Carbohydrate Polymers 103 (2014) 339 347

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Carbohydrate Polymers

jo ur nal homep age: www.elsev ier .com

Injectable pectin hydrogels produced by internaldepend ies

Helena R liniPedro L. a FEUP Faculd o, Portb Laboratorio d nit dMilano, Piazza c Dipartimentod Dept. of Mole f Paviae Dept. of Occu oundaf INEB Institu 80 Porg Instituto de C rtuga

a r t i c l

Article history:Received 20 May 2013Received in revised form10 December 2013Accepted 11 December 2013Available onlin

Keywords:BiomaterialsInternal gelatiNatural polymPectinTissue enginee

The production of injectable pectin hydrogels by internal gelation with calcium carbonate is proposed.The pH of pectin was increased with NaOH or NaHCO3 to reach physiological values. The determination ofthe equivalence point provided evidence that the pH can be more precisely modulated with NaHCO3 thanwith NaOH. Degradation and inability to gel was observed for pectin solutions with pH 5.35 or higher.

1. Introdu

In the injectable bimplantatioity to avoid2007; Salgadrug delivethe treatmemolecules btivation of lower theirenteral admInjectable m

Corresponica G. Natta, eda Vinci, 32, 2

E-mail add1 These auth

0144-8617/$ http://dx.doi.oe 24 December 2013

oners

ring

Therefore, pectin solutions with pH values varying from 3.2 (native pH) to 3.8 were chosen to producethe gels. The increase of the pH for the crosslinked hydrogels, as well as the reduction of the gelling timeand their thickening, was dependent upon the amount of calcium carbonate, as conrmed by rheology.Hydrogel extracts were not cytotoxic for L-929 broblasts.

On the overall, the investigated formulations represent interesting injectable systems providing anadequate microenvironment for cell, drug or bioactive molecules delivery.

2013 Elsevier Ltd. All rights reserved.

ction

elds of drug delivery and regenerative medicine,iomaterials hold great promise, mostly due to then by minimally invasive surgery and the possibil-

systemic administration (Kretlow, Klouda, & Mikos,do, Sanchez, Zavaglia, Almeida, & Granja, 2012). Inry, large drug or protein doses are usually required fornt of a specic site, due to the uptake of the activey the surrounding tissues. The degradation or deac-

drugs or proteins by enzymatic reactions may further therapeutic efcacy. Injectable systems provide par-inistration or localized injection to the affected site.aterials as cell carriers for regenerative medicine share

ding author at: Dipartimento di Chimica, Materiali e Ingegneria Chim-d Unit di Ricerca Consorzio INSTM, Politecnico di Milano, Piazza L.

0133 Milano, Italy. Tel.: +39 0223999511; fax: +39 0223993360.ress: fabiola.munarin@polimi.it (F. Munarin).ors contributed equally to this work.

the same advantages as those used in drug delivery and, in addi-tion, are able to conform to the shape of a tissue defect, avoiding theneed of specic scaffold prefabrication (Bidarra et al., 2011; Kretlowet al., 2007; Salgado et al., 2012).

Natural polymers possess highly organized structures, someof them containing functionalities capable of binding cell recep-tors, thus inducing cell adhesion (Cheung, Lau, Lu, & Hui, 2007;Swetha et al., 2010). Furthermore, anionic polysaccharides suchas alginate, carboxymethyl cellulose, gellan, hyaluronic acid andpectin are good mucoadhesive materials (Lee, Park, & Robinson,2000; Munarin, Tanzi, & Petrini, 2012), and therefore, as carriers,may prolong the residence and the exposure time of drugs, allow-ing their improved absorbance (Liu, Jiao, Wang, Zhou, & Zhang,2008). In addition, the crosslinking of anionic polysaccharides, theirpH and time of gelation can be controlled, leading to the fabri-cation of systems with tunable properties. The control of the pHplays a key role in tissue engineering and drug delivery. For drugdelivery applications, the modulation of the pH of polysaccharidesolutions may provide the binding of specic drugs, an optimizedrelease prole and the activation/inactivation of the drugs. In thecase of tissue engineering, maintaining the pH in the physiological

see front matter 2013 Elsevier Ltd. All rights reserved.rg/10.1016/j.carbpol.2013.12.057ence of gelling and rheological propert

. Moreiraa,b,1, Fabiola Munarinb,,1, Roberta GentiGranjaa,f,g, Maria Cristina Tanzib, Paola Petrinib

ade de Engenharia da Universidade do Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porti Biomateriali, Dipartimento di Chimica, Materiali e Ingegneria Chimica G. Natta and UL. da Vinci, 32, 20133 Milan, Italy

di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milan, Italycular Medicine, Center for Tissue Engineering (C.I.T.), INSTM UdR of Pavia, University opational Medicine, Ergonomy and Disability, Lab. Nanothecnology, Salvatore Maugeri Fto de Engenharia Biomedica,Universidade do Porto, Rua do Campo Alegre, 823, 4150-1incias Biomdicas Abel Salazar (ICBAS), Largo Prof. Abel Salazar, 2, 4099-003 Porto, Po

e i n f o a b s t r a c t/ locate /carbpol

gelation: pH

b,c, Livia Visaid,e,

ugali Ricerca Consorzio INSTM, Politecnico di

, 27100 Pavia, Italytion, IRCCS, 27100 Pavia, Italyto, Portugall

340 H.R. Moreira et al. / Carbohydrate Polymers 103 (2014) 339 347

range is required for the production of cytocompatible injectablesystems.

