Materials Science and Engineering C 34 (2014) 437–445

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PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: Morphology, mechanical properties and bioactivity Dajana Milovac a,⁎, Gloria Gallego Ferrer b,c, Marica Ivankovic a, Hrvoje Ivankovic a a b c

Faculty of Chemical Engineering and Technology, University of Zagreb, Croatia Center for Biomaterials and Tissue Engineering, Polytechnic University of Valencia, Spain Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain

a r t i c l e

i n f o

Article history: Received 15 May 2013 Received in revised form 23 September 2013 Accepted 28 September 2013 Available online 5 October 2013 Keywords: Poly(ε-caprolactone) Hydroxyapatite Scaffold Tissue engineering Mechanical properties Bioactivity

a b s t r a c t In the present study, poly(ε-caprolactone)-coated hydroxyapatite scaffold derived from cuttlefish bone was prepared. Hydrothermal transformation of aragonitic cuttlefish bone into hydroxyapatite (HAp) was performed at 200 °C retaining the cuttlebone architecture. The HAp scaffold was coated with a poly(ε-caprolactone) (PCL) using vacuum impregnation technique. The compositional and morphological properties of HAp and PCL-coated HAp scaffolds were studied by means of X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM) with energy dispersive X-ray (EDX) analysis. Bioactivity was tested by immersion in Hank's balanced salt solution (HBSS) and mechanical tests were performed at compression. The results showed that PCL-coated HAp (HAp/PCL) scaffold resulted in a material with improved mechanical properties that keep the original interconnected porous structure indispensable for tissue growth and vascularization. The compressive strength (0.88 MPa) and the elastic modulus (15.5 MPa) are within the lower range of properties reported for human trabecular bones. The in vitro mineralization of calcium phosphate (CP) that produces the bone-like apatite was observed on both the pure HAp scaffold and the HAp/PCL composite scaffold. The prepared bioactive scaffold with enhanced mechanical properties is a good candidate for bone tissue engineering applications. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Scaffold materials for use in bone tissue engineering (BTE) that mimic both the structure and mechanical properties of the natural bone still represent a great challenge for researchers. Scaffold should provide a highly porous matrix with interconnected pores that enable the transport of nutrients, oxygen and metabolic waste products. Its surface properties must be suitable for cell adhesion, proliferation and differentiation. Also, the scaffold should be bioresorbable with a controllable degradation rate to match the replacement by new tissue. Additionally, the scaffold should possess sufficiently high mechanical properties such as stiffness, strength and toughness. One group of materials that attempt to meet many of these requirements is that of the composites formed by biodegradable polymers and bioactive ceramics [1–4]. Synthetic calcium phosphates, in particular hydroxyapatite (HAp, Ca10(PO4)6(OH)2), are the most commonly used ceramics in dentistry and bone repair due to their chemical similarity to the inorganic matrix of natural bone, excellent osteoconductivity and bioactivity [5,6]. The production of porous HAp scaffolds is still a theme of high relevance in the field of ⁎ Corresponding author at: Dept. of Inorganic Chemical Technology and Non-Metals, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 20/I, HR-10000 Zagreb, Croatia. Tel.: +385 1 4597 226; fax: +385 1 4597 260. E-mail address: [email protected] (D. Milovac). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.09.036

biomaterials science and technology. Currently, porous HAp scaffolds have been prepared by a number of manufacturing techniques including polymer foam replication, sol–gel and freeze casting, solid free-form fabrication, etc. [7–13]. However, these methods are expensive and not well defined concerning the internal porous architecture. The major drawback of the HAp scaffolds is their poor mechanical properties, especially the brittleness and low fracture toughness. Therefore, they cannot be used in load bearing application. To overcome these disadvantages HAp has been combined with polymers that provide flexibility to the brittle system. A wide range of enzymatically and hydrolytically degradable polymers have been proposed for biomedical applications, either natural (gelatine, chitosan, alginate) or synthetic (poly(εcaprolactone) (PCL), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), etc. [14–16]. Synthetic polymers are preferable to natural-based polymers for their tailorable designs, reproducible degradation characteristics and wide range of mechanical properties. Natural resources basically comprised of calcium carbonate (corals, seashells, nacres, cuttlefish) are receiving growing interest because of their possible conversion to HAp. HAp prepared from natural sources is no stoichiometric, and has other ions incorporated, mainly CO32−, trace of Na+, Mg2+, Fe2+, F−, and Cl− [17]. Carbonated HAp is closer to the chemistry of natural human bone than stoichiometrically pure HAp [18] and has been shown experimentally to have enhanced biocompatibility [19,20]. In recent years, a number of studies on conversion

