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European Journal of Pharmaceutical Sciences 37 (2009) 249–256

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences

journa l homepage: www.e lsev ier .com/ locate /e jps

ailoring vancomycin release from �-TCP/agarose scaffolds

.V. Cabanas, J. Pena, J. Román, M. Vallet-Regí ∗

epartamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain

r t i c l e i n f o

rticle history:eceived 11 November 2008eceived in revised form 19 January 2009ccepted 18 February 2009vailable online 5 March 2009

eywords:caffoldsrug delivery systems (DDS)

a b s t r a c t

In this work a multifaceted approach to the fabrication of scaffolds is considered, that is, besides thepreparation technique, the introduction of substances that may contribute to enhance their final perfor-mance, as well as the techniques required to ensure the correct preservation of the so obtained scaffoldsare taken into account to tailor the release of vancomycin from �-tricalcium phosphate (�-TCP)/agarosescaffolds. These materials were prepared by a shaping technique that allows to obtain pieces at a tem-perature low enough to simultaneously include active substances susceptible of heat degrading such asvancomycin, the model drug considered in this work. In the first approximation poly(ethylene glycol)(PEG), a hydrophilic substance employed as a matrix capable of binding compounds such as proteins or

oom temperature shapingeinforced hydrogelsoly(ethylene glycol)reservation tools

peptides and release them in a controlled fashion, was included in the formulation. The second tool togovern the vancomycin liberation is based on the drying procedures employed to process and preservethe obtained scaffolds: freeze-drying and heat desiccation at 37 ◦C. These modifications resulted in thegeneration of different pore architectures and certain chemical interactions, such as the formation of anagarose–PEG–vancomycin complex that yielded different drug release patterns. The so obtained pieces

en im

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behave like a hydrogel whof the cancellous bone.

. Introduction

There is a growing interest on scaffolds capable not only of ful-lling the requirements demanded from this type of structures butlso with the ability to host and release certain substances, or cells,hat may help to avoid some negative effects that usually appearith the implantation of a material, i.e. infection, inflammation,

tc. At the same time, the inclusion of other type of substances suchs growing factors, angiogenesis inductors, guide cell proliferationifferentiation and migration factors could contribute to facilitateome of the many steps implied in the successful integration of araft and the, some times, required substitution by the patients ownissue (Kopecek, 2003). In fact, some of the techniques employedo prepare scaffolds, such as the rapid prototyping-based three-imensional printing, are being utilized in applications linked torug delivery systems (Katstra et al., 2000; Yu et al., 2008).

Hydrogels are considered appealing materials for tissue engi-eering since they mimic the physico-chemical properties of extra-

ellular matrix not only in terms of the chemical resemblance butlso due to their highly hydrated three-dimensional architectureLee and Mooney, 2001; Drury and Mooney, 2003). The polymerstudied and utilized to date in tissue engineering can be divided

∗ Corresponding author. Tel.: +34 91 3941861; fax: +34 91 3941786.E-mail address: vallet@farm.ucm.es (M. Vallet-Regí).

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928-0987/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.ejps.2009.02.011

mersed in a hydrated medium but show a consistency comparable to that

© 2009 Elsevier B.V. All rights reserved.

etween synthetic poly(vinyl alcohol), poly(ethylene oxide),oly(vinyl alcohol), poly(acrylic acid), poly(propylene furmarate-o-ethylene glycol), polyphosphazene, polypeptides, etc. (Lee andooney, 2001) and natural polymers (collagen, fibrin, alginate,

garose, hyaluronic acid, chitosan, etc.) (Jiyoung et al., 2006). Thisolymeric network with high water content mimics many roles ofxtracellular matrixes found in tissues by providing a place for cellso adhere, proliferate, and differentiate, allowing the diffusion ofutrients into the gel and cellular waste out of the gel and the trans-ission of chemical and physical stimuli via mechanical deforma-

ion of these matrixes. In addition, these materials can gel “in situ”nder physiological conditions thus facilitating the inclusion ofhermally labile substances or the encapsulations of cells (Jen et al.,996; Drury and Mooney, 2003; Hoffman, 2002; Zhang et al., 2008).