Anionic polysaccharides carrying carboxylic groups exhibitspecic physicochemical properties due to their ability to formionotropic Evangelistainherent bipolysaccharinjectable sgel-formatior cells (GutintermolecuAl3+, providand, therefo& Granja, 20

Among very prompolysaccharSuttangkakgelling ageKjniksen, posed of thrrhamnogalaHGA is the m-(14)-dtially methLow methothe presenform intermtion (Kastn2012; Ravansociated caknown as th& Altenbachelectrostatigen bonds sneighboringNunez, & VFraeye et apectin crossvalent counattribute mthe releaseet al., 2011)

Two appgels crosslition. The exachieved byalginate, peAlmeida & & Attwoodcrosslinkingcium chloriand beads, et al., 1996;Petrini, et asuitable formatrices foLee, & Hengmeric solutcalcium carication, wdissolutiongelation of glucono--lpH of the p

Bidarra et al., 2011; Fonseca, Bidarra, Oliveira, Granja, & Barrias,2011; Fonseca et al., 2013). In this work, we hypothesize the pos-sibility of using CaCO3 for the preparation of pectin hydrogels byinternal gelation, and to do this avoiding the use of GDL. At present,

orts osslia nonctin sen, ditivlling

gell. In thGDL eric

pH ire, ie reqns, ryzede ho

acid inje, diffis-etgrad

for HuntCO3pectimeri

formnicalryinginkin

gelleneo

gelsor thernat, to llingtin-bellingeneoiffer

thinnon tho sca

erim

ateri

me provwderaOH)Aldr

arac

Moletionrepagels (Bidarra, Barrias, Barbosa, Soares, & Granja, 2010; et al., 2007; Munarin, Tanzi, et al., 2012). Due to theocompatibility and soft tissue-like behavior of anionicides, these biomaterials may be exploited to designystems with slow crosslinking kinetics, allowing in situon for injectable therapeutic delivery of drugs, proteinsowska, Jeong, & Jasionowski, 2001). The crosslinking bylar attraction with multivalent cations, such as Ca2+ ores means of modulating viscoelastic properties of gelsre, their injectability (Munarin, Petrini, Tanzi, Barbosa,12; Munarin, Tanzi, et al., 2012).negatively charged natural polymers, pectin is aising biomaterial. Pectin is a biocompatible anionicide that constitutes 30% of plants cell wall (Harholt,ul, & Scheller, 2010), widely used as thickener,nt, stabilizer and emulsier in food products (Tho,Nystrm, & Roots, 2003). It is almost entirely com-ee polysaccharidic domains: homogalacturonan (HGA),cturonan-I (RG-I) and rhamnogalacturonan-II (RG-II).ajor component of pectic polysaccharides and contains

-linked galacturonic acids (1,4--d-GalA) that are par-yl-esteried and sometimes partially acetyl-esteried.xyl pectins (degree of methylation, DM < 50%), requiresce of calcium ions or other divalent metal ions thatolecular ionic junction zones responsible for gel forma-

er, Einhorn-Stoll, & Senge, 2012; Munarin, Tanzi, et al.,at & Rinaudo, 1980). The calcium bridges between dis-

rboxyl groups, thus forming well organized structurese egg-box model (Braccini & Prez, 2001; Naumenko, 2007; Morris, Nishinari, & Rinaudo, 2012). Besides thec interactions, van der Waals interactions and hydro-tabilize the chain when egg-boxes are formed between

chains (Alonso-Mougan, Meijide, Jover, Rodriguez-azquez-Tato, 2002; Braccini, Grasso, & Prez, 1999;

l., 2010; Munarin, Tanzi, et al., 2012). The solubility oflinked with Ca2+ ions can be controlled by other mono-terions (such as Na+) that can replace calcium ions. Thisakes pectin suitable for externally tunable systems for

of immobilized cells or therapeutic agents (Munarin.roaches can be followed for the preparation of pectinnked with calcium ions: the external or internal gela-ternal gelation is an ionotropic mechanism commonly

extruding droplets of the polyanionic polymers (i.e.ctin, gellan) (Ching, Liew, Heng, & Chan, 2008; FerreiraAlmeida, 2004; Miyazaki, Aoyama, Kawasaki, Kubo,

, 1999; Santucci et al., 1996; Sriamornsak, 1998) in a solution containing soluble calcium salts, such as cal-de. This mechanism allows the production of particlesdue to the extremely fast gelling reaction (Alhaique

