Carbohydrate Polymers 114 (2014) 458466

Contents lists available at ScienceDirect

Carbohydrate Polymers

j ourna l ho me pa g e: www.elsev ier .com/ locate /carbpol

Structu atibiomed

Dina M. S MoMaria Ro iscoa CEBCentre o ent ofCampus de Guab QOPNA, Depa

a r t i c l

Article history:Received 30 MReceived in reAccepted 2 AuAvailable onlin

Keywords:DextrinInjectable hydStructural analysisMALDI-MSSEC

ercias werf the, resp

dextrties ty of t

for biomedical applications. 2014 Published by Elsevier Ltd.

1. Introdu

Dextrinsproduced bor glycogenlose and thof amylopehydrolysis a measure Hudson, & Adifferencessweetness, may be assDepending hydrolysis displaying application

Dextrinssafe (GRAS)rial with a

CorresponE-mail add

1 Contribute

http://dx.doi.o0144-8617/ ction

are a class of low molecular weight carbohydratesy acid or/and enzymatic partial hydrolysis of starch, thus exhibiting the -(1 4)-Glc structure of amy-e -(1 4)- and -(1 4,6)-Glc branched structurectin, but with lower polymerization. The extent ofis expressed in terms of dextrose equivalent (DE),of the total reducing power (Chronakis, 1998; White,damson, 2003). Dextrins with the same DE can display

in terms of hygroscopicity, fermentability, viscosity,stability, gelation, solubility, and bioavailability, whichigned to distinct structural features (Chronakis, 1998).on the source of the native starch, as well as on theconditions, several types of dextrins can be obtained,different properties that can be suited for specics.

are an affordable raw material, generally regarded as (Alvani, Qi, & Tester, 2011). It is a widely used mate-great variety of applications such as adhesives, foods,

ding author. Tel.: +351 253 604 418; fax: +351 253 678 986.ress: fmgama@deb.uminho.pt (F.M. Gama).d equally to this study.

textiles and cosmetics (Gonc alves, Moreira, Carvalho, Silva, & Gama,2014). Regarding biomedical applications, dextrin is yet relativelyunexplored, being clinically used as a peritoneal dialysis solutionthat can also perform as a drug delivery solution (Peers & Gokal,1998; Takatori et al., 2011) and as wound dressing agent (DeBusk &Alleman, 2006). Although its limited number of current biomedicalapplications, dextrin exhibit a set of advantages that potentiatesits use specically in the biomaterials eld. It is a biocompatibleand non-immunogenic material, degradable in vivo by -amylasesand its molecular weight ensures renal elimination avoiding tis-sue accumulation due to repeated administration (Hreczuk-Hirst,Chicco, German, & Duncan, 2001; Moreira et al., 2010).

Hydrogels are three-dimensional, hydrophilic and polymericnetworks that are highly hydrated and are receiving attentionas scaffold materials (Drury & Mooney, 2003; Hoffman, 2002).The relevance of injectable hydrogels for biomedical purposes hasincreased in the last years since they generally are biocompati-ble, biodegradable, exhibit mechanical and structural propertiesthat resembles extracellular matrix, are processed under mildconditions and enable less invasive clinical procedures (Drury& Mooney, 2003; Hoffman, 2002; Van Vlierberghe, Dubruel, &Schacht, 2011). Hydrogels can be used alone or in combinationwith other components (e.g. cells, drugs, DNA, signalling molecules)(Jagur-Grodzinski, 2010; Peppas, Hilt, Khademhosseini, & Langer,2006).

rg/10.1016/j.carbpol.2014.08.0092014 Published by Elsevier Ltd.ral analysis of dextrins and characterizical hydrogels

ilvaa,1, Cludia Nunesb,1, Isabel Pereiraa, Ana S.P.srio M. Dominguesb, Manuel A. Coimbrab, Francf Biological Engineering, IBBInstitute for Biotechnology and Bioengineering, Departmltar, 4710-057 Braga, Portugalrtment of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

e i n f o

arch 2014vised form 24 June 2014gust 2014e 17 August 2014

rogels

a b s t r a c t

The characterization of several commhydrogels for biomedical applicationdextrins allowed the determination o6 to 17 glucose residues and 2 to 13%further characterization.