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of natural aragonite (CaCO3) structures to HAp have been reported [21–31]. Of these, cuttlefish bone has been the most extensively studied [24–31]. Cuttlefish bone is a low cost, worldwide available natural material possessing an extreme porosity (~90%), ideal pore size (200– 600 μm) and interconnectivity. The preservation of the overall scaffold structure of the cuttlefish bone is essential for its potential application as bone substitute. Rocha and coworkers [24–27] were the first who performed the hydrothermal transformation of aragonitic cuttlefish bone to produce hydroxyapatite scaffolds retaining the cuttlebone architecture. The studies included tests for biocompatibility with osteoblasts and in vitro bioactivity. The carbonate substitution in the hydrothermally produced HAp structure and the kinetics of transformation of cuttlefish bones into HAp as a function of temperature and time of hydrothermal treatment were studied by Ivankovic and coworkers [28,29]. Battistella et al. [30] performed biological characterization of cuttlefish bone scaffolds using the preosteoblastic cell line MC3T3-E1. The expression of osteogenic markers as ALP and osteocalcin and the cell proliferation was investigated. However, concerning the mechanical properties of scaffolds the existing literature is very limited. Recently, Sarin et al. [31] reported on compressive strength (2.38 MPa) and noncatastrophic failure behavior of porous biphasic calcium phosphate scaffolds obtained from cuttlefish bone. It is reasonable to hypothesize that the mechanical properties of scaffolds may be negatively affected by the hydrothermal process. Impregnation/coating of scaffold with polymer is a very simple and economical method that can improve the mechanical properties of porous HAp scaffold while preserving the connectivity of the pores which is crucial for its application in BTE. Due to its biodegradability, biocompatibility, appropriate mechanical properties, and low emission of harmful byproducts PCL has been widely used in the biomedical field for the manufacture of tissue engineering supports [16]. PCL-coated HAp scaffolds have been investigated in the literature [32,33]. Most of the applied methods use HAp powder in scaffold preparation. Kim et al. [32] and Zhao et al. [33] obtained porous HAp scaffolds by impregnating polyurethane (PU) foams with the slurry containing HAp powder. The obtained bodies were heat-treated at 600°C to burn out the PU foams, and ultimately sintered at 1200 or 1300°C. Finally, HAp porous scaffolds were coated with PCL [30] or HAp/PCL composite coatings [32]. As reported by Kim et al. [32] the scaffold possessed high porosity (87%) and controlled pore size (150–200) μm). With the composite coatings, the compressive strength of the scaffolds between 0.24 and 0.45 MPa and elastic modulus between 1 and 1.43 MPa were obtained. The results of Zhao et al. [33] indicated that as the concentration of PCL solution increases (5%, 10% and 20% (w/v)) the compressive strength increased from 0.09 MPa to 0.51 MPa while the porosity decreased from 90% to 75% for the HAp/PCL composite scaffolds. The published data on the use of microporous HAp ceramics obtained from natural sources to prepare HAp/PCL composite scaffolds are limited. The present work reports on preparation of PCL-coated highly porous HAp scaffolds derived from cuttlefish bone using very simple and inexpensive methods. First, hydrothermal transformation (HT) of aragonitic cuttlefish bone into hydroxyapatite was performed at 200°C retaining the cuttlebone architecture. Then, the scaffold was coated with the PCL polymer using vacuum impregnation technique. The composite scaffold was exposed to Hank's balanced salt solution (HBSS), in order to investigate its ability to induce the precipitation of biologically active bonelike calcium phosphate layer on its surface. Its mechanical properties, composition and morphology were also examined and compared with those of the pure HAp scaffold. 2. Experimental 2.1. Hydrothermal synthesis of porous hydroxyapatite Cuttlefish bones (Sepia officinalis L.) from Adriatic Sea were used as starting material for the hydrothermal synthesis of hydroxyapatite