Evangelista et al., 2007; Grellier et al., 2009; Munarin,l., 2012; Munarin et al., 2011). The internal gelation is

the preparation of scaffolds for tissue engineering andr drug release, as it produces homogeneous gels (Chan,, 2006). In the case of the internal gelation, the poly-ion is mixed with a poorly soluble calcium salt, such asbonate (CaCO3), and the crosslinking starts after acid-ith H+ ions promoting the progressive pH-controlled

of CaCO3. For this reason, when used for the internalanionic polysaccharides, CaCO3 is combined with d-actone (GDL), a chemical compound able to lower theolymeric solutions (Baldursdttir & Kjniksen, 2005;

no repgels crGDL is and peKjnikthe adself-ge

Theof GDLlated: polymwhosethermoGDL arsolutiohydrolor to th

Thetion asthe pHTEA (trand deline pH2002; of NaHpH of depoly

ThemechapH, vacross-l

Thehomogloadeding prithe intcontencontro

Pecslow ghomogtailor dshear-injectibility t

2. Exp

2.1. M

Lowkindlydry poide (NSigma

2.2. Ch

2.2.1. Solu

were pexist so far regarding the preparation of pectin hydro-nked with CaCO3 without the addition of GDL. Though-toxic compound already employed to produce alginatehydrogels for biomedical applications (Baldursdttir &2005; Fonseca et al., 2011, 2013), avoiding the use ofe can be more convenient, due to the formation of a

system and to the reduced time of production.ing kinetic could be easily controlled avoiding the usee presence of GDL, two phenomena are partially corre-time-dependent hydrolization to lower the pH of thesolution, and the solubility of CaCO3 in the solution,s changing with time due to the hydrolysis of GDL. Fur-n order to obtain fast gelling kinetics, higher amounts ofuired, but this leads to a severe acidication of pectineaching its maximum values when GDL is completely, that may cause damages to the cells possibly entrappedst tissues.ic pH of pectin solutions and gels can limit their applica-ctable systems for biomedical applications. To increaseerent bases can be employed, including NaOH, NaHCO3,hanol-amine) and others. NaOH is known to precipitatee the poly-GalA chains of pectin, when pectin is at alka-long time (Eder & Ltz-Meindl, 2008; Hotchkiss et al.,er & Wicker, 2005; Renard & Thibault, 1996). The use, as a pH-modier, could be an alternative to raise then solutions avoiding the occurrence of polysaccharidezation.ation of hydrogels with different gelling kinetics and

properties may be obtained by nely modulating pectin it over a specic pH range, prior to the addition of theg agent.ing kinetic is a fundamental parameter in designingus gels and composite gels, injectable systems, cell, in vivo gelling systems, and for an effective drug load-e formation of the gel. To this end our aim was to obtainl gelation of pectin by ne- tuning of NaHCO3 and CaCO3keep a tight control over the pH of the hydrogels thus

their gelling kinetics.ased injectable systems were developed to: (i) have

kinetics, to avoid premature gelling and allow to obtainus gels; (ii) possess tunable crosslinking kinetics, toent surgical procedures and anatomical sites; (iii) areing, possessing low viscosity at high shear to allow therough a needle; (iv) are easily prepared with the possi-le-up the preparation process to the industrial setting.

ental

als

thoxyl pectin from citrus fruits (CU701, DE = 38%) wasided by Herbstreith & Fox (Neuemberg, Germany) in a

form. Sodium bicarbonate (NaHCO3), sodium hydrox- and calcium carbonate (CaCO3) were purchased fromich (Milan, Italy) and used without further purication.

terization of pectin

cular weight by gel permeation chromatography (GPC)s of 2.4% (w/v) pectin with a pH ranging from 3.2 to 11.0red by dissolving pectin powder in solutions of NaHCO3

H.R. Moreira et al. / Carbohydrate Polymers 103 (2014) 339 347 341

Table 1Experimental set up of the rheological characterization.

Experiment Measured parameters Temperature (C) Stress (Pa) Strain (%) Frequency (Hz) Time (min)

Oscillation amplitude G , G , tan , LVR (linear viscoelastic region) 25 0.1100 0.1 Time sweepFrequency sTemperature mp) Creep

and NaOH under stirrimined usinHPLC pump2414). The mUltrahydrog0.8 mL/min(w/v) pectitration of 0(0.45 m pGPC appara

2.2.2. DeterThe equi

tion, as prev2012), withsion (0.1% wneutralize tcarboxylic gwere addedpH were evsequential iapproximatvolume (fropoint.

The rstcurves was regions of t

2.3. Prepara

Pectin hadjusting thpH), 13 mMobtained ad(correspond2 and 4, respectin conc(1100 rpm)solutions wtemperatur

2.4. Charac

2.4.1. RheoRheolog

an AR 150with paragap = 1200 quency andon hydrogeparameters

2.4.2. SwellTo deter

were incuba

vesticent

[wt

wt iweig

The ted aanda

pH m pH o

Cyb pen

tocot cell

use F (Rocand nted

pyrat 37l culmpaater), waded cubather n (RPeasuad La

All ated b

atisti

mea as med

valu

ults

fect oG , G , tan , time of gelling (gel point) 25 weep G , G , tan 25, 37

sweep G , G , tan 2060 (2.5 /min raStrain 25

with different molarity (023 mM) and left overnightng. The molecular weight of pectin solutions was deter-g a GPC system consisting of a Waters 1515 isocratic

with a differential refractive index detector (Watersobile phase (0.1 M NaNO3) was eluted through WaterselTM 1000, 500 and 250 columns with a ow rate of

at 25 C. Samples were prepared by dissolving 2.4%n solutions in 0.1 M NaNO3 for a nal pectin concen-.2% (w/v). After ltration through RC syringe ltersorosity), 200 L of the solutions were injected in thetus.