The combination of hydrogel with acompromising the mechanical propeviability, conrms the biocompatibilion of dextrin-based

reirab, M. Gamaa,

Biological Engineering, University of Minho,

l dextrins and the analysis of the potential of dextrin derivede performed in this work. The structural characterization of

polymerization and branching degrees, which ranged fromectively. Tackidex, a medical grade dextrin was choosen for

rin nanogel and urinary bladder matrix was achieved withoutor microstructure. The encapsulation of cells, preserving itshe injectable hydrogels, which have therefore great potential

D.M. Silva et al. / Carbohydrate Polymers 114 (2014) 458466 459

Several dextrin-based hydrogels, obtained by radical polymer-ization have been reported (Carvalho, Goncalves, Gil, & Gama,2007; Carvalho, Coimbra, & Gama, 2009; Das, Das, Mandal,Ghosh, & Pal, 2014; Moreira et al., 2010). Also, an oxidizeddextrin hyddescribed bAdipic acidtion of poly(Maia, Ferr(Schramm guluronate)Despite thereported a hydrogels. non-crosslicomprehendextrins, wgels reticulthe crosslinglutaraldehsidered cytoSawamura,biomedical

2. Materia

2.1. Materi

All reageSigma-Aldrgift from Taw28, w35, D4894 werechemicals acommercia

2.2. Structu

Neutral by Nunes e1 M at 100acetylated chromatogrElmer, Claru225 (J&W Sof 0.25 mmgram used wa rate of 40rate of 20 CThe injector230 C. The

2.2.1. MethLinkage

by Ciucanudissolved insodium hymortar to gNaOH (abowith CH3I (also carboxby Nunes ehydrolyzedacetylated apartially manalyzed by

(GCMS) (Agilent Technologies 6890N). The GC was equipped witha DB-1 (J&W Scientic, Folsom, CA, USA) capillary column (30 mlength, 0.25 mm of internal diameter and 0.15 m of lm thick-ness). The samples were injected in splitless mode (time of splitless

, withper

C/mifollog fo

C/mis helre ofpole

at 70can m

Matrmetr

MAF Ap

temsgen nol, oxybn (LDI

ren 60

was d Bi

, Sim

Size e deterfored b. Then. AcolumPLaq, 300stemhe erate ullul00 kD

ll cu

use ebeccrn ctrepting

withediu

Evalud-cyt

cytotara

inc, 24, orho, cell, durrogel cross-linked with adipic acid dihydrazide wasy our group (Molinos, Carvalho, Silva, & Gama, 2012).

dihydrazide may be used to promote the reticula-sacharide-based hydrogels, such as oxidized dextraneira, Carvalho, Ramos, & Gil, 2005), hyaluronic acidet al., 2012);(Su, Chen, & Lin, 2010), poly(aldehyde

(Bouhadir, Hausman, & Mooney, 1999), among others. overall good biocompatibility, Schramm et al. (2012)mild cytotoxic effect of the dihydrazide-reticulatedHowever, information about the biocompatibility ofnked monomer is very scarce. This work provides asive structural characterization of several commercialhich were used to produce oxidized dextrin hydro-ated with adipic acid dihydrazide. The cytotoxicity ofking agent was evaluated and compared with that ofyde. The later is a widely used crosslinker, often con-toxic (Huang-Lee, Cheung, & Nimni, 1990; Mcpherson,

& Armstrong, 1986), but still used for reticulation ofproducts (Furst & Banerjee, 2005).

ls and methods

als

nts used were of laboratory grade and purchased fromich, unless stated otherwise. Koldex 60 dextrin was ate & Lyle. Tackidex was a gift from Roquette. Dextrinsw60 and w80 were a gift from Avebe. Icodextrin and

obtained from Baxter and Sigma, respectively. All othernd solvents used in this work were of the highest puritylly available.

ral analysis of dextrins

sugars were determined as alditol acetates as describedt al. (2012). The hydrolysis was performed with H2SO4C during 2.5 h. Monosaccharides were reduced andand alditol acetate derivatives were analyzed by gasaphy (GC) with a ame ionisation detector (Perkins 400). The GC was equipped with a 30 m column DB-cientic, Folsom, CA, USA) with i.d. and lm thickness

and 0.15 m, respectively. The oven temperature pro-as: initial temperature 200 C, a rise in temperature at

C/min until 220 C, standing for 7 min, followed by a/min until 230 C and maintain this temperature 1 min.

and detector temperatures were, respectively, 220 andow rate of the carrier gas (H2) was set at 1.7 mL/min.

ylation analysisanalysis was carried out by methylation as described

and Kerek (1984). Dextrin samples (12 mg) were 1 mL of anhydrous dimethylsulfoxide (DMSO). The

droxide pellets were ground with a pestle in a dryet a ne powder in a N2 atmosphere, then powderedut 40 mg) was added and samples were methylated80 L) during 20 min. The methylated fractions wereyl-reduced by a modication of the method describedt al. (2012). The methylated oligosaccharides were

with 2 M TFA at 121 C for 1 h, and then reduced ands previously described for neutral sugar analysis. The

ethylated alditol acetates (PMAA) were separated and gas chromatography with mass spectrometry detector