However, scaffolds based on hydrogels generally exhibit a nar-ow and limited range of mechanical properties. To overcome thisrawback, composites or mixtures with ceramics have been pre-ared (Rezwana et al., 2006). On the other hand, the addition ofiodegradable polymers to a bioresorbable and bioactive ceramican improve the mechanical weakness, i.e. poor toughness andrittleness and contribute to regulate the degradability. For com-

osites containing active substances drug release profiles can beltered depending on the nature and properties of the polymericomponents.

In a previous work a scaffold fabrication technique at roomemperature (Cabanas et al., 2006) was employed to prepare

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idEriSGeA Hg porosimetry study was carried out using a MicromeriticsAutoPore III 9410 porosimeter. N2 adsorption was carried out on aMicromeritics ASAP 2010 instrument; surface area was obtained byapplying the BET method to the isotherm and the pore size distribu-

Table 1Samples notation and nominal composition (wt/vol.%) of the hydrogels synthesized.

Samplea �-TCP Agarose PEG Vancomycin

A� 20 2.5 – –

50 M.V. Cabanas et al. / European Journal o

garose/�-TCP pieces loaded with vancomycin (Román et al.,008), a hydrophilic antibiotic drug, commonly used for treatingsteomyelitis and preventing osseous staphylococcal infectionsfter surgery. Besides its traditional uses (Rinaudo, 2008) agaroseas gained a new appreciation in tissue engineering applicationsuch as cell–hydrogel hybrids (Prashant et al., 2006), nerve guid-nce scaffolds (Dodla and Bellamkonda, 2006), matrix materialor dental and bone replacement (Tabata et al., 2003; DiMiccot al., 2007), beads and microcarriers for drug delivery or cellulture (Liu and Li, 2005; Meilander et al., 2003), micropatternedtamping arrays (Mayer et al., 2004) and molecularly imprintedembranes for protein recognition (Lin et al., 2008). �-Tricalcium

hosphate (Ca3(PO4)2, �-TCP) has been observed to resorb in vivo,ith new bone growth replacing the original graft (Dorozhkin

nd Epple, 2002; Vallet-Regí and González-Calbet, 2004). Thisroperty imparts significant advantage to �-TCP compared withther biomedical materials not as easily resorbed and replaced byatural bone.

The scaffolds obtained by the above-mentioned prepara-ion method behave like a reinforced hydrogel whose swellingehaviour and drug release rate depend on their composition, i.e.n the agarose and ceramic percentages (Román et al., 2008). More-ver, it must be stressed out one of the most outstanding featuresf this preparation technique, that is, the relatively mild pH andemperature conditions used, which allow the introduction of sub-tances or organisms susceptible to enhance the performance of theo obtained scaffolds. The inclusion of this type of additives duringhe fabrication process, however, is not an easy task since many ofhe preparation methods imply an ulterior thermal treatment or anggressive medium that generally ruins their properties (Tsang andhatia, 2004).

The purpose of the present work is to tailor the vancomycinelease through the inclusion of additional substances such asoly(ethylene glycol) (PEG). PEG is a non-toxic, non-immunogenicnd non-antigenic hydrophilic hydrogel (Manta et al., 2003). Themportance and possibilities of this substance lies on the capabilityf PEG molecules to bind to different compounds such as proteinsr peptides in order to control their delivery to the desired targetPeppas et al., 2000; Veronese, 2008). The technique of covalentoupling of poly(ethylene glycol) to pharmaceutical proteins, com-only named PEGylation, has become the dominant protein drug

elivery system for the biotech industry, with considerable sales ofEGylated protein drugs such as Adagen®, Oncaspar®, PEGIntron®,EGASYS®, Neulasta® and Somavert® (Veronese and Harris, 2002;asut and Veronese, 2007).

A final point to be taken into account, considering an integralverview of scaffolds fabrication for tissue engineering, is the toolshan enable preservation which permits the “off the shelf” avail-bility of engineered scaffolds and tissues (Pancrazio et al., 2007).oreover, the drying technique of the hydrated pieces has been

onsidered as a possible route to control the pore architecture ofhese scaffolds which should decisively influence on the swellingehaviour and on the release of active substances included withinhem. In fact, freeze-drying has been utilized not only to tailor theorosity by controlling the freezing time and temperature (Zmorat al., 2002), but also to build complex composites (Deville et al.,006).