mination of the equivalence pointvalence point of pectin was evaluated by chemical titra-iously reported (Farris, Mora, Capretti, & Piergiovanni,

slight modications. Briey, pectin aqueous disper-/v) was treated with 7.5 mL of 0.1 N HCl, to completelyhe negative charge distributed along the unprotonatedroups of pectin backbone. Then, 0.1 N NaHCO3 or NaOH

under fast stirring. The electrical potential and thealuated with a Jenway 4510 conductivity meter afternjections of NaHCO3 or NaOH. The titrant was droppedely every 60 s to allow solution equilibrium, reducing itsm 0.5 to 0.1 mL) while approaching to the equivalence

derivative of the data on all points of region 2 of titrationcalculated to access the slope between the two bufferingitration curves, for titration curves comparison.

tion of pectin hydrogels

ydrogels were prepared using calcium carbonate ande pH of 2.4% (w/v) pectin solutions with 0 mM (native

and 23 mM NaHCO3. Calciumpectin hydrogels wereding 12.5 mM, 25 mM and 50 mM CaCO3 suspensionsing to the stoichiometric ratio R = [Ca2+]/2[COO] of 1,pectively) to pectin solutions, to reach a 2% (w/v) nalentration. Each mixture was kept under fast stirring

at room temperature until a gel started to form. Then,ere transferred to Petri dishes and left overnight at roome to stabilize the gel networks.

terization of pectin hydrogels

logical characterizationical characterization of hydrogels was performed with0ex rheometer (TA Instruments, Italy), equippedllel-plate geometry (diameter = 20 mm, workingm). Oscillatory experiments (amplitude, time, fre-

temperature sweeps) and creep tests were performedls, to evaluate their injectability and stability. The

was inthe per

W =

whereinitial ration.presenwith st

2.4.3. The

using aspecial

2.5. Cyindirec

Molectionasks plemesodiumbated the celcytocotilled wextractthe seethen infor fursolutiowas m(Bio-R2013).calcula

2.6. St

All sentedperformwhen p

3. Res

3.1. Ef set up for each experiment are indicated in Table 1.

ing studiesmine the swelling capacity of pectin hydrogels theyted at room temperature in distilled water and swelling

Gel permmine the mpH values, the GPC anof the pectthe pH of th5 0.7 305 0.01100 0.1 0.1 16

10 10

gated at different time points, up to 24 h, by calculating weight variation using the following equation (Eq. (1)):

w0w0

] 100 (1)

s the wet weight of gels at time point t and w0 is theht of the gels, measured immediately after their prepa-measurements were made in triplicate, the results ares average weight variation and the error was calculatedrd deviation.

easurementsf pectincalcium carbonate hydrogels was determinederscan pH 110 pHmeter (Eutech Instruments) with aetration electrode (Hamilton double pore slim).

mpatibility evaluation of pectin hydrogels through viability assay

ibroblasts L929, from the American Type Culture Col-kville, MO) below passage 15, were cultured in 75 cm2

grown in RPMI-1640 culture medium (pH = 8.2), sup-with 10% (v/v) fetal bovine serum, 1% l-glutamine, 1%uvate and 1% penicillin/streptomycin and were incu-C, in 5% CO2 atmosphere. The reagents employed forture were provided by Gibco (Invitrogen). An indirecttibility test was performed on pectin gels, where dis-, previously incubated with gels for 24 h or 7 days (gels diluted (1:10) with fresh medium and then added tocells. Fibroblasts seeded at density 104 cells/well wereted with each diluted culture medium of gel extracts24 h. At the end of incubation, 200 L 10% (w/v) MTTMI serum free) were added for 3 h, and the absorbance

red at 595 nm in a iMark Microplate Absorbance Readerboratories) (Munarin, Bozzini, Visai, Tanzi, & Petrini,ssays were done in triplicate and their signicance wasy Students t-test.

cal analysis

surements were made in triplicate, and data are pre-ean values standard deviation. Students t-test was

and differences were considered statistically signicantes resulted lower than 0.05.

f the pH on pectin molecular weighteation chromatography (GPC) was performed to deter-olecular weight (Mw, Mn) of pectin solutions at differentadjusted or not with NaHCO3 and NaOH. The results ofalyses are shown in Table 2. The values of MW and Mnin solution at its native pH increased when increasinge solution both with NaOH and NaHCO3, and this effect

342 H.R. Moreira et al. / Carbohydrate Polymers 103 (2014) 339 347

Table 2Molecular weight analysis of pectin solution as determined by GPC.