5 min)ing temof 10

ature, standinof 15

gas wapressuquadrumode a full s

2.2.2. spectro

TheTOF/TOBiosysa nitro(dithradihydrsolutiothe MAtive ionbetweesition (ApplieNunes

2.2.3. The

was pdescrib(2014)solutioa pre-series (15 mtion sy36 C. Ta ow with p5.816

2.3. Ce

Moin Dulnewbocillin/scontainvestedsame m

2.3.1. induce

Theand gluviouslyAfter 0by sulfpoints20 C the injector operating at 220 C, and using the follow-ature program: 45 C for 5 min with a linear increasen up to 140 C, and standing for 5 min at this temper-wed by linear increase of 0.5 C/min up to 170 C, andr 1 min at this temperature, followed by linear increasen up to 280 C, with further 5 min at 280 C. The carrierium with a ow rate of 1.7 mL/min and a column head

2.8 psi. The GC was connected to an Agilent 5973 mass selective detector operating with an electron impact

eV and scanning the range m/z 40500 in a 1 s cycle inode acquisition.

ix-assisted laser desorption/ionization massy (MALDI-MS)LDI-MS mass spectra were acquired using MALDI-plied Biosystems 4800 Proteomics Analyzer (Applied, Framingham, MA, USA) instrument equipped withlaser emitting at 337 nm. A volume of 4 L matrix10 mg/mL in methanol and 0.1% TFA solution, or 2,5-enzoic acid) were mixed with 2 L of phatlocyanine10 mg/mL) and 1 L of this mixture was deposited onplate. Spectra were subsequently acquired in the posi-ector mode using delayed extraction in the mass range0 and 4500 Da with ca. 1500 laser shots. Data acqui-carried out using a 4000 Series Explorer data systemosystems, Framingham, MA, USA) (Moreira, Coimbra,es, & Domingues, 2011).

xclusion chromatographyermination of the average molecular weight (Mw)med by size exclusion chromatography (SEC) asy Passos, Moreira, Domingues, Evtuguin, and Coimbra

samples were dissolved in an aqueous 0.1 M NaNO3 PL-GPC 110 chromatograph was equipped withn PLaquagel-OH 15 m and two SEC columns in

uagel-OH40 15 m, 300 7.0 mm and PLaquagel-OH60 7.0 mm). The pre-column, the SEC columns, the injec-, and the refractive index detector were maintained atluent (aqueous 0.1 M NaNO3 solution) was pumped atof 0.9 mL/min. The analytical columns were calibratedan standards (Polymer Laboratories, UK) in the range ofa.

lture

mbryo broblasts 3T3 (ATCC CCL-164) were grownos modied Eagles media supplemented with 10%alf serum (Invitrogen, U.K.) and 1 g/mL peni-avidin (cDMEM) at 37 C in a 95% humidied air5% CO2. At 80% conuency, 3T3 broblasts were har-

0.05% (w/v) trypsin-EDTA and subcultivated in them.

ation of adipic acid dihydrazide and glutaraldehydeotoxicitytoxicity of the un-cross linked adipic acid dihydrazideldehyde was assessed in a 3T3 broblasts culture pre-ubated for 24 h in a 24-well plate (2 104 cell/well).48 and 72 h of incubation, cell viability was determineddamine B assay (Skehan et al., 1990). At different times were xed in a methanol:acetic acid (1%) solution ating, at least, 30 min. Then, a sulforhodamine B solution

460 D.M. Silva et al. / Carbohydrate Polymers 114 (2014) 458466

(0.5% w/v) in 1% acetic acid was added to the cells for 1.5 h at 37 C.After several washes with 1% acetic acid solution, to remove theexcess dye that did not bind to the proteins, the bound sulforho-damine B was dissolved with a 10 mM Tris solution. The absorbanceat 540 nm w

2.3.2. Cell eTo perfo

blasts wereof 3.75 10dihydrazidebottom of twas added.(Invitrogenmodicatioby DMEM w(2 M)/ethiwell and increscence mused as con

2.4. Extract

This proVelankar, Lders were 0.9% sodiumserosa, musurothelial cunder agitasisting of thlamina proaqueous soethanol, forbiomaterial15 min in pwashes of 1was freeze-

2.4.1. EnzymOne gram

ysed with solution, duwas achieve

2.5. Prepara

2.5.1. DextrDextrin

(2012). Briewith a 2 mLdegree of oxin the dark.dropwise aunreacted pagainst watcut-off 100

2.5.2. DeterThe deg

number of quantied uelsewhere (spectra wasas a peak ar

DO (%) = (X/Y) 100 where, X is the average integral correspond-ing to the peak at 7.3 ppm and Y is the average integral of theanomeric protons at 4.8 ppm and 5.4 ppm.

