording and interpretation of AFM images isalso acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.actbio.2013.10.003.


Appendix B. Figures with essential colour discrimination

Certain figures in this article, particularly Figs. 1–4, 6, 7 andScheme 1, are difficult to interpret in black and white. The full col-our images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2013.10.003).

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Oxidized dextrins as alternative crosslinking agents for polysaccharides: Application to hydrogels of agarose–chitosan