Solutions pH Mw (kDa) Mn (kDa) Mw/Mn

Pectin 3.22 317 74 4.3

Pectin + NaHCO3

3.62 387 164 2.33.88 341 117 2.94.63 344 107 3.26.25 99 32 3.18.07 93 44 2.1

Pectin + NaOH

3.63 398 151 2.63.86 n.a. n.a. n.a.4.21 355 68 5.14.72 374 131 2.85.35 328 135 2.4

11.0 118 28 4.1

was retained up to pH 5.35. This result suggests that the hydro-dynamic volume of pectin chains increased due to the salicationof the carboxyl groups and the consequent electrostatic repulsionof the carboxylates (Kong, Kaigler, Kim, & Mooney, 2004; Munarinet al., 2013). Above a certain limit (Table 2), such as an increaseof pH above 5.35, a reduction of Mw occurs, due to depolymer-ization by -elimination of the pectin backbone (Munarin, Tanzi,et al., 2012), leading to chain scission responsible for polysaccha-ride degradation. A 50% Mw decrease was previously reported insimilar conditions (Hunter & Wicker, 2005).

3.2. Determination of the equivalence point

The equivalent point, dened as the equal stoichiometric num-ber of moles of the titrant and the analyte, provides a quantitativemeasure of the volume of titrant required to deprotonize the car-boxyl groupversus the vare shown i

Fig. 1. Titratio(B) rst deriva

Table 3Equivalent point and derivative properties.

Titrant Equivalent point Derivative line

Concentration (m Eq) pH Slope Angle ()

NaOH 0.94 6.59 46.9 88.8NaHCO3 0.98 6.59 12.7 85.6

The curves clearly display three distinct regions (Fig. 1B). As theequivalent point occurs when the electrical potential is zero, the pHof the equivalent point of pectin titrated with NaHCO3 is the sameof that of pectin titrated with NaOH (Table 3). Absolute values ofslope and angle of the rst derivative of the curves in region 2 aresteeper in the case of NaOH than in the case of NaHCO3 (Fig. 1B andTable 3), meaning that the full neutralization of the acid groupsalong the pectin backbone is faster when adding NaOH. As a result,modifying the pH of pectin solutions becomes easier with NaHCO3due to lower pH buffering of pectin solutions (pH 8 for NaHCO3and pH 11 for NaOH) and slower neutralization of carboxyl groups,leading to tighter control of the pH of pectin solutions.

3.3. pH and gel point of pectin solutions as function of CaCO3concentration

Accordinsolutions tbackbone aronment. Tconcentrati

Increasiof pectin sosolid-like m

The timeor is he gmpo996)gel t

caseed afounn, isO3 is

2), aion os in pectin backbone. Plots of electrical potential and pHolume of titrant for dilute aqueous pectin dispersionsn Fig. 1.

behaviG at ttwo coet al., 1sol-to-in thisobservThe amsolutioof CaC(Tablereductn curves of (A) pectin solutions titrated with NaHCO3 and NaOH andtive of titration curves.

Fig. 2. Pectintions of CaCO3g to the previous results, NaHCO3 was added to pectino partially neutralize the carboxyl groups of pectinnd to reach an adequate pH for the physiological envi-he evolution of the pH of the gels with the NaHCO3on is shown in Table 4.ng the concentration of NaHCO3, an increase of the pHlutions can be observed, as well as a decrease of theorphology of the gels (Fig. 2).-point when materials start to exhibit viscoelastic solidrheologically dened as gel point. The values of G andel point are the same because of the cross-over of thenents (tan 1) (Munarin, Petrini, et al., 2012; Santucci. Aqueous low methoxyl pectin (LMP) solutions undergoransformation in the presence of crosslinking cations,

Ca2+ derived from CaCO3. An increase in the pH waster the addition of CaCO3, when gel formation occurred.t of Ca2+ available in solution, derived from CaCO3 dis-

correlated with the nal increase of pH, as solubility pH-dependent (Table 4). As seen in the GPC results

pH raise over a certain limit (pH > 5.35) leads to a highf the molecular weight and, therefore, chain scission.calcium carbonate hydrogel formulations with different concentra-and NaHCO3.

H.R. Moreira et al. / Carbohydrate Polymers 103 (2014) 339 347 343

Table 4Gelling characteristics of pectin formulations.

Name NaHCO3 (mM) pH of pectin solution CaCO3 (mM) pH of the hydrogels Gelling time

PNa0Ca12.50 3.23

12.5 3.78 0.10 8 50 12PNa0Ca25 25.0 3.98 0.14 3 48 6PNa0Ca50 50.0 4.41 0.12 1, the prevails, as observed for PNa0, PNa13 and PNa23 solu-not shown). Gels with higher tan values resulted to behe gels exhibited a shear-thinning behavior, associatedase of viscosity with the increase of frequency (Fig. 5C).hen the dynamic measurements are repeated on gels

mechanical spectra showed the same results (data notarding storage, tan and viscosity values, being G fre-ependent and always higher than G.

teststs were set up to evaluate the behavior of gels underress. According to previous studies on the viscoelas-r of alginate and pectate gels (Mitchell & Blanshard,ell & Blanshard, 1976a, 1976b; Clark & Rossmurphy,ion amplitude test results of PC1, P13C1 and P23C1 hydrogels. Filledsent G , void symbols represent G .