(HAp). The bones were carefully cut into small pieces (2cm3) and treated with aqueous solution of sodium hypochlorite (NaClO, 13% active chlorine, Gram-mol) for 12 h to remove the organic components. The pieces of cuttlefish bone were then sealed with the required volume (respecting the molar ratio of Ca/P = 1.67) of a 0.6 M aqueous solution of ammonium dihydrogenphosphate (NH4H2PO4, 99%, Scharlau) in a TEFLON lined stainless steel pressure vessel at 200 °C for 72 h. The pressure inside the reactor was self-generated by water vapor and reached 18 bars. After hydrothermal treatment the resulting pieces of HAp scaffold were washed with boiling demineralised water and dried at 105 °C. 2.2. Preparation of the HAp/PCL composite scaffold HAp scaffolds were impregnated with PCL solution in chloroform using the vacuum impregnation unit (CitoVac, Struers). The purpose of impregnation in vacuum is to remove air out of the pores, thus ensuring that the PCL solution can easily flow into the pores. A homogeneous 20 w/v % solution of poly(ε-caprolactone) (PCL, Mn = 45000, SigmaAldrich) was prepared by intensive stirring of PCL pellets in chloroform (CHCl3, p.a., Kemika) and poured into a cup connected to the vacuum chamber through the tube. The specimens of porous HAp were put in a beaker and placed in the vacuum chamber. The pressure was set to 0.11 bars. After 10 min the valve was open to suck the PCL solution through the tube filling the beaker over the porous specimens. The specimens were soaked for 10 min. Then, the vacuum was stopped to allow the air pressure to force the PCL solution into pores in the specimens. The beaker was removed from the vacuum chamber. The soaked scaffolds were put on a net and placed in the vacuum chamber again. The vacuum was restored in order to remove the excess PCL solution away from the scaffolds and to dry the specimens. The experimental details of the HAp/PCL composite scaffold preparation are shown in Scheme 1. 2.3. In vitro bioactivity test Hank's balanced salt solution (HBSS; H1387, Sigma-Aldrich) with an ionic composition similar to that of human blood plasma was used as the immersion solution for in vitro assessment of the scaffolds' bioactivity. This solution consisted of 0.1396 g CaCl2, 0.09767 g MgSO4, 0.4 g KCl, 0.06 g KH2PO4, 8.0g NaCl, 0.04788 g Na2HPO4, 1.0 g D-Glucose and 0.35 g NaHCO3 in 1 L distilled H2O with an initial pH of 7.4. The HAp and HAp/PCL composite samples were soaked in HBSS at 37 °C in an orbital shaker at 100 rpm for 3, 7, 14 and 28 days with a solid/liquid ratio of 2 mg/mL. The scaffolds were kept in vertical position inside closed plastic flasks containing HBSS. The solution was changed at three-day intervals and stirred continuously. After the immersion period the samples were filtered and carefully washed with distilled water to remove residual HBSS and dried at room temperature. 2.4. Characterization X-ray diffraction analysis was performed with an X-ray diffractometer (Shimadzu XRD 6000) equipped with CuKα radiation source generated at 40 kV and 40 mA in the range of 5° b 2θ b 70° at a scan speed of 0.2°/s. Identification of the phases was performed by comparing the experimental XRD patterns to standards compiled by the Joint Committee on Powder Diffraction Standards (JCPDS). The Fourier transform infrared (FTIR) spectra of investigated materials were recorded by attenuated total reflectance (ATR) spectrometer for solids with a diamond crystal (Bruker Vertex 70) at room temperature. An amount of material was placed onto diamond crystal without any additional sample preparation. 64 scans were collected for each measurement over the spectral range of 400–4000 cm−1 with a resolution of 4 cm−1.

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Cuttlefish bone HAp/PCL

NaClO (aq.)

CitoVac unit

scaffold

PCL/CHCl3 20 w/v %

0.11 bar

Hydrothermal reactor NH4H2PO4 (aq.)

washing

HAp

200°C, 72 h, 18 bar

drying, 105°C

scaffold

Scheme 1. Preparation of HAp/PCL composite scaffolds.