. Materials and methods

.1. Components

The ceramic powder, �-tricalcium phosphate, �-Ca3(PO4)2,as synthesized by a crystallization method (Rodríguez-Lorenzo

nd Vallet-Regí, 2000) from aqueous solutions of Ca(NO3)2·4H2O

AAA

a

aceutical Sciences 37 (2009) 249–256

0.9 M) and (NH4)2HPO4 (0.6 M), which were dropped simultane-usly at a flow rate of 22 mL/min, being the reaction temperature0 ◦C, and the reaction time 3 h. Throughout the mixing process, theeaction pH was maintained between 5.6 and 6.0 by the additionf an ammonia solution. The precipitated powder was stored for0 h at room temperature, then washed with de-ionized water andreeze-dried. The resulting powder was thermally treated at 900 ◦Cor 1 h. Agarose (Sigma–Aldrich, Steinheim, Germany, for routinese) and poly(ethylene glycol) 4000 (PEG 4000 from Sucesorese José Escudero, S.L., Terrasa, Barcelona) were commercially pur-hased. Vancomycin was kindly supplied by Normon Laboratories.A. (Madrid, Spain).

.2. Scaffolds preparation

The fabrication procedure consists, briefly, on the followingteps (Cabanas et al., 2006; Román et al., 2008): (i) the agaroseowder is suspended in de-ionized water (2.5 wt/vol.%) and heatedt 80 ◦C, (ii) once a translucent sol is achieved, the working tem-erature is progressively decreased to a value near to 40 ◦C, thusllowing the introduction of thermally labile substances such ashe vancomycin considered in this study and (iii) �-TCP powder20 wt/vol.%) is added under continuous stirring. The so obtainedlurry can be easily injected into cylindrical polystyrene moulds;fter a few minutes the complete consolidation of the bodies allowso unmold the pieces and shape them by means of a ordinary cut-er. The so obtained pieces were dried by two different procedures:n an oven at 37 ◦C for 24 h (termed as samples O) or freeze-driedtermed as samples F).

Scaffolds containing polyethylene glycol were prepared in a sim-lar way by introducing PEG 4000 into the agarose water solutionefore adding the ceramic. For antibiotic loaded pieces vancomycinas mixed with the �-TCP powder, before adding to the suspension.

The synthesized samples by this procedure are listed in Table 1.he naming convention stands for the presence of vancomycin (V)r polyethylene glycol (P). An additional character, O or F indicateshen the sample has been dried in and oven at 37 ◦C or by freeze-rying, respectively.

.3. Characterization

The obtained pieces were analyzed by X-ray diffraction (XRD)n a Philips X-Pert MPD diffractometer. Thermogravimetry andifferential thermal analysis (TGA/DTA) were performed in a Perkinlmer Pyris Diamond TG/DTA analyser, with 10 ◦C/min heatingamps, from room temperature to 600 ◦C. Fourier transformnfrared (FTIR) spectra were obtained in a Nicolet (Thermo Fishercientific) Nexus spectrometer equipped with a Smart Goldenate ATR accessory. Surface morphology was analyzed by scanninglectron microscopy (SEM) in a JEOL 6400 Electron microscope.

�V 20 2.5 – 1�P 20 2.5 2.5 –�PV 20 2.5 2.5 1

a An additional type, O or F indicates when the sample has been dried in and ovent 37 ◦C or by freeze-drying, respectively.

M.V. Cabanas et al. / European Journal of Pharm

Fi(

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ig. 1. Photographs showing the ease of manipulation and shaping (a, b), a compar-son between freshly prepared (as obtained), freeze-dried and heat treated samplesc).

ion was determined by the BJH method from the desorption branchf the isotherm. Compressive strength was determined by usingMTS Bionix®858 Test System with a load cell of 2.5 KN (Charge

imit 2000 N). Test StarII® for general control and Test Works4®

or data acquisition were used as software. Ten cylinders of eachample of 10 mm × 7 mm were prepared for mechanical testing.