Prepadizedd saazidebaseorigid du

Prepatrin . Theing, aa, 20ex),sionhe ohydrose d to

Prepaationdized

temacid takinl dex

2 h.

echa

sssnatiossessydro2 an

etallutione ex/minined

ranged.

yo-sc

topoEM. Hn (slf the, subhe frring finto tion SyoSE

350

egrad

r besed ias read in a multiplate reader (Bio-Tek, Synergy HT).

ncapsulation in the hydrogelrm cell encapsulation in the tackidex hydrogel, bro-

mixed in the oxidised dextrin solution at a nal density5 cell/mL of hydrogel solution. Then, the adipic acid

was added and the hydrogel was left to gelify at thehe wells for 20 min, after which the culture medium

The cell viability was assessed by Live and Dead assay, UK) as indicated by the manufacturer with a slightn. After 4.5 h of incubation the medium was replacedithout serum or phenol red and 200 L of a calcein AMdium homodimer-1 (4 M) solution was added to eachubated for 1.5 h. The hydrogels were visualized by uo-icroscopy. The polystyrene plate and DMSO (20%) weretrols.

ion of extracellular matrix from porcine bladder

cedure has been reported before (Freytes, Martin,ee, & Badylak, 2008). Briey, after harvesting, the blad-washed to remove urine residues and stored in a

chloride solution at 4 C until removing the tunicacularis externa, submucosa and muscularis mucosa. Theells were then removed in a solution of 1.0 M NaCl,tion, in order to obtain the urinary bladder matrix con-e basement membrane of urothelial cells and subjacentpria. The urinary bladder matrix was immersed in anlution of 0.1% (v/v) of peracetic acid and 4% (v/v) of

2 h, under agitation, to promove the disinfection of the. Peracetic acid residues were removed in two washes ofhosphate buffered saline (PBS) pH 7.4, followed by two5 min with sterile water. The urinary bladder matrixdryed (LaboGene, Lynge, Denmark) until use.

atic hydrolysis of urinary bladder matrix of the lyophilized urinary bladder matrix was hydrol-

100 mg of pepsin in 100 mL of 0.01 M chloridric acidring 48 h, at 37 C, under stirring. Pepsin inactivationd by raising the pH to 8.0.

tion of hydrogels

in oxidationoxidation was performed as described by Molinos et al.y, aqueous solutions of dextrin (2% w/v) were oxidized

sodium m-periodate solution, to yield the theoreticalidation of 40%, at room temperature, with stirring, and

After 20 h, the oxidation reaction was stoped by addingn equimolar amount of diethyleneglycol, to reduce anyeriodate. The resulting solution was dialyzed for 3 dayser, using a dialysis membrane with a molecular weight0 Da, and then lyophilized for 10 days.

mination of aldehyde groups by 1H NMR analysisree of oxidation of oxidized dextrin is dened as theoxidized residues per 100 glucose residues, and wassing the tert-butylcarbazate (tBC) method, as describedBouhadir et al., 1999; Molinos et al., 2012). The 1H NMR

used to determine the degree of oxidation, calculatedea ratio in the NMR spectra according to the equation:

2.5.3. Oxi

bufferedihydrmolar in the procee

2.5.4. Dex

(2012)scatter& Gamtrin (oDsuspenThen, tacid diof glucallowe

2.5.5. combin

Oxiat roomadipic basis, originaduring

2.6. M

Strecombiwere aEach h123 mmallel mdistribpressiv0.5 mmdetermstrain analys

2.7. Cr

TheCryo-Snitrogestage oturatedfrom tsputteferred resoluand CrEnergy

2.8. D

Afteimmerration of dextrin-adipic acid dihydrazide hydrogels dextrin was dissolved in PBS buffer (phosphate

line) (30% w/v) at room temperature and an adipic acid solution (prepared separately) was added at 5%, in, taking into account the number of glucose residuesnal dextrin. The crosslinking reaction was allowed toring 2 h.

ration of hydrogelnanogel combinationsnanogel was prepared as described by Molinos et al.

nanogel formation was conrmed by dynamic lights described previously (Carvalho et al., 2010; Goncalves08; Goncalves, Martins, & Gama, 2007). Oxidized dex-

DO 40%, (30% w/v) was dissolved in PBS (pH 7.4) or in a of nanogel for approximately 16 h at room temperature.xidized dextrin suspensions were mixed with 5% adipicazide (in molar basis taking into account the numberresidues in the original dextrin). The crosslinking wasproceed at room temperature for about 2 h.