Scanning electron microscopy (SEM) with energy dispersive X-ray (EDX) analysis (SEM ISIDS-130) was used to examine the morphology of the hydrothermally treated cuttlefish bone and the composite scaffolds before and after HBSS immersion and to obtain the elemental composition of the calcium phosphate deposit on their surfaces. The samples were gold-coated. In order to determine the amount of PCL in the HAp/PCL composite scaffold thermogravimetric analysis (TGA) was performed on a Perkin Elmer thermobalance TGS-2. Samples were heated from room temperature to 1000°C at a heating rate of 10°C/min with the constant synthetic air flow of 150 cm3/min. The compression tests were carried out by a Microtest standard compression machine (Microtest S.A.) with a 15 N maximum load at a crosshead speed of 0.2 mm/min in ambient conditions. The porous scaffolds of raw cuttlefish bone, HAp and HAp/PCL composite were cut into cube blocks of 6 mm edge length. A compressive load was applied perpendicular to lamellae on each specimen. The elastic modulus and the compressive strength were calculated from the stress–strain curves. Ten to fifteen specimens for each sample group were used for the compressive testing.

for ν2 mode, 563cm−1 and 598cm−1 for ν4 mode, 958cm−1 for ν1 mode, and 1022 and 1086 cm−1 for ν3 mode. Stretching and vibrational modes of OH− groups appear at 3572 cm−1 and 630 cm−1, respectively. There are two types of carbonate substitution in HAp. The carbonate substitute either at the phosphate tetrahedral sites (B-type) or at the hydroxyl sites (A-type) or both (AB-type) [20,35]. Carbonated HAp is closer to the chemistry of natural human bone than stoichiometrically pure HAp [18] and has been shown experimentally to have enhanced biocompatibility derived bands at 872 cm−1 for ν2 [19,20]. High intensity of the CO2− 3 mode, and 1413 cm−1 and 1456 cm−1 for ν3 mode indicates that B-type substitution occurred during HAp synthesis. Careful analysis of spectra indicates the presence of small amount of the A-type substituted band at 879 cm−1 and the very weak HAp as suggested by ν2CO2− 3 2− −1 ν3CO3 band at 1549 cm [28,36]. Biological apatites, which constitute bone mineral, feature mixed AB-type substitutions [37,38]. This analysis indicates the formation of AB mixed type carbonated HAp after hydrothermal transformation of the cuttlefish bone. Therefore, the produced scaffolds may have a great potential as bone substitutes. The PCL spectrum shows characteristic peaks of C_O stretching vibrations at 1726 cm−1, CH2 bending modes at 1473, 1397 and

3. Results and discussion 3.1. X-ray analysis The powder X-ray diffraction pattern of cuttlefish bone after HT process (HAp in Fig. 1) showed well-resolved peaks indicating a high degree of crystallinity of the inorganic material. Comparing the experimental XRD pattern to JCPDS standards the crystalline phase was identified as HAp mineral (JCPDS; 09-0432). In the XRD pattern no peaks corresponding to any CaCO3 crystalline phase were found indicating that aragonite conversion into HAp was complete. Besides the diffraction peaks belonging to HAp, the diffraction pattern of HAp/PCL composite showed two additional strong diffraction peaks at Bragg angles 2θ = 21.2° and 23.6° attributed to the (110) and (200) crystallographic planes of semicrystalline PCL [34]. 3.2. FTIR studies FTIR spectra of HAp, PCL and composite HAp/PCL samples are shown −1 in Fig. 2. The spectrum of HAp shows typical bands for PO3− 4 at 473 cm

Fig. 1. XRD patterns of HAp and HAp/PCL scaffolds.

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Absorbance (a.u)

440

HAP/PCL -

OH

CO3 CO3

HAP 4000

3500

3000

2500

2000

2000

1800

1600

PO4

2-

1400

2-

3-

1200

PO4

3-

800

400

800

400

C-O

C=O Absorbance (a.u.)

C-O-C CH2

CH2 CH2

PCL

4000

3500

3000

2500

Wave number (cm-1)

2000

2000

1800

1600

1400

Wave number (cm-1)

1200

Wave number (cm-1)

Fig. 2. Characteristic infrared spectra of PCL, HAp and HAp/PCL composite scaffolds. Spectra are vertically shifted for the sake of clarity.