344 H.R. Moreira et al. / Carbohydrate Polymers 103 (2014) 339 347

Fig. 5. Averag(A) Storage moof frequency.

1987), incras decreasidecreased, robservation

Fig. 7. Linear relationship between the reciprocal of creep compliance measured at10 s at different NaHCO3 and CaCO3 concentrations.

frequency sweep tests (Fig. 5). Fig. 6b gave the evidence of the pres-ence of slow changes in creep compliance at long times, especiallyfor PNa23 hydrogels. This may indicate that the crosslinks are notpermanent, but that they can break or reorganize when the gelis deformed. Considering the values of creep compliance obtained

wor gels 976

7 shin thispectin1974, 1

Fig.e frequency sweep test results of pectincalcium carbonate hydrogels.dulus, (B) tan and (C) complex viscosity are represented as function

easing the amount of the cross-linker (Fig. 6), as wellng the pH of the gels, strain and complex complianceesulting in an improved stiffness of the material. Theses were consistent with the results of the oscillatory

reciprocal oof the lineaof pectin so

The creetted with series with

J(t) = J0 +

where J(t) nian viscosVoigt elemproduced h

The recople, shown

Jr(t) = J(t)

where t1 is 6ery was calparametersresults and

As show1976b), thethe model,lower than elements ar

Fig. 6. Creep test results of pectincalcium carbonate hydrogels. Strain (a) and crek, the produced hydrogels appeared stiffer than otherproduced with GDL and CaHPO4 (Mitchell & Blanshard,a).ows an approximately linear relationship between thef 10 s compliance and CaCO3 concentration. The sloper interpolation was found to decrease increasing the pHlutions.p curves, obtained as reported in Section 2.4.1, werea viscoelastic model constituted by Maxwell element in

three Voigt elements, as described by Eq. (2).

i

Ji(1 e1/i ) +t

N(2)

is the measured creep compliance, N is the Newto-ity and i are the retardation times associated with theents. The parameters obtained tting the curves of theydrogels are reported in Table 5.very was calculated according to the Boltzman princi-in Eq. (3):

J(t t1) (3)

00 s, the time at which the stress is removed. The recov-culated using J(t) as obtained with Eq. (2) and with the

of Table 5. A comparison between the experimentalthe calculated model is reported in Fig. 8 as an example.n in previous works (Mitchell & Blanshard, 1976a,

rst part of the experiment is in good agreement with while in the recovery phase the exponential curve isthe calculated results. This may suggest that more Voigte required for an optimal tting of the curves.ep compliance (b) of the produced formulations.

H.R. Moreira et al. / Carbohydrate Polymers 103 (2014) 339 347 345

Table 5Rheological parameters calculated from creep compliance time responses.

Parameters PNa0Ca12.5 PNa0Ca25 PNa0Ca50 PNa13Ca12.5 PNa13Ca25 PNa13Ca50 PNa23Ca12.5 PNa23Ca25 PNa23Ca50

J0 (Pa1) 2.5 103 8.0 104 5.0 104 2.9 103 2.5 103 1.9 103 1.7 102 1.3 102 1.1 104J1 (Pa1) 1.6 104 8.0 105 3.6 105 1.9 104 2.3 103 1.5 104 2.0 103 7.5 104 1.4 102J2 (Pa1) 1.8 104 1.3 104 5.9 105 6.9 105 4.7 104 1.2 104 8.0 104 1.5 103 2.5 103J3 (Pa1) 8.9 104 3.0 106 4.7 105 4.3 104 5.0 106 1.4 104 4.5 103 3.0 106 1.0 105T1 (s) 6 6 8 4 7 6 6 6 6T2 (s) 26 43 82 121 233 339 222 205 26T3 (s) 151 509 115 344 567 544 501 355 152N (Pa s) 1.05 106 1.41 106 9.60 106 3.02 106 1.43 106 1.90 106 2.95 105 4.61 105 1.05 106

Fig

3.4.4. TempThe vari

the same trewith a platdecrease ofin the elastliquid-like bging from 5formulationperatures hmacromoleduring heat

3.5. Swellin

All of th(Fig. 10) dueand PNa13retained upweight slighlations inste

Fig. 9. Evolutiuated by temp

Swell

totox

otoxiry exels ware u

initels (batio

extrlues cultu

cussi

gelation kinetics and the mechanism involved in the. 8. Measured and calculated creep and recovery curves.

erature sweep testation of G modulus with temperature (Fig. 9) followednd applicable for any polysaccharide gel, characterizedeau region during an extended temperature range. The

G and viscosity with temperature, meaning a decreaseic behavior of the samples and an approximation toehavior, was visible from a critical temperature, ran-

5 C for PNa0 gels, to 36 C for PNa23 gels (Fig. 9). Somes showed an increase of the storage modulus for tem-igher than 50 C, which can be due to changes in thecule conformation or different solubilization of CaCO3ing (Gouva et al., 2009).

g studies

e hydrogel formulations showed an initial swelling to water absorption. PNa0Ca25, PNa0Ca50, PNa13Ca25

Fig. 10. ature.

