International Journal of Pharmaceutics 486 (2015) 30–37

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Tuning dual-drug release from composite scaffolds for bone regeneration J.L. Paris a,b , J. Román a , M. Manzano a,b , M.V. Cabañas a, * , M. Vallet-Regí a,b, * a Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, UCM, Instituto de Investigación Sanitaria Hospital 12 de Octubre i+12, 28040 Madrid, Spain b Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 January 2015 Received in revised form 20 March 2015 Accepted 21 March 2015 Available online 23 March 2015

This work presents the tuning of drug-loaded scaffolds for bone regeneration as dual-drug delivery systems. Two therapeutic substances, zoledronic acid (anti-osteoporotic drug) and ibuprofen (anti-inflammatory drug) were successfully incorporated in a controlled manner into three dimensional designed porous scaffolds of apatite/agarose composite. A high-performance liquid chromatography method was optimized to separate and simultaneously quantify the two drugs released from the dualdrug codelivery system. The multifunctional porous scaffolds fabricated show a very rapid delivery of anti-inflammatory (interesting to reduce inflammation after implantation), whereas the antiosteoporotic drug showed sustained release behaviour (important to promote bone regeneration). Since ibuprofen release was faster than desired, this drug was encapsulated in chitosan spheres which were then incorporated into the scaffolds, obtaining a release profile suitable for clinical application. The results obtained open the possibility to simultaneously incorporate two or more drugs to an osseous implant in a controlled way improving it for bone healing application. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Dual-drug delivery system Designed porous scaffold Ibuprofen Zoledronic acid Co-delivery release Tissue engineering

1. Introduction In recent decades, social development has led to an aging population as well as a considerable increase in the number of traffic accidents. Both factors, as a consequence of the increased life expectancy and the way of life in developed countries, have contributed significantly to an exponential increase in different bone diseases. Consequently, treatment of bone defects continues generating a considerable effort in the scientific community (Arcos et al., 2014; Drosse et al., 2008). In this sense, tissue engineering (TE) (Fisher and Mauck, 2013; Langer and Vacanti, 1993) has played a key role as it has introduced new strategies based on the development of composite systems that integrate cells, growth factors, and scaffolds, (Drosse et al., 2008; Eisenbarth, 2007; ValletRegí and Ruiz-Hernández, 2011) avoiding major drawbacks associated to autografts and allografts (O’Brien, 2011). TE develops substrates that restore, maintain or improve the function of

* Corresponding authors at: Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, UCM, Instituto de Investigación Sanitaria Hospital 12 de Octubre i+12, 28040 Madrid, Spain. Tel.: +34 913941843; fax: +34 913941786. E-mail addresses: [email protected] (M.V. Cabañas), [email protected] (M. Vallet-Regí). http://dx.doi.org/10.1016/j.ijpharm.2015.03.048 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

damaged tissues (Langer, 2000). In case of damaged bone, TE scaffolds with added drug delivery function, have emerged as an attractive approach in recent years (Catauro et al., 2015a; Mouriño et al., 2013). In bone TE, biocompatible, biodegradable and interconnected highly porous scaffolds in a three dimensional (3D) geometry are desirable. In this sense, composite scaffolds which combine biopolymers and bioactive ceramics are being developed for this application (Catauro et al., 2015b; Garg and Goyal, 2014; Mallick and Cox, 2013; Mouriño et al., 2013). Great attention has been focused on hydrogels as biopolymers because of its resemblance to the extracellular fluids (Fisher et al., 2014; Malda et al., 2013). They have been studied for different medical applications, such as drug delivery systems and bionanotechnology (Ankareddi and Brazel, 2007; Gaharwar et al., 2014; Peppas et al., 2006). In particular, the natural polysaccharide agarose, which is a thermo sensitive hydrogel, has been successfully used in tissue engineering and other biological applications (Cheng et al., 2007; Gruber et al., 2006; Luo and Shoichet, 2004; Marras-Marquez et al., 2014; Rotter et al., 1998). Inclusion of hydroxyapatite (the mineral phase present in bone) on hydrogels improves both the mechanical stability and bioactivity (Juhasz et al., 2010) and also induces an osteoinductive and osteoconductive behaviour. In this sense, carbonate-substituted hydroxyapatite shows better bioactivity in