1361 cm−1 and CH2 stretching at 2942 and 2862 cm−1. The C\O\C stretching vibrations yield peaks at 1233, 1107 and 1042 cm−1. The bands at 1160 and 1290 cm−1 were ascribed to C\O and C\C stretching in the amorphous and in the crystalline phase, respectively [39]. FTIR spectrum of prepared HAp/PCL composite appears as a superposition of the spectra of HAp and PCL. No other bands or band shifts were observed in the spectrum indicating that no chemical reactions occurred between HAp and PCL. Similar findings are reported in the literature [32] where a porous HAp scaffold obtained by a polymeric foam reticulate method was coated with PCL and HAp composite coatings.

cellular quasi-periodic microstructure consisting of lamellae separated by numerous pillars (Fig. 4(a)). In the internal matrix each of the individual lamellae and pillars are coated with organic material composed primarily of β-chitin [42,43]. The pillars in the lamellar matrix form channels of width between 100 and 200 μm which progress through the material along a meandering, convoluted path (Fig. 4(b)). As shown in Fig. 4(c) and (d) the interconnected structure of the cuttlefish bone is maintained after the hydrothermal conversion into HAp. At higher magnification irregularly shaped microspheres of HAp crystals

3.3. TG analysis Thermal stability of the HAp is evident from the TGA curve obtained up to 1000 °C (Fig. 3). The total weight loss of the HAp sample of about 4 wt.% can be attributed to the loss of physically and chemically absorbed water and CO2 elimination as a result of the decarbonation process in the range 600–1000 °C. Weight loss of the HAp/PCL composite scaffold until 550 °C can be ascribed to the three-step degradation process of PCL which decomposes to carbon dioxide, water, carbon monoxide and short-chain carboxylic acids [40]. The results showed that the composite HAp/PCL scaffold contains 48.7 ± 0.1 wt.% of PCL. 3.4. Scanning electron microscopy Representative scanning electron micrographs of the raw cuttlefish bone, HAp and HAp/PCL composite scaffolds are shown in Figs. 4 and 5. The structure of cuttlefish bone is systematically and in detail described by Birchall et al. [41]. Briefly, it consists of two regions: a thick external wall or dorsal shield and internal regions with a layered,

Fig. 3. Thermal decomposition of HAp and HAp/PCL scaffolds.

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Fig. 4. SEM micrograph of raw cuttlefish bone (a, b) and cuttlefish bone after HT conversion into HAp (c–f). (a) Detail of lamellar matrix transverse cross-section (b) channels formed by convoluted pillar (c, d) transverse cross-section clearly displaying interconnected channeled structure maintained after HT conversion, (e) roughly spherical aggregates of HAp crystals and (f) dandelion-like structures.

formed on the surface of lamellae and pillars are seen (Fig. 4(e)). The quasi-spherical particles merge and form a continuous layer that resembles the cauliflower morphology. The enlarged view (Fig. 4(f)) indicates the existence of dandelion-like structures. These globular-shaped structures enhance the surface roughness and surface area and should provide an efficient bonding of PCL coating to the HAp scaffold. SEM micrographs shown in Fig. 5(d) and (e) clearly display a PCL layer on the HAp aggregates forming the cauliflower-like morphology. A qualitative analysis of SEM images given in Figs. 4(f) and 5(e) shows that the surface roughness of PCL-coated HAp is different compared to uncoated HAp. As seen, PCL covered the dandelion-like polycrystalline structures of HAp resulting in the smoother surface and decreased roughness of the composite scaffold. As seen from Fig. 5(d) and (e) the roughness of the cauliflower-like microstructures is still present in the PCLcoated HAp scaffolds. Surface roughness is an important factor influencing cell attachment and cell growth on the material. In general, surface roughness increases cell adhesion, migration and production of

extracellular matrix. Cells sense and respond to topographical features in a dimension-dependent way [44]. Surfaces with varying heights that are less than 1 μm different are often considered to be nanostructured, or nanorough. These surfaces have been used with a variety of cell types and surface chemistries, and a range of responses has been observed in terms of cellular adhesion, proliferation, and morphology [45]. Deligianni et al. [46] reported that human bone marrow cell adhesion and proliferation were surface roughness sensitive and increased as the roughness of HAp increased. As shown in Fig. 5(a)–(c) after polymer impregnation the interconnectivity of the pores in the HAp scaffold is maintained. No blocking of pores is observed. Well interconnected structures, as shown by the scaffolds prepared in this work, are of critical importance in tissue engineering applications. They allow the migration of cells into the scaffold and permit the adequate diffusion of nutrients and eventually signaling substances throughout the structure, which represents a basic condition for tissue regeneration [47].