3.6. Cy

Cytliminahydrogviable

Thehydrogof incuPNa23ing vatissue

4. Dis

The

Ca50 reached a plateau phase and their weight was

to 24 h, whereas PNa0Ca12.5 and PNa13Ca12.5 wettly decreased with time. The weight of PNa23 formu-ad was rapidly decreasing with time.

on of the storage modulus (G) of pectin hydrogel formulations eval-erature ramp test.

ionotropic pectin soluavailabilitymolecular properties.

Among tis importanof the start

Fig. 11. Indireactivity read fvalues were caing behavior of pectin hydrogels incubated in dH2O at room temper-

icity studies

city tests were performed on pectin hydrogels as pre-periments to future trials as injectable systems, whereith the ability to gel in situ, entrap cells and keep themsually required.ial cytotoxicity may be due to the acidic pH of theTable 4), which is probably equilibrated after 7 daysn. An increase of the cytocompatibility is observed for

acts after 7 days of incubation, with cell viability reach-comparable with the control (i.e. cells seeded on there plate) (Fig. 11).

ongel formation are strictly dependent upon the pH oftions. pH regulates the dissociation and therefore the

for ionic complexation of carboxylic groups, and theweight of the polysaccharides, affecting the nal gel

he properties of pectin solutions, the equivalence pointt because it constitutes the point at which all the H+

ing solution are neutralized by the titrant. The curves

ct cytocompatibility of 24 h and 7d hydrogels extracts. The metabolicor the control (TCPS) was taken as the reference (100%). Signicancelculated by t-test (*p 0.05).

346 H.R. Moreira et al. / Carbohydrate Polymers 103 (2014) 339 347

of electrical potential and pH (Fig. 1) clearly display three distinctzones. First buffering phase (region 1) corresponds to the neutral-ization of H+ ions by HCl. Region 2 is characterized by reaching theequivalence point, where the pH of pectin progressively increasesand hence the carboxy(OH and Hpersion. Thhydrogen g

PectinCOO

PectinCOO

When thelectrical p(Bochek, ZaThe lower psibly due tothe lower vof NaOH, is and suggesboxyl groupwith NaHCO

The chosrespond to tthe nal hyconcentratition is relateIncreasing tgelling timegel states isFraeye et al

Frequenfollowed bhigher amogels (Iglesiacan be expare typicallpectin pKa. hydrogen bhydrogel. Achains chanbonds dimcharacteristcrosslinking& Heilshorn(Table 3), sMw occurspectin backresponsiblepreviously r

The formdynamic bethe pectin nciation of ggroups (Sria

The hydmodulus (F(Fig. 5C). A2012) valueof each hydical temper

hydrogels (G and complex viscosity) remained stable (Fig. 9), thusconrming the suitability of the injectable hydrogels as implantablecarriers for regenerative medicine.

Focusing on the injection procedure, a viscoelastic behaviorotecty thh ths PNue toand 012

andere oe gelen, Sriateing a

hydroftethe be con) forse setrati

alkaties.

clus

tincationindu

kineoverotes hybilittin s

highe degels. A

its biew menstratincaspecirmoruld ds. Ins exhg th

wled

s womio dnd Mi.

autherm

The acal sugy suthe electrical potential decreases as deprotonation oflic groups proceeds, due to the increase of titrant ionsCO3 from NaOH and NaHCO3. respectively) in the dis-ese ions derive from the neutralization of dissociatedroups of pectin backbone according to Eqs. (4) and (5).

H + Na+ + OH PectinCOO + Na+ + H2O (4)

H + Na+ + HCO3 PectinCOO + Na+ + H2O + CO2(5)

e carboxylic groups are completely titrated, pH andotential reach another plateau phase (region 3) (Fig. 1)bivalova, & Petropavlovskii, 2001; Farris et al., 2012).H was reached when titrating pectin with NaHCO3, pos-

the release of CO2 that is known to lower the pH. Also,alue of the slope of NaHCO3, if compared with the slopetypical for a titration of a weak base with a strong acid,ts that the conversion of the acidic form of pectin car-s into the salied form can be more easily controllable3.en concentrations of CaCO3 (12.5, 25 and 50 mM) cor-he stoichiometric ratios, R = [Ca2+]/2[COO], desired fordrogels of 1, 2 and 4, respectively. The inuence of theon of calcium ions on low methoxyl (LM) pectin gela-d with the amount of non-methoxylated GalA residues.he concentration of CaCO3 resulted in a reduction of the, conrming that the transition rate between the sol and

a function of calcium concentration (Dobies et al., 2004;., 2010; Gigli, Garnier, & Piazza, 2009).cy sweep results (Fig. 5) showed a linear behaviory the hydrogels and. Regarding the calcium content,unts of CaCO3 available in solution resulted in strongers & Lozano, 2004). The stiffness of P hydrogels (Fig. 5A)lained by the pectin pKa of 3.5. LM pectin hydrogelsy spreadable, with rigidity increasing as pH falls belowBelow the pKa, less dissociation of H+ results in greateronding of the pectin chains, thus originating a stifferbove the pKa, acid groups are ionized, the polymericge their conformation and the number of hydrogen