ration of hydrogelurinary bladder matrixs

dextrin was dissolved in PBS buffer pH 7.4 (30% w/v)perature. Urinary bladder matrix was dissolved in an

dihydrazide solution which was added at 5%, in molarg into account the number of glucose residues in thetrin. The crosslinking reaction was allowed to proceed

nical analysis

train experiments of crosslinked hydrogels and theirns with a dextrin nanogel and urinary bladder matrixed using a Shimadzu-AG-IS universal testing machine.gel disc, with a supercial area ranging from 103 tod a thickness 0.45 mm, was placed between two par-

ic circumferential plates, in order to promote a uniform of the compressive force along the sample. The com-periments were carried out at a deformation rate of

and at room temperature. The elastic modulus was by the average slope of the stressstrain curve over thee 020%. For each condition samples in triplicate were

anning electron microscopy (Cryo-SEM) analysis

graphy and porosity of the hydrogels were studied byydrogel samples were rapidly immersed in sub-cooled

ush nitrogen) and transferred under vacuum to the cold preparation chamber. The frozen samples were frac-limated for 120 s at 90 C (to partially sublimate wateractured hydrogel surface) and coated with Au/Pd byor 50 s with a 12 mA current. Samples were then trans-the SEM chamber and analysed at 150 C using a Highcanning Electron Microscope with X-ray MicroanalysisM experimental facilitiesJEOL JSM 6301F/Oxford INCA/Gatan Alto 2500.

ation of oxidized dextrin hydrogels

ing prepared and weighted (Wi), the hydrogels weren PBS pH 7.4 (diffusion medium), and incubated at 37 C.

D.M. Silva et al. / Carbohydrate Polymers 114 (2014) 458466 461

Table 1Glycosidic-linkage analysis of the dextrins (mol%) and estimated average degree of polymerisation and percentage of branching (average of three replicates), and dextrinsoxidation degree, gelation and degradation times of the hydrogels based on the different commercial dextrins.

w28 w60 D4894 w35 w80 Tackidex Icodextrin Koldex 60a

T-Glcp 14.8 0.3 14.4 1.2 19.1 2.1 11.2(1 4)-Glcp 80.3 0.9 73.7 1.9 68.7 3.9 85.3(1 6)-Glcp 3.3 0.5 0.3 0.1 (1 4,6)-Gl 4.9 1.2 8.6 0.8 11.9 1.6 3.5DP 10 16 13 13Branching (% 5 9 13 4Oxidation de 30.1 3.58 31.7 1.95 30.9 1.56 33.2 2.82Gelation tim

462 D.M. Silva et al. / Carbohydrate Polymers 114 (2014) 458466

Table 3MALDI-MS analysis of the dextrins (values are indicative of the relative abundance of the glucosyl oligosaccharides within each sample observed as sodium adducts [M + Na]+).

m/z [M + Na]+ DP3 DP4 DP5 DP6 DP7 DP8 DP9 DP10 DP11 DP12 DP13 DP14 DP15 DP16 DP17 DP18527 689 851 1013 1175 1337 1499 1661 1823 1985 2147 2309 2471 2633 2795 2957

w28 100 95 73 55 35 25 18 13 10 4 3 2w60 55 100 93 78 62 45 48 28 20 20 12 8 5 4 3D4894 74 96 100 85 76 71 53 50 41 28 29 15 13 6 6 6w35 79 99 100 74 64 45 35 25 20 16 14 8 8 3 2 2w80 86 100 100 84 67 49 35 23 20 10 9 5 5Tackidex 57 100 98 80 72 50 35 24 20 10 10 6 4 2Icodextrin 100 26 11 6 7 8 16 5 5 5 4 4Koldex 60 100 68 30 25 20 15 10 8 4 2 1

molecular weight compounds, with DPs comparable to those esti-mated by methylation analysis. These results suggest that the SECanalysis does not in this case provide an accurate estimation of themolecular weight, probably due to aggregation of the poor watersoluble dextrin molecules.

The different dextrins were used to prepare hydrogels. Thesewere obtained using oxidized dextrins reticulated with adipic aciddihydrazide, as comprehensively described in a previous work(Molinos et al., 2012). Despite the structural differences concerningthe polymerization and branching degrees of the dextrins, thehydrogels produced from the different dextrins performed fairlysimilarly in terms of gelation and degradation time, exceptionmade for Koldex 60, which exhibited a very slow degradation ratecombined with a faster gelation. Since no signicant differences,apart from Koldex, were observed between the dextrin hydro-gels, Tackidex, was choosen for further characterization. This isa medical grade dextrin, therefore better suited for the aimeddevelopment of biomedical applications. As compared to some ofthe other dextrins, it bears a suitable gelication rate, convenientfor its use as an injectable device, easy to handle by the surgeonduring the procedures.