J.L. Paris et al. / International Journal of Pharmaceutics 486 (2015) 30–37

vitro and in vivo than stoichiometric hydroxyapatite (Porter et al., 2005). Moreover, nanoscale particulate apatites are being increasingly considered in composite scaffolds to closely mimic the nanosized features of natural bone. Following the implantation of a scaffold, the material is still not vascularized. Therefore, systemic administration of drugs would not be effective at this stage. Drug release from the scaffolds could provide an adequate therapeutic concentration of the drug in the target site. Moreover, the simultaneous release of two or more therapeutic substances with different pharmacological activity may improve the outcome in different pathologies (Aderibigbe et al., 2015; Lee et al., 2008). Most of the fabrication processes to obtain scaffolds with 3D designed porosity employ organic solvents, high temperature or acidic conditions that preclude the incorporation of bioactive molecules and drugs (Fierz et al., 2008; Martínez-Vázquez et al., 2013). In previous work we have described an easy and inexpensive method to obtain agarose/hydroxyapatite scaffolds with hierarchical porosity at room temperature for bone repair application (Peña et al., 2010; Román et al., 2011). Biocompatibility studies demonstrate that these scaffolds allow the culture of osteoblasts inside and outside the material (Alcaide et al., 2010; Cabañas et al., 2014). Recently, these scaffolds were investigated as protein release matrices with different release kinetics depending on the strategy used to incorporate the protein into the scaffold (Cabañas et al., 2014). This work aims to improve the functionality of these biodegradable scaffolds, turning them into dual-drug delivery systems (DDDS) for bone regeneration. In this sense, we have designed a DDDS for bone regeneration with two drugs widely used in traumatology: zoledronic acid (a bisphosphonate antiresorptive drug) and ibuprofen (a nonsteroidal anti-inflammatory drug). Bisphosphonates are used in standard clinical practice for the treatment of diseases associated with increased bone resorption, such as osteoporosis, and might enhance bone regeneration when included in scaffolds (Cattalini et al., 2012; Coleman et al., 2011; Giger et al., 2013; Nancollas et al., 2006; Rosenqvist et al., 2014). On the other hand, ibuprofen is commonly prescribed to relieve pain due to inflammation that arises from different osseous pathologies or surgical treatments (Mouriño and Boccaccini, 2010; Rainsford, 2013), and it is also commonly prescribed simultaneously with bisphosphonates in order to minimize one of its main side effects: the “flu-like syndrome”(Coleman et al., 2011). This DDDS should have a long-term release of zoledronic acid (to enhance bone regeneration) and a relatively fast delivery of ibuprofen (to reduce inflammation).

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2. Materials and methods 2.1. Materials Nanocrystalline hydroxycarbonateapatite (nHCA) was prepared by a precipitation method from Ca(NO3)2
4H2O, (NH4)2HPO4 and (NH4)2CO3 aqueous solutions at pH 9.2 (NH4OH solution) and 37  C (Padilla et al., 2008). Agarose polymer (for routine use) was purchased from Sigma– Aldrich, Steinheim, Germany; as well as Chitosan (from crab shells, min. 85% deacetylated) and Sodium Tripolyphosphate, TPP (Tech., 85%). Zoledronic acid (ZOL) monohydrate and ibuprofen (IBU) antinflamatory were kindly provided by Novartis Pharmaceuticals AG (commercial name Zometa) and by Normon Laboratories S.A. (Madrid, Spain), respectively. 2.2. Dual-drug delivery systems preparation and characterization Three dimensional interconnected porous agarose/nHCA scaffolds containing drugs were fabricated using a shaping process patented by the authors: GELPOR3D (Peña et al., 2010; Vallet-Regí et al., 2010). The scaffolds were loaded with two drugs in two different steps (during the scaffold fabrication and after its consolidation). Briefly, the fabrication method consists of the following steps (Fig. 1): the agarose powder was suspended in deionized water (3.5% w/v) and heated to 90  C with continuous stirring; once a translucent sol was achieved, temperature was gradually decreased to 45  C and then the ceramic powder (nHCA) was added. In this step, one of the drugs (ZOL or IBU alternatively) was added as powder to the suspension (drug 1), which was incorporated through the whole volume of the material. The slurry so obtained was poured into a designed mold (a cube constituted by rigid filaments of stainless steel 1 mm in diameter arranged parallel to each other). After five minutes at room temperature, complete consolidation of the bodies was reached. After withdrawal of the designed mold, the scaffolds, freshly prepared, were easily cut and shaped (Fig. 1). Then, the pieces were freeze-dried (Heto Drywinner, freezing temperature and sublimation pressure of 86  C and 0.05 mbar, respectively). Once the scaffolds containing drug 1 were dried, the same amount of another drug (drug 2, IBU or ZOL alternatively) was incorporated into the scaffolds by injection of an aqueous solution, exploiting its hydrogel-like behaviour (Fig. 1). Afterwards, the systems were freeze-dried again. The composition employed to prepare the DDDS (50% ceramic and 50% hydrogel) was chosen according to previous results