.4. In vitro swelling and delivery assays

The in vitro swelling behaviour and vancomycin delivery assaysere performed in parallel by immersing disc-shaped samples

xtracted from a freshly prepared cylindrical block (Fig. 1a) into0 mL of a phosphate buffer solution (pH 7.4) at 37 ◦C under con-inuous stirring (200 rpm).

Disc swelling was monitored by gravimetrically measuring theater intake as a function of time. Disc weights were recorded byeriodically removing them from the swelling media, blotting withbsorbent tissue and weighing the pieces. Three pieces extractedrom each sample were measured to calculate the average value.

The swelling ratio, S, which corresponds to the average hydra-

ion degree, has been determined according to the followingquation (Taguchi et al., 1999):

= Wt − W0

W0(1)

sto

i

aceutical Sciences 37 (2009) 249–256 251

here Wt is the weight of the discs after immersion time t, and W0s the weight of the dry discs.

The concentration of vancomycin release was followed by UVpectroscopy, using an UNICAM UV500 (Unicam Instruments) spec-rophotometer. Absorbance values were taken at a wavelength of= 281 nm, where the antibiotic shows an absorbance maximum.ancomycin concentration was determined from the average of

he readings of three different discs. The calibration curve of van-omycin was determined between 0.1 and 0.5 mg mL−1; within thisnterval, the calibration curve fits the Lambert and Beers law.

The vancomycin release data were analyzed using the followingquation applicable to porous hydrogel matrices (Korsmeyer et al.,983; Lee, 1985; Peppas et al., 2000):

Mt

M∞= ktn (2)

here Mt/M∞ is the fractional drug release, t is the release time, ks a kinetic constant and n is an exponent which characterizes the

echanism of drug release.

. Results and discussion

.1. Characterization of the scaffolds

Pieces of �-TCP/agarose with or without PEG and vancomycinTable 1) have been prepared at low temperature by a simple proce-ure based on agarose thermal gelation. The addition of PEG and/orancomycin into the agarose matrix did not measurably affect to theelation time, thus not altering the preparation procedure. Onceonsolidation has occurred, the resulting gels can be shaped intoiscs, cubes or strips by punching out or cutting them with a razorFig. 1); moreover also the above preparations can be easily formu-ated into injectable shapes.

The agarose component entraps water thereby providing a rub-ery consistency to the hydrated samples. Once desiccated, theseieces show a glassy consistency. The samples dried in the ovent 37 ◦C (samples O) undergo a considerable shrinkage (ca. 50–60%f initial volume) while for those freeze-dried the shrinkage wasuch lower (ca. 5–10% of initial volume). Despite the considerableeight loss, higher than 80%, no significant change in body shapeas observed before and after the drying process.

The ceramic component, a single phase �-Ca3(PO4)2 (�-CP—JCPDS card 9-169), as deduced from X-ray diffraction, withparticle size lower than 1 �m (SEM), and a specific surface area

qual to 4.3 m2/g (BET), is responsible of all the diffraction max-ma detected by XRD, being difficult to identify any other maximattributable to the rest of the components. In this sense, the XRDatterns of samples prepared without �-TCP show some diffrac-ion maxima that can be attributed to PEG 4000 (JCPDS card9-2095) within an amorphous background corresponding to thegarose and vancomycin components. The predominance of the �-CP diffraction maxima can be explained by taking into accounthe weight proportion of this ceramic in the pieces (∼80–90%).his value has been determined by thermogravimetry: the residueeft after heating at 600 ◦C corresponds to the inorganic compo-ent. Additionally, this technique allows to estimate the amount ofater in the hydrated pieces, i.e. the mass lost below 150 ◦C cane attributed to water elimination (around 80%). In all cases theo obtained water content is slightly lower than the theoreticallyxpected (about 5–7%) and therefore, the content of solids results

lightly higher compared to the theoretically expected; �-TCP con-ents near to the theoretical value (between 77% and 89%) werebtained.