3.2. Evaluation of adipic acid dihydrazide and glutaraldehydeinduced-cytotoxicity

As can be seen in Fig. 1A, a marked cytotoxic effect of adipicacid dihydrazide related with cell death at e.g.72 h can beobserved only for the highest concentrations tested (24% w/v).In the production of the hydrogel a concentration of about 1%(w/v) is used, which according to the results induce an inhibitoryeffect on cell proliferation. However, the actual free dihydrazideconcentration in the hydrogel is much lower, since the dihy-drazide quickly reacts with the aldehyde groups of dextrin chains.When comparing the two crosslinkers (Fig. 1B), it can be seenthat glutaraldehyde has a much higher cytotoxicity. Some stud-ies on the biocompatibility of glutaraldehyde-crosslinked polymers chitosan-based scaffolds for cartilage tissue (Hoffmann, Seitz,Mencke, Kokott, & Ziegler, 2009), chitosan/poly(vinyl alcohol)blends (Costa, Barbosa-Stancioli, Mansur, Vasconcelos, & Mansur,2009), collagen scaffolds (Lee, Grodzinsky, & Spector, 2001; Sheu,Huang, Yeh, & Ho, 2001) have demonstrated a satisfactory in vitroresponse in terms of cell adhesion and proliferation. On the otherhand, other works reported some cytotoxicity, as in the case of

Fig. 1. Cell prsulforhodamincompared witoliferation of 3T3 cells in the presence of different concentrations of adipic acid dihydr B assay. Results presented as mean SD (n = 3). Two-way Anova analysis was performeh the T0 control) and ### P < 0.001 (when compared with the DMEM control0%).azide (A) and glutaraldehyde (B) for 24, 48 and 72 h, determined byd; statistical differences were reported as * P < 0.05; ** P < 0.01 (when

D.M. Silva et al. / Carbohydrate Polymers 114 (2014) 458466 463

Fig. 2. Cr atrix

glutaraldeh2003), or inmercial bioinammatio

Overall, occurs onlytaraldehydeAs shown iglutaraldeh10 000 timeand adipic study, it cacytotoxic thtaraldehyde

3.3. Characcombined de

The oxidnanogel anderived exttive propergroup (Gonobtained bybone, whicwith a hydret al., 2010;Pereira, Schachieved uduced by ceyo-SEM images from cross-sections of hydrogel (A) and hydrogelurinary bladder myde-crosslinked collagen membranes (Marinucci et al., the study of Furst and Banerjee (2005), used as a com-medical glue containing glutaraldehyde, which inducesn, edema and necrosis, in vivo.

our results showed that dihydrazide-induced cell death at a concentration 300 times higher the one of glu-. Genipin is reputed a highly biocompatible crosslinker.n a study comparing the cytotoxicity of genipin andyde, the former showed toxic effects at concentrationss superior to those of glutaraldehyde. Although genipinacid dihydrazide were not directly compared in thisn be concluded that adipic acid dihydrazide is morean genipin (by about 30 fold) but much less than glu-

(Sung, Huang, Huang, & Tsai, 1999).

terization of nanogel and urinary bladder matrixxtrin hydrogels

ized dextrin hydrogel was combined with a dextrind with an enzymatic hydrolizate of porcine bladderracellular matrix, to improve its functional and bioac-ties. The dextrin nanogel, previously developed in ourcalves & Gama, 2008; Goncalves et al., 2007) was

grafting hydrophobic molecules to the dextrin back-h self-assembles in water, originating nanoparticlesophobic core. The incorporation of proteins (Carvalho

Molinos et al., 2012) and hydrophobic drugs (Gonc alves,ellenberg, Coutinho, & Gama, 2012) was successfullysing these nanogels. The extracellular matrix is pro-lls of each tissue and organ and consists of 3D structure

composed bronectinscaffold forerative meBenders etcal analysis(hydrogel, below.

3.3.1. Cryo-The mo

as their coby Cryo-SEstructure isconnective Mithieux, WTang, Bowyhighly heteand 400 nmthe un-swoan increase

3.3.2. DegraFig. 3 de

gel incubatdextrin nanFor all combwith an inby gradualswelling ph (B) before (1) and after (2) immersion on distilled water for 6 h.generally by type I collagen, glycosaminoglycans,, laminin and a variety of growth factors. It is a natural

tissue regeneration, thus highly valuable for regen-dicine applications (Badylak, Taylor, & Uygun, 2011;

al., 2013). The structural characterization, mechani- and degradation proles of the combined materialsurinary bladder matrix and nanogels) are described