Fig. 1. Fabrication method of dual-drug delivery systems.

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(Román et al., 2011): compositions with low contents of agarose result in an insufficient ceramic particle-polysaccharide binder interaction that may cause the particles migration once implanted. On the other side, slurries containing high ceramic loads may result too viscous to be poured in the mould and fill the interstices between the rigid filaments. Thus, two types of systems containing 45% nHCA, 45% agarose and 10% drugs (5% IBU + 5% ZOL) (data are expressed in weight %) were fabricated: AH-zol-ibu sample (where zoledronic acid was incorporated during the scaffold preparation, and the ibuprofen was added after scaffold consolidation) and AH-ibu-zol sample (in which ibuprofen was added before consolidation and the zoledronic acid was incorporated after) (Fig. 1). The fabricated DDDS were characterised by X-ray diffraction (XRD) with a Philips X-Pert MPD diffractometer, scanning electron microscopy (SEM) in a JEOL 6400 and Hg intrusion porosimetry using a Micromeritics AutoPore III 9410 porosimeter. Fourier Transform Infrared (FTIR) spectra were obtained in a Nicolet Nexus spectrometer equipped with a Smart Golden Gate Attenuated Total Reflectance (ATR) accessory. 2.3. Chitosan spheres preparation and characterization Ibuprofen-loaded chitosan spheres were prepared by the following procedure(Agnihotri et al., 2004): ibuprofen (150 mg) was dispersed in 10 mL of 1.5% chitosan solution in 0.5% acetic acid in deionized water. This suspension was added drop wise to a 10% Sodium Tripolyphosphate (TPP) solution in deionized water under gentle stirring. Chitosan spheres formed almost immediately and were cured for 20 min. Then they were filtered and dried in an oven at 37  C overnight (sample CT-ibu). The spheres containing IBU were then introduced in the composite scaffolds following the same procedure previously described to introduce drug 1 (sample AH-CT-ibu). The systems were characterised by SEM and FTIR. 2.4. In vitro drugs release study In vitro delivery assays were performed by soaking scaffolds of 1 cm side in 10 mL saline solution (NaCl 0.9%) at 37  C. The pH of the release medium was adjusted to 7.4 (physiological pH) with TrisBuffer. To avoid limitation of the delivering rate by external diffusion constrains, continuous stirring was maintained during the delivery assays. The volume of release medium employed was chosen to ensure sink conditions: The concentration of the drugs in the medium was never higher than 20% of their solubility in that medium. Aliquots of 150 mL were extracted at different times. The concentration of released drugs from the loaded scaffolds was monitored by reverse-phase high-performance liquid chromatography (RP-HPLC) in a Waters Alliance automatic analysis system with a Model #2695 separations module coupled to a Model #2996 photo-diode array detector. Chromatographic separations were carried out with a 250  4.6 mm prepacked analytical column Mediterranea Sea18 (Teknokroma Inc.) containing 5 mm C-18 functionalized silica beads. In order to separate and quantify the drugs introduced into the scaffolds, specific analytical conditions were developed and the following method was employed. The gradient mobile phase was a mixture of aqueous phosphate buffer 10 mM at pH 3 and methanol (Vallet-Regí et al., 1998; Vila et al., 2013). The best resolution of two drugs was achieved with the following mobile composition: the initial mixture composition was 90:10 (v/v) for the first 5 min, and then was changed to 15:85 (v/v) over a period of 10 min, keeping the flow with the mobile phase for other 25 min. The flow rate of the mobile phase was 0.7 mL min1 and the injection volume was 10 mL. The column temperature was maintained at 37  C.