Besides, the differential thermal analysis (DTA) allows tonfer the formation of complexes between the agarose, PEG and

252 M.V. Cabanas et al. / European Journal of Pharmaceutical Sciences 37 (2009) 249–256

dried

vaccFctttmtptl(

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Fig. 2. Differential thermal analysis of samples freeze-

ancomycin components. This hypothesis is based on the appear-nce of an exothermic peak, around 175–225 ◦C, in samplesontaining both agarose and PEG (samples A�P and A�PV) whichannot be appreciated in samples without PEG (A� and A�V,ig. 2a) nor in the individual components (agarose, PEG or van-omycin, Fig. 2b). The presence of this peak could be related withhe formation of an agarose–PEG complex which decomposes atemperatures around 180 ◦C. This peak is shifted towards higheremperatures in presence of vancomycin, thus indicating the for-

ation of a triple complexation agarose–PEG–vancomycin with ahermal stability higher than the agarose–PEG complex, decom-osing at around 225 ◦C. The presence of �-TCP does not seemo influence in the formation of these complexes, because simi-ar results were observed in samples synthesized without �-TCPFig. 2b).

The formation of polymer/polymer complexes has been estab-ished in many polysaccharide systems in aqueous solutionsRaimundo, 2006). The cooperative hydrogen bonds allow thetabilization of the helical conformation, and are also involvedn the hydration and dissolution of polysaccharides. Interchainydrogen bonds allow the formation of original cooperativeolymer–polymer complexes. Despite PEG being a neutral poly-er, strong interactions between the PEGs and polymer network

y formation of hydrogen bonds in the PVA gels have beenescribed (Masaro et al., 1999). In addition, strong interactions

etween PEG and chitosan, fribinogen or drugs has been observed

n other hydrogels systems (Almany and Selikter, 2006; Wang etl., 2007). Consequently, similar interactions can be expected in theEG–agarose system through the many hydroxyl groups present onhe agarose gel network (Weng et al., 2005).

tffop

(a) and prepared without the ceramic component (b).

The formation of these complexes has also been detected byTIR as can be deduced from the differences observed between thepectra of the individual components and those of the samples pre-ared. The examination of these samples showed, mainly, bandsttributable to phosphate groups, thus hindering the appreciationf bands of the minority components. For this reason, samples with-ut �-TCP, whose FTIR spectra are displayed in Fig. 3, were prepared.n samples containing agarose–PEG–vancomycin or agarose–PEGesides the bands attributable to PEG, agarose or vancomycin, it cane observed the appearance/intensification or definition of weakands, even shoulders at 1181, 1068 and 787 cm−1. A similar phe-omenon, based also on the modification of the FTIR spectrum, wasescribed for a chitosan, PEG and ciprofloxacin system (Wang et al.,007).

The samples prepared show, by scanning electron microscopy,pparently a homogeneous morphology being constituted by a con-inuous polymeric matrix that embeds the ceramic particles (Fig. 4).owever, the micrographs at a higher magnification reveal theppearance of several pores in freeze dried samples (Fig. 4b) thato not appear in those treated at 37 ◦C (Fig. 4a). On the other hand,he introduction of PEG or vancomycin does not seem to have aignificant effect on the samples morphology.

In order to quantify the differences, in terms of porosity and den-ity, that can be easily observed macroscopically with the naked eyend confirmed by scanning microscopy, the samples were charac-

erized by means of a mercury intrusion porosimeter. The main dif-erences between samples desiccated in an oven (Fig. 5a) and thosereeze-dried (Fig. 5b) lies on the appearance for the latter of a sec-nd pore distribution zone around 50 �m and up to 100 �m. Theseores cause a considerable increase in the porosity percentage

M.V. Cabanas et al. / European Journal of Pharmaceutical Sciences 37 (2009) 249–256 253

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ig. 3. FTIR spectra of the individual components: agarose, PEG, vancomycin as wellhat of an agarose–PEG–vancomycin-containing sample.

rom 54% to 82% in the presence of PEG (samples A�PO and A�PF,espectively) and from 48% to 70% without this component (sam-les A�O and A�F, respectively). No differences were observedetween samples containing or not vancomycin.