inishes. As a consequence, the polymer acquires theics of a polyelectrolyte, thus allowing for more efcient

by calcium ions (Aguado, Mulyasasmita, Su, Lampe,, 2012). Above a certain limit, as shown by GPC resultsuch as an increase of pH above 6.25, a reduction of, due to depolymerization by -elimination of thebone (Munarin et al., 2012b), leading to chain scission

for polysaccharide degradation. A 50% Mw decrease waseported, in similar conditions (Hunter & Wicker, 2005).ation of a three-dimensional network is shown by thehavior of pectin hydrogels. The entangled structure ofetwork is formed by intermolecular electrostatic asso-alacturonans and ionic complexation of the carboxylmornsak, 2003; Strm & Williams, 2003).

rogels showed no frequency-dependence of the storageig. 5A) but decreasing viscosity at higher frequenciesccording with the literature, (Munarin, Petrini, et al.,s of tan lower than 1 indicated the solid-like behaviorrogel formulation (Fig. 5B). When applying physiolog-atures (i.e. 3542 C), the rheological properties of the

can praged bthrougsuch acells dow) et al., 2gellinggels wof thes(VoragappropprovidPNa23being s

On it can b(Fig. 11

Theconcention inproper

5. Con

Pecnal gelmight gellingsively to promgeneoucell via

Pecof 6 orcharidform gdue to

In ventrapdemonof pectto the Furtheand comethothe gelallowin

Ackno

ThiRisparTanzi aL. Visa

Theburg, Gwork. technirheolo the cells and/or bioactive molecules from being dam-e mechanical forces experienced during the extrusione needle. The ow of stiffer or more compliant gels,a0 gels through the syringe needle, could damage the

pressure drop, shearing forces (due to linear shearstretching forces (due to extensional ow) (Aguado). Injectable systems for localized delivery require slow

tunable crosslinking kinetics. Despite homogeneousbtained with PNa23 formulations, the evident softnesss caused lower resistance to frequency and temperaturechols, & Visser, 2003). While PNa0 hydrogels appear

injectable formulations for the repair of hard tissues, three-dimensional structural support for the host cells,ogels seems more indicated for soft tissue regeneration,r and easier conforming to the anatomical site.asis of the results obtained with the cytotoxicity assay,cluded that pectin hydrogel extracts were not cytotoxic

L929 cells.veral factors, solubility and concentration of CaCO3,on and pH effect of NaHCO3, as well as pectin degrada-line pH affect cell viability, as well as all the investigated

ions

alcium carbonate hydrogels were prepared by inter-, avoiding the addition of D-glucono--lactone, whichce difculties in the control of the nal pH and of thetics of the hydrogels. The tight control achieved exclu-

the amounts of NaHCO3 and CaCO3, was determinant appropriate gelling kinetics, required to obtain homo-drogels, as well as to reach pH values compatible withy.olutions, modied with NaHCO3 and NaOH, with a pHer, undergone chain scission, responsible for polysac-radation and consequently they were not adequate tolso, the control of the pH resulted easier with NaHCO3uffer capability.of developing pectin-based injectable systems for cellt in biomedical applications, the results of this worked that the gelling kinetics and rheological propertieslcium carbonate hydrogels can be modulated accordingc agent to be entrapped and to the tissue to be treated.e, the considered formulations were cytocompatiblebe obtained with inexpensive and easy preparationjectability was conrmed by rheological analyses, asibited a viscoelastic and shear-thinning behavior, thuseir injection through a needle.

gements

rk was supported by grants from Fondazione Cassa dii Trento e Rovereto, SG2329/2011-2011/0206 to M.C.IUR, PRIN 2010-11 project (prot. 2010FPTBSH 009) to

ors wish to acknowledge Herbstreith & Fox (Neuen-any) for kindly providing the LM pectin used in thisuthors are also thankful to Monica Moscatelli for herpport on GPC analyses and Marco Coletti for theoreticalpport.

H.R. Moreira et al. / Carbohydrate Polymers 103 (2014) 339 347 347

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Injectable pectin hydrogels produced by internal gelation: pH dependence of gelling and rheological properties1 Introduction2 Experimental2.1 Materials2.2 Characterization of pectin2.2.1 Molecular weight by gel permeation chromatography (GPC)2.2.2 Determination of the equivalence point

2.3 Preparation of pectin hydrogels2.4 Characterization of pectin hydrogels2.4.1 Rheological characterization2.4.2 Swelling studies2.4.3 pH measurements

2.5 Cytocompatibility evaluation of pectin hydrogels through indirect cell viability assay2.6 Statistical analysis

3 Results3.1 Effect of the pH on pectin molecular weight3.2 Determination of the equivalence point3.3 pH and gel point of pectin solutions as function of CaCO3 concentration3.4 Rheological characterization3.4.1 Oscillation amplitude test3.4.2 Frequency sweep test3.4.3 Creep test3.4.4 Temperature sweep test

3.5 Swelling studies3.6 Cytotoxicity studies

4 Discussion5 ConclusionsAcknowledgementsReferences