The detection of the released drug was performed by UV at different wavelengths: 210 nm for ZOL and 219 nm for IBU. Chromatograms were recorded using Millenium Software. Under these conditions, the retention time of the ZOL was 4  0.5 min, whereas the IBU retention time was 25  0.6 min (Fig. 2). Several solutions at concentrations ranging from 0.003 to 0.5 mg mL1 (ZOL) or 0.003 to 0.125 mg mL1 (IBU) in aqueous NaCl 0.9% buffered at pH 7.4 were used as standards. In addition, the chromatographic peaks were stable appearing at the same retention time during the analysis and no degradation peaks were present in the chromatograms. Results were processed with the software OriginPro8 (OriginLab Northampton, MA) 3. Results and discussion Multifunctional 3D scaffolds based on nano-hydroxycarbonateapatite/agarose composites have been fabricated at room temperature through a shaping method named GELPOR3D (Peña et al., 2010; Román et al., 2011; Vallet-Regí et al., 2010). These scaffolds were loaded with two drugs, (ZOL and IBU), in two different steps. In these systems, the drug included during scaffold fabrication, drug 1, is mentioned first and the drug included after scaffold consolidation, drug 2, is stated last, giving two different materials: AH-zol-ibu and AH-ibu-zol. The method used to obtain these DDDS allows a complete control over the amount of drugs present in the scaffolds. This constitutes a significant advantage versus other designed porosity systems that depend on immersing the scaffolds in highly concentrated solutions of the drugs to load them into the material (a very inefficient process). The addition of the drugs (10 wt.%) during the scaffold fabrication does not affect the body consolidation process neither the textural features of the scaffolds as demonstrated by SEM and Hg porosimetry studies. These DDDS show a macroporous structure constituted by a designed 3D network of interconnected pores of around 900 mm, and a connected porosity due to freezedrying with diameters between 100 and 200 mm (Fig. 3). Scaffolds fabricated with similar composition and drying method to those used in this work (Román et al., 2011) showed a porosity around 95%, with 30% for the pores with the highest diameter, around 65% for the 100–200 mm pores and less than 3% for pores around 50– 100 nm (due to the structure resulting from the coating of nHCA particles by the agarose).

Fig. 2. Typical HPLC chromatogram of ZOL and IBU standard mixture at 219 nm.

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Fig. 3. AH-zol-ibu scaffold: photograph (a); SEM micrographs at different magnifications (b,c); schematic representation of the dual matrix (d).

The presence of loaded drugs into the scaffolds was confirmed by FTIR spectra. Fig. 4 shows FTIR spectrum corresponding to AH-ibu-zol scaffold. (The FTIR spectrum corresponding to AH-zolibu was very similar and it is not included in the figure for the sake of clarity). The spectra corresponding to IBU, ZOL and the scaffold without any loaded drug (AH) or containing just zoledronic acid (AH-zol) are also included for comparative purposes. FTIR spectrum of AH-ibu-zol shows the presence of characteristic bands associated to functional groups corresponding to nHCA and agarose (see FTIR spectrum of sample AH) and additional bands due to the drugs were also detected. The most intense band of ibuprofen, at 1720 cm1, that corresponds to carboxylic acid group, -COOH, (Szegedi et al., 2012) is not observed. However, a new band at 1545 cm1, corresponding to carboxylate ion COO, can be appreciated in the co-loaded scaffold. This indicates the deprotonation of the acid group of IBU during the synthesis process, due to the basic environment provided by the nHCA, as it has been observed in other basic materials (Krupa et al., 2010). This deprotonation was not observed in scaffolds prepared without nHCA (Supporting Information). On the other hand, the bands corresponding to the aromatic ring chains of zoledronic acid (Pascaud et al., 2012), ca. 1548 and 1578 cm1, can be observed in the fabricated scaffolds with ZOL, indicating that aromatic rings remain unmodified. Moreover, the vibration bands of ZOL at 1300 and 1321 cm1 attributed to P—O bond (Waghe et al., 2006) are shifted to 1286 and 1307 cm1, respectively, in the spectra corresponding to scaffolds containing ZOL (AH-zol and AH-ibu-zol scaffold). This displacement can be attributed to the interaction between the ZOL and the nHCA, as the bisphosphonates possess a pyrophosphatelike chemical structure that confers a strong affinity of calcium by a chelate formation (Bujoli et al., 2006; Juillard et al., 2010; VargasBecerril et al., 2013). Systems containing ZOL (forming a chelate with Ca from the ceramic component) and/or IBU (with its carboxylic group deprotonated) were immersed in aqueous solution to study the co-release of the drugs. To quantify zoledronic acid and ibuprofen, a RP-HPLC method was developed in order to determine the concentration of both drugs (since they are being released simultaneously, and their UV–vis spectra overlap). Firstly, the specific analytical conditions were studied to separate both drugs by optimizing the composition of the mobile phase (Fig. 2): methanol and aqueous phosphate buffer in various ratios. After