In addition, a sharper distribution at narrower pore size2–0.1 �m) can be observed for all samples, independently eithern the components or the drying method. These pores can bettributed to the spaces generated between the particles mainlyomposed by the ceramic component (�-TCP). However, the pres-nce of PEG influences on this distribution by generating a bimodalistribution whose higher maximum is shifted towards wider poresnd maintains a smaller maximum that overlaps the monomodalistribution observed in the absence of PEG. Besides, as above-entioned, the presence of PEG causes an increase in the porosity

ercentage.Regarding one of the major disadvantages of hydrogel-based

caffolds, i.e. the weak mechanical properties, the ceramic compo-ent provides a superior consistency that enables the manipulationnd processing of the scaffolds. In this sense, in order to estimatehe mechanical performance of these pieces, a preliminary studyas carried out. The compressive strength of the samples is modi-ed by the drying procedure varying between 1.7 (±0.15) MPa forhe freeze-dried sample (A�F) and 17.7 (±1.9) MPa for sample driedn the oven (A�O). The introduction into the system of PEG and/or

ancomycin slightly decreases the compressive strength to valuesround 8.5 (±0.7) MPa. The compressive modulus was calculateds 110 (±20) MPa in all the samples. These values are in the sameange of magnitude to that observed in different calcium phos-hate/natural polymers systems such as: collagen/hydroxyapatite

ao

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Fig. 4. SEM micrographs of A�PO and A�PF.

TenHuisen et al., 1995), calcium phosphate/chitosan (Liu et al.,006) or the collagen-chitosan/�-TCP composites (Yin et al., 2003).oreover, the failure strength of the prepared scaffolds is quite

omparable to human cancellous bone, which has a typical com-ressive strength between 0.5 and 14.6 MPa (Goulet et al., 1994).

.2. In vitro swelling behaviour and vancomycin delivery assays

In spite of the predominant presence of the ceramic componenthich, as already mentioned, confers a mechanical performance

omparable to similar inorganic/organic systems including cancel-ous bone, these scaffolds behave like an hydrogel, i.e. hydrophilicetwork polymers which are glassy in the dehydrated state and

n the presence of water absorb a significant amount of fluid toorm elastic or rubbery gels. This behaviour can be attributed tohe hydrogel components since, due to the hydrophilic nature ofheir chains, the network is able to absorb water within its struc-ure, swell without destruction, and to maintain its overall structureYalpani, 1988; Harris, 1990).

Fig. 6 shows the variation of the swelling ratio (S) with the dura-ion of disc immersion in a phosphate buffer solution at pH 7.4nder stirring. As it can be observed, both the swelling behaviournd the swelling ratio, depend on the drying procedure as well as

n the presence of PEG into the sample composition.

Freeze-dried samples reach the swelling equilibrium in a fewinutes after an initial fast weight uptake, whereas the swelling

rocess of samples O (dried in an oven) is significantly slower

254 M.V. Cabanas et al. / European Journal of Pharm

F

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tpmmodel drug used in this work, vancomycin. In vitro release profiles

ig. 5. F and O samples pore size distribution obtained by Hg intrusion porosimetry.

equiring a much longer time of immersion to reach the equilib-ium. The experimental swelling data corresponding to samples O

ould be fitted to the theoretical model for the diffusion-controlledwelling proposed by Schott (1992), which indicates that thewelling behaviour of these dried systems would fit to a second-rder diffusion kinetic.

ig. 6. Swelling ratio as a function of immersion time in the phosphate medium.

oct

aceutical Sciences 37 (2009) 249–256

On the other hand, PEG-containing samples reach higherwelling ratios (275% and 175%, samples A�PL and A�PO, respec-ively) when compared to those observed in the absence of thisolymer (190% and 125%, samples A�L and A�O, respectively). Sim-

lar swelling profiles to that shown in Fig. 6 were observed forancomycin-containing samples, that is, the presence of the van-omycin does not modify, apparently, the swelling behaviour.