SEM analysisrphology of the oxidized dextrin hydrogels as wellmbination with urinary bladder matrix was assessedM analysis (Fig. 2). A characteristic three-dimentional

observed for all samples, with a network of inter-pores, often found in this kind of materials (Annabi,eiss, & Dehghani, 2009; Zhang & Chu, 2002; Zhang,

er, Eisenthal, & Hubble, 2005). The pore diameter isrougeneous within samples and can vary between 200. Despite the pore size heterogeneity, when comparingllen hydrogels with the ones immersed in water for 6 h,

in the pore size can be observed.

dation proles of combined dextrin hydrogels.picts the mass loss proles of oxidized dextrin hydro-ed in PBS (pH 7.4) at 37 C, alone and combined withogel (1 mg/mL) and urinary bladder matrix (4 mg/mL).inations, a similar degradation prole can be observed,itial swelling phase during the rst 4 h, followed

mass loss until complete dissolution (1224 h). Thease is caused by the hydrolysis of the hydrazone bonds

464 D.M. Silva et al. / Carbohydrate Polymers 114 (2014) 458466

5 2-30

-10

10

30

50

70

Mas

s los

s (%

) 90110

HydrogelHydrogel-urinary bladder matrix

nd ur

(Molinos eturinary bladdation ratethe aldehydsites. Howeences werecombinatioby a bulk ethe matrix,degradation

Hydrogeapproach hreticulated to get fullytion used (Sdextran hyddegrades incentration. hydrogel isbone regenperform as drug delivebrings bioaeffective inobtained usapatite grannot shown)ally slower In the case regeneratiolated matrixThe very prmulations, degradation

3.3.3. MechThe com

hydrogel a(1 mg/mL) lar stressssamples hafor higher sindicates thples.

The comfor hydrogefound for hhydrogelnthe maximbehaviour bthat the incnary bladde

its m (Figcrostctly h, Bo

nantantaziderable009;

terac

rderere . The

soluhe co

poteas teo 3T3izedazideter 2cencis war ma

in t blahe reel, siing ar tis

encan deel m

clus

strusourengt2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.Time (h)

Fig. 3. Degradation proles of hydrogel and combinations with nanogel a

al., 2012) and osmotic effects. The combination withder matrix seems to results in a slightly higher degra-

. This is probably because the peptides may react withe groups of oxidized dextrin, lowering the cross-linkingver, through Cryo-SEM analysis, no structural differ-

detected between hydrogelurinary bladder matrixn and hydrogel alone. The degradation seems to occurrosion process, that is, by permeation of water into

leading to the network desintegration by a non-linear prole (von Burkersroda, Schedl, & Gopferich, 2002).ls made of different polysaccharides using a similarave been reported. Oxidized hyaluronic acid hydrogelswith adipic acid dihydrazide take from 3 to 30 days

solubilized, depending on the dihydrazide concentra-u et al., 2010). Maia et al. (2005) described an oxidizedrogel cross-linked with adipic acid dihydrazide, which

10 to 23 days, also depending on the crosslinker con-The relatively quick degradation rate of the Tackidex

convenient for some applications, such as for instanceeration. In this particular case, the hydrogel is aimed toan injectable carrier of hydroxyapatite granules and as ary system. The incorporation of urinary bladder matrixctivity to this formulation, which proves to be very

supporting cell adhesion and proliferation. The resultsing formulations of tackidex hydrogels with hydroxy-ules, in bone regeneration studies in the rabbit (data, demonstratates that the in vivo degradation is actu-than in vitro yet compatible with a swift cell invasion.of hydrogels with much slower degradation rates, then is hampered by the physical presence of the reticu-, which avoids the cell proliferation in the bone defect.

omising results obtained in vivo using the tackidex for-namely demonstrating the suitability of the observed

rates, will be published elsewhere.

anical analysispressive mechanical properties of oxidized dextrin

s well as of its combinations with dextrin nanogeland urinary bladder matrix (4 mg/mL) showed simi-train proles. Mechanical measurements revealed thatve a linear behaviour at deformations below 30% andtrains the stressstrain curve became non-linear, which

changeresultslar miis dire(Ansetdextrinto subsdihydrcompaPark, 2

3.4. In

In otions wstartedplasmafrom t

Thetem wembryof oxiddihydrand afuoresanalysbladdesulatedurinarycells. Thydrogbecomused foon theistratiohydrog

4. Con

Theferent chain lat plastic deformation occurred in the hydrogel sam-

pression modulus calculated at 20% deformationl alone was 0.023 0.012 MPa, similar to the value

ydrogelnanogel, hydrogelurinary bladder matrix andanogelurinary bladder matrix combinations. Finally,um compressive strength also showed a similaretween the different samples. These results suggestorporation of the different materials (nanogel and uri-r matrix) in the hydrogel matrix does not signicantly

trins is muccommonly seem to inThe combinwas succesdihydrazideproliferatiogel crosslinwas attesteto endure t5.0 27.5