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Fig. 5. SEM micrographs of PCL coated HAp scaffold. After polymer impregnation the interconnectivity of the channels in HAp scaffold is maintained, (a)–(c). A PCL layer on the HAp aggregates that resemble the cauliflower morphology is evident, (d) and (e).

3.5. In vitro evaluation of bioactivity After 28days of immersion in HBSS solution both HAp and HAp/PCL scaffolds exhibited mineral formation as shown in Fig. 6. No uniform coating but particles or aggregates of different size and regularity, distributed on the substrate surface are seen. The SEM image of the HAp surface (Fig. 6(a)) clearly illustrates the growth of the particles from the initial layer into the final hollow microspheres. A higher magnified image (Fig. 6(b)) shows highly crystalline background different from that seen in Fig. 4(f) indicating that during mineralization dandelionlike HAp structures transformed into hexagonal prismatic crystals. From Fig. 6(a) crack-like defects on the scaffold surface typical for brittle ceramics are seen. The mineralization on the HAp/PCL composite surface resulted in particles (microspheres) without hollow and with an outer fibrous layer as seen from Fig. 6(d). The top layer of growing particles seems quite amorphous. A well defined porous sponge-like microsphere about 3 μm in diameter nicely illustrates Fig. 6(e). The spherical

morphology is often observed for in vitro formed apatite [48,49]. Aggregates of merged microspheres are seen in Fig. 6(f). EDX analysis of the precipitated particles on the HAp surface revealed the presence of P, Ca, O and minor amounts of Na+, Mg2+, K+ and Cl− ions, similar to the composition of natural apatite from human bone [50,51]. The Ca/P molar ratio values obtained from eight EDX analyses range from 1.3 to 1.8 indicating the presence of calcium phosphate (CP) phases that produce the bonelike apatite. The mechanism of biomineralization of bone-like apatite on synthetic HAp was studied by Kim et al. [52]. On immersion and soaking in simulated body fluid, the synthetic HAp was found to induce the formation of bone-like apatite on its surface through the formation of Ca-rich amorphous calcium phosphate (ACP) in the early soaking period and the formation of Ca-poor ACP in the late soaking period. According to Kim et al. [52], on immersion in SBF the HAp could reveal a negative surface charge due to the hydroxyl and phosphate groups that gather the positive calcium ions forming the Ca-rich ACP. Then, the Ca-rich layer formed on the HAp surface interacts

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Fig. 6. SEM micrographs of the scaffolds' surface after soaking in HBSS for 28 days: Mineralization on the HAp (a, b) and HAp/PCL substrates (c–f).

with the negative phosphate ions in the fluid to form Ca-poor ACP which eventually crystallizes into bonelike apatite. Regarding the calcium phosphate mineralization on the HAp/PCL surface it is believed that nucleation occurs heterogeneously by complexation of the calcium ions and the negatively charged carboxylate groups formed during hydrolysis of the ester groups in the polymer [53,54]. 3.6. Mechanical properties To investigate the influence of PCL impregnation on the mechanical properties of HAp scaffold compression tests were performed on raw cuttlefish bone, HAp and HAp/PCL composite specimens. The compressive stress–strain curves shown in Fig. 7 are characterized by three different regions typical for porous structures [55]. The initial increase in stress at low strain (linear-elastic region) is followed by a long stress plateau region. The fluctuations observed in this region could be attributed to the layer by layer collapse of the microstructure under compression. Further increase in load did not result in a catastrophic failure but in a densification region accompanied by a steep increase in stress where the specimens crumbled into powder. Two stress–strain curves shown in Fig. 7(b) and (c) are two