Fig. 4. FTIR spectra corresponding to (from top to bottom): AH scaffold; AH-ibu-zol scaffold; AH-zol scaffold; zoledronic acid and ibuprofen.

separation they were quantified by UV at different wavelengths: 210 nm for ZOL and 219 nm for IBU. An important concern regarding drug release from scaffolds is to ensure the drug stability during its incorporation and release. Drug stability in this study was assured by the unmodified UV/vis spectra of both drugs throughout the analysis time. Again, this proves the versatility of the scaffold preparation procedure, based on the gelation of agarose, carried out at mild conditions, allowing the incorporation of active molecules during the process without modifying them. Similar systems rely on the setting reaction of cements, an exothermic process that can actually reduce the activity of the incorporated drugs (Vorndran et al., 2010). Fig. 5 shows the release profiles of IBU and ZOL as a function of the incorporation method of the drug at pH 7.4. Data depicted in this figure show that ibuprofen has a short-term release while zoledronic acid has a long term and sustained release behaviour. Moreover, independent release behaviours of the drugs were observed, since the presence of a drug does not influence the release of the other (single drug systems have also been studied as reference, data not shown). In a physiological pH saline solution, IBU is practically released in 3 h, for both types of incorporation, whereas for this time, just around a 3% of ZOL is released when it is added during the scaffold

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Fig. 5. Release profiles of ZOL (5) and IBU (*) from the DDDS: AH-zol-ibu (—) and AH-ibu-zol (- - - -) at pH 7.4.

Fig. 6. Photograph and SEM micrograph of CT-ibu spheres (a, b); SEM micrograph of AH-CT-ibu scaffold with arrows pointing CT-ibu spheres (c); schematic representation of the new dual matrix (d).

fabrication (AH-zol-ibu) and 9% when it was incorporated after fabrication (AH-ibu-zol). Data collected in this figure show that, at 72 h, only 20% (AH-ibu-zol) or 10% (AH-zol-ibu) of zoledronic acid has been released. Contrarily to what one would expect, both loading modes (during and after scaffold manufacture) resulted in rather similar in vitro release profile of ibuprofen: a very fast release of the drug, related to the rapid swelling of the scaffolds in aqueous solution observed in previous studies. (Cabañas et al., 2014) On the other hand, the retention of antiosteoporotic drug is related to the complexation between the phosphonate groups of zoledronic acid and the Ca ions of nHCA, previously mentioned. This bonding nHCA-ZOL has been found to yield protection against the observed cytotoxic effects of ZOL in vitro. (Murphy et al., 2014) The lower release observed for AH-zol-ibu indicates that the complexation reaction is facilitated when ZOL is introduced during the fabrication process. Even though these results match the initial objective of this work (a very slow release of zoledronic acid and a very fast release of ibuprofen), ibuprofen release is too fast for the clinical application we are aiming at. The drug should be released during a short period of time (a few days) in order to yield a relevant antiinflammatory effect. However, ibuprofen release should not last much longer, since prolonged anti-inflammatory treatment might difficult the regeneration process and, therefore, deteriorate the clinical outcome (Thomas and Puleo, 2011). In order to extend ibuprofen release, and because of the versatility of the scaffold fabrication process, the drug was encapsulated in chitosan-TPP spheres (CT) (648.016  22.03 mm in diameter) before being introduced in the scaffold. Chitosan is a biodegradable natural polysaccharide widely used in drug delivery systems (Agnihotri et al., 2004). Drug-loaded chitosan spheres, containing a 26 wt.% of IBU (CT-ibu) were introduced in the slurry before its addition to the 3D-mold. These CT-ibu spheres isolated, or included in the composite scaffolds (AH-CT-ibu) together with a schematic representation of the system are shown in Fig. 6. FTIR spectrum of CT-ibu spheres shows that IBU is present with its carboxylic group protonated (Supporting information). Ibuprofen release studies from spheres isolated (CT-ibu) and scaffolds with spheres within them (AH-CT-ibu) were carried out in the same conditions as those used previously. The results are shown in Fig. 7. We can see a slower release from the sphereloaded scaffolds than from the isolated chitosan spheres (and, in