The influence of the drying procedure can be explained by takingnto account the macroscopic phenomena that can be appreciated

ith the naked eye, in particular the variation of the pieces dimen-ions. The freeze-dried samples remain almost constant whenompared to freshly prepared pieces, that is, the evacuated waterreates the porous structure characterized by porosimetry and SEM.nce one of these samples is immersed in the phosphate buffer, it

mmediately reaches the swelling equilibrium recovering its initialolume. On the other hand, oven desiccated pieces, which have suf-ered a considerable decrease on their dimensions, do not recoverompletely their initial state. The presence of higher porosity inamples F allows for a fast convective mass transport, that is anasy penetration of water, rather than a slow diffusion swelling, ast occurs for samples O. This open structure justifies an easy and fastater penetration and, consequently, shorter swelling stabilization

imes for samples F. On the contrary, samples dried at 37 ◦C suffern important shrinkage that could hinder the rehydration process,herefore increasing the stabilization times and decreasing the finalater uptake.

Moreover, the chemical structure of the polymer also affects thewelling ratio of the hydrogel. Hydrogels containing hydrophilicroups swell to a higher degree when compared to those containingydrophobic groups (Peppas et al., 2000; González et al., 2008). Inhis sense, the higher hydrophilic nature of PEG component justifieshe higher swelling ratio observed in these samples compared tohe pieces without PEG (Fig. 6). Besides, the introduction of the PEGnto the agarose matrix could diminish the cross-linking density ofhe agarose thus increasing the swelling degree of the hydrogelWang and Wu, 1997).

Once studied the influence of the drying procedure, as well ashe introduction of PEG, on the piece fabrication, on their mor-hology and microstructure and on their behaviour in an hydratededium, all these parameters should be related to the release of the

f vancomycin from the samples are shown in Fig. 7. Two main con-lusions can be immediately deduced: (1) the presence of PEG inhe formulation decreases the drug release, regardless of the drying

Fig. 7. Cumulative fraction of vancomycin released (Mt/M∞) versus time.

M.V. Cabanas et al. / European Journal of Pharm

Table 2Kinetic parameters of vancomycin release from different samples. The confidencelimits for any parameters are 95%.

Sample Kinetic constanta k (h−n) Release exponenta n r2b �2c

A�V-O 0.69 (±0.02) 0.545 (±0.05) 0.995 0.0005A�V-F 0.522 (±0.006) 0.493 (±0.03) 0.996 0.00019A�PV-O 0.506 (±0.008) 0.509 (±0.03) 0.995 0.0003A�PV-F 0.34 (±0.01) 0.568 (±0.03) 0.990 0.00086

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a Standard deviations are given between branches.b Values of correlation coefficients.c Goodness of fit to Eq. (2).

ethod and (2) for the same composition, the drug release inreeze-dried samples is slower than in the samples O.

The vancomycin release data were analyzed using the expres-ion previously shown in Eq. (2) (Mt/M∞ = ktn). Table 2 summarizeshe kinetic parameters resulting from the fit of vancomycin releaserofiles.

The release exponent n is indicative of the release mecha-ism and depends on the matrix carrier geometry (Ritger andeppas, 1987). Cylinder-shaped devices give values of n = 0.45,.45 < n < 0.89, n = 0.89 or n = 1 indicating Fickian release, anoma-

ous (non-Fickian) transport, Case-II release, or independent ofime, respectively. In the present work, the mean n values (Table 2)ndicate that the release mechanism of the obtained scaffolds cane described as an anomalous diffusion. The drug-loaded hydro-els are usually stored in the dry glassy state before usage dueo stability and dosage form requirements. The release of wateroluble drugs from such dehydrated hydrogel matrices generallynvolves the simultaneous absorption of water and desorption ofrug via a swelling-controlled diffusion mechanism (Lee, 1985).hus, as water penetrates a glassy hydrogel matrix, the dispersedrug diffuses through this swollen rubbery region into the exter-al releasing medium. Such diffusion generally does not followFickian diffusion mechanism. In addition to diffusion, the exis-

ence of molecular relaxation processes within the matrix duringolvent penetration is believed to be responsible for the observedon-Fickian behaviour (Korsmeyer et al., 1983; Lee, 1985).