“extreme” results obtained by testing ten to fifteen specimens of HAp and HAp/PCL composite scaffolds. The compressive strength and elastic modulus of the porous scaffolds were quantified from the maximum stress and the initial slope of the stress–strain curve, respectively. The average values of ten to fifteen measurements are summarized in Table 1. The comparison of Fig. 7(a) and (b) and the results given in Table 1 indicate that the removal of the organic component from the cuttlefish bone and the HT transformation of aragonitic cuttlefish bone into HAp had a negative effect on the mechanical properties of the scaffold. The HAp scaffold is much more fragile with low and irregular resistance of its lamellae to applied loads as compared to the raw cuttlefish bone scaffold. It may be hypothesized that the role of the organic component (β-chitin) in the raw cuttlefish bone scaffold is to redistribute compressive stress and dissipate energy during deformation along the organic layer, rather than through the inorganic crystals, inhibiting propagation of cracks. The similar role was expected from PCL coating on the HAp scaffold. As seen from Table 1. PCL coating on HAp was very effective in increasing the mechanical properties of the scaffold. The HAp/PCL composite scaffold displayed the highest compressive strength (0.88 MPa) and the elastic modulus (15.5 MPa) compared to the raw

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surface. The mechanical properties of the prepared scaffolds are comparable to or even better than those reported in the literature [32,33] for the HAp/PCL scaffolds prepared with more expensive and quite complex procedure. Typically, the modulus of human trabecular bone can vary tremendously, in the range of 10 to 3000 MPa depending on the porosity, position and direction, as well as testing methods. The strength is generally two orders of magnitude lower than modulus and is usually in the range of 0.1 to 30 MPa [56]. Therefore, the mechanical properties of the HAp/PCL composite scaffold obtained in this study are within the lower range of properties reported for human trabecular bones, presumably due to the high porosity. In this respect, the studied HAp/PCL composite scaffolds may have potential for bone tissue engineering applications under some load-bearing conditions. Further improvement of the mechanical properties of the scaffold could be realized by applying a thicker PCL coating by repeated polymer solution dipping while keeping the open porosity from interconnected pores. 4. Conclusions Natural aragonite from cuttlefish bone was hydrothermally transformed into hydroxyapatite (HAp) at 200 °C preserving the natural well interconnected porous structure. The obtained HAp scaffold was coated with a poly(ε-caprolactone) (PCL) using vacuum impregnation technique. The in vitro mineralization of calcium phosphate (CP) that produces the bone-like apatite was observed on both the pure HAp scaffold and the HAp/PCL composite scaffold. PCL coating on HAp was found to be very effective in increasing the mechanical properties of the scaffold. The compressive strength (0.88 MPa) and the elastic modulus (15.5 MPa) of the HAp/PCL composite scaffold are within the lower range of properties reported for human trabecular bones. The scaffold could be suitable for cell attachment, proliferation and differentiation and could be used in bone tissue engineering applications. Acknowledgments The financial support of the Ministry of Science, Education and Sports of the Republic of Croatia (project 125-1252970-3005: “Bioceramic, Polymer and Composite Nanostructured Materials”) and from the Spanish Ministry project DPI2010-20399-C04-03 is gratefully acknowledged. Rocío Ochoa-Fernández is also acknowledged for her help with the mechanical tests. References

Fig. 7. Stress–strain curves of the raw cuttlefish bone (a); HAp (b) and HAp/PCL composite scaffolds (c). Two stress–strain curves shown in (b) and (c) are two “extreme” results obtained by testing ten to fifteen specimens of HAp and HAp/PCL composite scaffolds.

cuttlefish (0.46MPa; 6.2MPa) and the HAp scaffold (0.15MPa; 0.7MPa). The applied vacuum infiltration of PCL probably facilitates the filling of the crack-like defects inhibiting crack propagation on HAp scaffolds' Table 1 Elastic modulus and compressive strength of porous scaffolds. Mechanical properties

Raw cuttlefish bone

HAp

HAp/PCL

Elastic modulus (MPa) Compressive strength (MPa)

6.2 ± 1.8 0.46 ± 0.06

0.7 ± 0.3 0.15 ± 0.09

15.5 ± 1.2 0.88 ± 0.11

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