both cases, slower than non-encapsulated ibuprofen-loaded scaffolds). In the case of the scaffold with encapsulated ibuprofen, with minimised burst effect, its release lasts more than 2 days, what would be suitable for bone healing. The release profiles of Figs. 5 and 7 were fitted to different kinetic models, in order to try to determine the mechanism driving drug release (Costa and Sousa Lobo, 2001). Table 1 summarizes the kinetic parameters resulting from the better fittings of zoledronic acid and ibuprofen release. For the release of zoledronic acid, the best fit was obtained by the power law equation known as Korsmeyer–Peppas equation (Peppas, 2000) (Eq. (1)): Mt ¼ ktn M

(1)

where Mt/M is the accumulative amounts of drug release at time t; k is a kinetic constant and n is an exponent which characterised the mechanism of drug release and it ranges between n = 0.5 (for Fickian diffusion), and n = 1 (for Case II transport), respectively, being the inter-mediate values indicative of anomalous transport.

AH- ibu

100

% Drug released

80

CT-ibu

60

AH-CT-ibu 40

20

0 0,0

10,0

20,0

30,0

40,0

50,0

Time (h) Fig. 7. Release profiles of IBU from non-encapsulated IBU-loaded scaffolds (AHibu), IBU-loaded chitosan spheres (CT-ibu) and composite scaffolds containing drug-loaded spheres (AH-CT-ibu) at pH 7.4.

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Table 1 Kinetic parameters of zoledronic acid (ZOL) and ibuprofen (IBU) released from different materials. Drug

Material

Model

Kinetic parameters

Fit parameters

ZOL

AH-ibu-zol

Korsmeyer–Peppas Mt/M = ktn Korsmeyer–Peppas Mt/M = ktn First order release Mt/M = A(1  eKt) First order release Mt/M = A(1  eKt) Korsmeyer–Peppas Mt/M = ktn Korsmeyer–Peppas Mt/M = ktn

k = 2.5 (hn)  0.2 n = 0.24  0.01 k = 1.03 (hn)  0.04 n = 0.206  0.005 A = 96.1  1 K = 0.056 h1  0.004 A = 97.23  2 K = 0.051 h1  0.004 k = 9.69 (hn)  2.85 n = 0.43  0.06 k = 1.28 (hn)  0.215 n = 0.525  0.023

R2 = 0.993

AH-zol-ibu Non-encapsulated IBU

AH-ibu-zol AH-zol-ibu

Encapsulated-IBU

CT-ibu AH-CT-ibu

In our case the value of the exponent n obtained was lower (ca. 0.2). This low value may be attributed to a non-typical Fickian diffusion mechanism with some physico-chemical interference (Chen and Zhu 2012; Tamimi et al., 2008). These data agree with the nHCA and ZOL interaction described above, the formation of a chelate and detected by FTIR spectra. Comparing the kinetic fitting for ZOL release, a higher value of k when ZOL is introduced after the scaffold consolidation can be observed, what is in agreement with the higher amount of the drug released from this scaffold. On the other hand, the release of non-encapsulated ibuprofen from the scaffolds showed in Fig. 5 follows a first order exponential decay model (according to the Noyes–Whitney equation) (Peppas, 2000) (Eq. (2)): Mt ¼ Að1  eKt Þ M