According to the k values, which reflect the vancomycin releaseinetic, the delivery process is faster in samples without the PEGomponent as well as for samples O, that is, for samples dried inn oven at 37 ◦C. This behaviour seems to be in contradiction withhe idea that systems with a higher degree of porosity or thoseith hydrophilic components should release a water soluble drugore easily, i.e. following a similar pattern to that observed in the

welling study (Fig. 6).However, these liberation patterns (Fig. 7) can be explained

y considering other elements that determine the liberationf a substance. First of all, in PEG-containing samples (A�PVamples) vancomycin is forming a complex that undoubtedly hin-ers the release of this drug as has been already observed ingarose-containing analogous systems by Sjoberg et al. (1999).n this sense, the DTA peak around 200 ◦C, attributable togarose–PEG–vancomycin complex, is not observed in samples�PV after the release test thus indicating the disappearance of

he complex as a consequence of the vancomycin liberation.In addition, the dissolution of the PEG may induce the forma-

ion of voids filled with a rather viscous solution characteristic ofissolved polymers. The high viscosity in the pores contributes toetard the diffusion of the drug, decreasing the release rate as has

een already observed in other polymeric systems (Korsmeyer etl., 1983).

Another point to be considered is the size of pores/channelsince the release rate of substances from hydrogels is generallyiffusion-controlled through these aqueous channels. However,

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onsidering the significant differences between the large size ofhe pores, specially those generated by the freeze-drying proce-ure, and that of the effective molecular size of the vancomycinVallet-Regí, 2006), the drug molecule moves easily and freely inhe water-filled pores in the agarose scaffold.

In addition, it must be taken into account the low temperatures−80 ◦C) employed during the freezing which could modify thetructure/texture of the hydrogel, thus affecting to the drug diffu-ion through the space available between macromolecular chains.n this sense, it has been observed, that the freezing temperaturend rate affect to the pore size and shape in hydrogels containingystems (Zmora et al., 2002; Stokols and Tuszynski, 2006), and morepecifically to the crystallization of PEG4000 as extended or foldedhains (Urbanetz and Lippold, 2005).

Finally, not only the overall volume fraction of water, but alsots energetic state can influence on the solute diffusion in gelsWisniewski and Kim, 1980; Bromberg et al., 1987; Bromberg andon, 1998). Water in polymers can be either energetically identicalo the bulk water (“free”) or associated with the polymer chains“bound”) and the transport of solutes does not occur through theels containing only bound water (Bromberg et al., 1987). In thisense, the drying conditions may induce different water content inerms of energy states for samples F and O, thus contributing toxplain their vancomycin release patterns.

All these phenomena contribute to explain how can a moreorous hydrogel containing an hydrophilic substance, PEG, releaseancomycin slower than other non-porous or without PEG. In anyase, four different �-TCP/agarose scaffolds showing diverse van-omycin release rates have been tailored in this work.

. Conclusions

The preparation technique employed allows to prepare �-CP/agarose-containing scaffolds that behave like an hydrogelhen immersed in an hydrated medium. In spite of this behaviour

hese pieces show enough consistency to be manipulated, pro-essed and shaped thanks to the presence of the bioceramic. Thisethod results flexible enough to admit, even under mild tempera-

ure and pH conditions, the inclusion of additional substances suchs PEG and vancomycin considered in this study, without affectinghe preparation procedure or the mechanical performance com-arable to that of cancellous bone or to similar ceramic/naturalolymer systems. Consequently, in addition to those applicationselated to hard tissue engineering, this fabrication method can beonsidered a low-cost and simple-to-use alternative for the prepa-ation of drug delivery systems.

The combination of both approaches, i.e. PEG introduction andreservations tools, allows to tailor different vancomycin releaseatterns which range from that usually described for most of theydrogel systems—characterized by a great dependence on thewelling behaviour—to a markedly slower release pattern, wherehe higher porosity percentage or the presence of an hydrophilicubstance seems to play a subdued role. This tailoring approachllows also to obtain intermediate release patterns.

cknowledgements

Financial support by Spanish CICYT MAT2008-00736 and CAM-Mat-000324-0505 is acknowledged. The XRD and SEM mea-urements were performed at C.A.I Difracción de Rayos X and

icroscopia Electrónica (UCM), respectively. Normon Laboratories

.A. (Madrid, Spain) kindly supplied vancomycin. Dr. X. Gil and P.evilla (Centre de Recerca en Enginyeria Biomedica, CREB, UPC) arecknowledged for their contribution in the characterization of theechanical properties.

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