Hydrogel-urinary bladder matrix-nanogelHydrogel-nanogel

inary bladder matrix in PBS (pH 7.4) at 37 C.

echanical performance and corroborates the Cryo-SEM. 2), which shows that different samples had simi-ructure. Since the compressive modulus of hydrogelsproportional to the intermolecular cross-link densitywman, & Brannon-Peppas, 1996), the incorporation ofogel, urinary bladder matrix or both does not appearially interfer in the linkages between aldehydes and the. The compressive properties of our biomaterial were

to those of others reported in literature (Liu & Chan- Tan & Hu, 2012).

tion with cells and blood

to mimick the clinical procedure, the percursor solu-mixed and transferred to a syringe were the gelicatinn, the hydrogel was injected in the human blood andtions. Hydrogel gelied as usual, no differences arisingntact with the blood/plasma proteins.ntial of oxidized dextrin hydrogel as a cell carrier sys-sted by live and dead assay of encapsulated mouse

broblasts. A cell suspension was mixed in the solution tackidex dextrin and reticulated with the adipic acid. The hydrogel was left gelifying in a multiwell plate0 min culture medium was added. The live and deade was analysed after 6 h of encapsulation. The sames performed for the hydrogel combined with urinarytrix. The majority of the cells that were kept encap-he tackidex hydrogel (98.2 1.9%) and hydrogel withdder matrix (99.5 0.7%) were calcein positive (viable)sults conrm the cytocompatibility of oxidized dextrinnce the cells could endure the cross-linking stage, thus

suitable platform for the incorporation of cells to besue engineering purposes. Indeed, preliminary studiespsulation of mesenchymal stem cells for in vivo admin-monstrate that the cells keep their viability; thus, thisay be a valuable platform for cell therapy applications.

ions

ctural characterization showed that dextrins from dif-ces displayed structural differences, namely regardingh and branching degree. The molecular weight of dex-

h lower than the values often reported in the literature,obtained by SEC. However, these differences do notuence the gelation and degradation of the hydrogels.ation with a dextrin nanogel and urinary bladder matrixsfully achieved. The cytotoxicity of the free adipic acid

was evaluated and only a mild inhibitory effect on celln was observed for the concentration used in the hydro-king. The biocompatibility of oxidized dextrin hydrogelsd through the encapsulation of cells, which were ablehe gelation process. Overall, these results demonstrate

D.M. Silva et al. / Carbohydrate Polymers 114 (2014) 458466 465

that the injectable dextrin hydrogels are a promising choice forbiomedical applications, namely for regenerative medicine and celltherapies.

Acknowled

D.M.S. wFundac o pfunding thracknowledgEstratgica project Nor

QOPNA cia e a TFEDER, and01-0124-FESpectrometAna S. P. (SFRH/BD/8Prof. Dmitry

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Structural analysis of dextrins and characterization of dextrin-based biomedical hydrogels1 Introduction2 Materials and methods2.1 Materials2.2 Structural analysis of dextrins2.2.1 Methylation analysis2.2.2 Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)2.2.3 Size exclusion chromatography

2.3 Cell culture2.3.1 Evaluation of adipic acid dihydrazide and glutaraldehyde induced-cytotoxicity2.3.2 Cell encapsulation in the hydrogel

2.4 Extraction of extracellular matrix from porcine bladder2.4.1 Enzymatic hydrolysis of urinary bladder matrix

2.5 Preparation of hydrogels2.5.1 Dextrin oxidation2.5.2 Determination of aldehyde groups by 1H NMR analysis2.5.3 Preparation of dextrin-adipic acid dihydrazide hydrogels2.5.4 Preparation of hydrogelnanogel combinations2.5.5 Preparation of hydrogelurinary bladder matrix combinations

2.6 Mechanical analysis2.7 Cryo-scanning electron microscopy (Cryo-SEM) analysis2.8 Degradation of oxidized dextrin hydrogels2.9 Hydrogel behaviour in human blood and plasma2.10 Data analysis

3 Results and discussion3.1 Structural dextrin analysis3.2 Evaluation of adipic acid dihydrazide and glutaraldehyde induced-cytotoxicity3.3 Characterization of nanogel and urinary bladder matrix combined dextrin hydrogels3.3.1 Cryo-SEM analysis3.3.2 Degradation profiles of combined dextrin hydrogels.3.3.3 Mechanical analysis

3.4 Interaction with cells and blood

4 ConclusionsAcknowledgmentsReferences