(2)

with A = (Mt/M)max being the maximum number of biomolecules released. The release rate constant, K, gives information about the solvent accessibility and the diffusion coefficient through the scaffold channels. In this case, no appreciable difference is observed in the kinetic fitting of IBU release introduced before or after scaffold consolidation (Table 1). To determine the mechanism underlying ibuprofen release, a scaffold AH-ibu was immersed in saline solution at pH 6.5, and the release profile of the drug was analyzed (Supporting information). IBU release at pH 6.5 was slower than at pH 7.4, as can be observed in the release profile and in the kinetic fitting to Eq. (2) (A = 94  4%, K = 0.0044 h1  0.0005, R2 = 0.987). Given that solubility of IBU is lower at acidic pH, and that the dissolution rate is a function of the solubility of a molecule (Costa and Sousa Lobo, 2001), the change in the release profile of IBU between the two cases (at pH 7.4 and pH 6.5) might indicate that the mechanism driving non-encapsulated ibuprofen release from this scaffolds is merely the dissolution of the drug. The slower ibuprofen release from chitosan spheres (inside a scaffold or isolated) can be described using, again, the Korsmeyer– Peppas equation (Table 1). In both cases, the n value is close to 0.5; this implies that the mechanism behind drug release is diffusion through the walls of the chitosan spheres. Even though in both cases, the same kinetic model can be used, the kinetic constant (k) is higher in the isolated spheres, what agrees with the faster release observed in that case, since in the spheres embedded inside a scaffold, ibuprofen has to get out of the scaffold once it has diffused out of the chitosan spheres. According to the drug release data, the proposed strategy with the dual pharmacological effect could be a versatile option for the treatment of bone injuries that would require bone replacement. The apatite/agarose scaffold itself could allow bone regeneration, since previous in vitro studies (Cabañas et al., 2014) showed that the material stimulated osteoblast proliferation and the cells colonized the interconnected macroporous scaffolds. At the same

R2 = 0.997 R2 = 0.991 R2 = 0.992 R2 = 0.987 R2 = 0.992

time, the drugs included in it would provide an environment where bone regeneration would be enhanced (by the slow release of ZOL) and the inflammation in the area would be reduced in the short-term (by the fast release of IBU).

4. Conclusions Multifunctional 3D designed porous scaffolds for bone regeneration were fabricated by a simple conformation method carried out in mild conditions that allow introducing many different biomolecules avoiding their degradation during scaffold manufacture. Two biological active substances, zoledronic acid (an antiresorptive drug) and ibuprofen (an antiinflammatory drug), have been incorporated into the scaffolds and were co-delivered to the medium following different tuneable release profiles. The composition of the scaffolds allows the bisphosphonate to be retained by the ceramic, nHCA, and the inclusion of ibuprofenloaded chitosan spheres enables the adjustment of its release behaviour. The development of a liquid chromatographic method allowed the separation and simultaneous quantification of both drugs which are released following different mechanisms, which can be described employing several kinetic models. The results obtained show the potential of these agarose/ nanohydroxycarbonateapatite scaffolds to retain and slowly release zoledronic acid, as well as to quickly deliver ibruprofen to the surrounding tissue. Although these DDDS have been studied for simultaneous delivery of drugs with antiinflamatory and antiosteoporotic properties, this approach allows the development of multifunctional scaffolds to deliver multiple pharmaceutical or biological agents for bone tissue regeneration.

Acknowledgments Financial support from Ministerio de Economía y Competitividad, Spain (Project MAT2012-35556 and Project CSO2010-11384E, Ageing Network of Excellence) is gratefully acknowledged. The XRD and SEM measurements were performed at C.A.I Difracción de Rayos X and Microscopia Electrónica, Universidad Complutense, respectively. We thank Novartis Pharmaceuticals AG and Normon Laboratories S.A. (Madrid, Spain) for providing the zoledronic acid and ibuprofen, respectively.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2015.03.048

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