Materials Science and Engineering C 55 (2015) 373–383

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

A novel squid pen chitosan/hydroxyapatite/β-tricalcium phosphate composite for bone tissue engineering Amin Shavandi a,c,⁎, Alaa El-Din A. Bekhit a, Zhifa Sun b, Azam Ali b, Maree Gould d a

Department of Food Sciences, University of Otago, Dunedin, New Zealand Department of Physics, University of Otago, Dunedin, New Zealand Department of Applied Sciences, University of Otago, Dunedin, New Zealand d Department of Anatomy, University of Otago, Dunedin, New Zealand b c

a r t i c l e

i n f o

Article history: Received 20 February 2015 Received in revised form 30 March 2015 Accepted 8 May 2015 Available online 12 May 2015 Keywords: Hydroxyapatite Tri-calcium phosphate Composite Squid pen chitosan

a b s t r a c t Squid pen chitosan was used in the fabrication of biocomposite scaffolds for bone tissue engineering. Hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) obtained from waste mussel shells were used as the calcium phosphate source. The composite was prepared using 2.5% tripolyphosphate (TPP) and 1% glycerol as a crosslinker and plasticizer, respectively. The weight percent (wt.%) ratios of the ceramic components in the composite were 20/10/70, 30/20/50 and 40/30/30 (HA/β-TCP/Chi). The biodegradation rate and structural properties of the scaffolds were investigated. Scanning electron microscopy (SEM) and microCT(μCT) results indicated that the composites have a well defined lamellar structure with an average pore size of 200 μm. The porosity of the composites decreased from 88 to 56% by increasing the ratio of HA/β-TCP from 30 to 70%. After 28 days of incubation in a physiological solution, the scaffolds were degraded by approximately 30%. In vitro investigations showed that the composites were cytocompatible and supported the growth of L929 and Saos-2 cells. The obtained data suggests that the squid pen chitosan composites are potential candidates for bone regeneration. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Significant progress in tissue engineering has been made in recent years. Many studies have focused on engineering human bone tissue using cells and biocomposites to repair or replace bone [1–3]. Biocomposites are essential to support the damaged tissue and cells allowing them to proliferate and differentiate. These composites act as a template replacing defected bone and are gradually replaced by new bone tissue. An ideal composite must be biocompatible and have proper mechanical and microstructure properties. It must also support cell adhesion and proliferation [4]. Squid pen chitosan may be more suitable for developing composite materials than ordinary crab or shrimp chitosan owing to its higher reactivity [5]. Bone is a natural biocomposite that contains organic and inorganic phases. In this regard, chitosan can be used as the organic phase in bioceramics and biocomposites, but it is necessary to optimize the mechanical and biological properties of chitosan scaffolds for application. Chitosan can be utilized in combination with other bioactive inorganic ceramics, especially hydroxyapatite (HA), to further enhance tissue regenerative efficacy and osteoconductivity. Chitosan is a natural linear polysaccharide, which has been a subject of interest for tissue ⁎ Corresponding author at: Department of Food Sciences, University of Otago, Dunedin, New Zealand. E-mail address: [email protected] (A. Shavandi).

http://dx.doi.org/10.1016/j.msec.2015.05.029 0928-4931/© 2015 Elsevier B.V. All rights reserved.

engineering. Chitosan is isolated from chitin by hydrolyzing acetyl groups (COCH3) of this monomer forming D-glucosamine (DG). Chitosan has been suggested as a natural polymer for use in orthopedic applications and is widely used for bone tissue engineering in various forms [6–8] due to its nontoxic, biocompatibility, and biodegradation properties. The cytocompatibility of chitosan is due to its chemical structure, which has a similar backbone to glycosaminoglycan, the major component of the extracellular matrix of bone [8,9]. The natural bone inorganic fraction is a form of calcium deficient hydroxyapatite [10]. Hydroxyapatite (Ca10(PO4)6(OH)2) and beta-tricalcium phosphate (β-TCP) have high biomimetic properties and have been successfully used in different orthopedic applications. The HA and β-TCP have been used as osteoconductive materials and can enhance bone formation on the implant [11,12]. Incorporations of calcium phosphate compounds into the chitosan matrix were found to improve the biocompatibility and hard tissue integration and enhance the resorption and degradation of composite [13]. Alpha-chitin and chitosan are commercially available products and are produced normally from shrimp or crab shell. Chitin/chitosan from the squid pen has a β-structure which is low packed and has weak intermolecular hydrogen bonds, compared to the heavily packed and strong molecular structure of the α-chitin/chitosan from shrimp and crab shells [5,14,15]. Most of the published studies were focused on αchitin/chitosan for scaffold fabrication and less attention has been paid to β-chitin/chitosan. Table 1 shows some studies, which utilized βchitin/chitosan for bone tissue engineering. Squid pen chitosan shows

374

A. Shavandi et al. / Materials Science and Engineering C 55 (2015) 373–383

Table 1 Published works that used squid pen β-chitin/chitosan for bone tissue engineering. Product

Preparation method

Characterization & application

Ref

β-Chitin scaffolds

CaCl2·6H2O/EtOH used as chitin solvent. Lyophilization technique.

[19]

β-Chitin–HAp composite

Membranes were prepared by alternate soaking of β-chitin membranes in CaCl2 (pH 7.4) and Na2HPO4.

β-Chitin hydrogel/nano hydroxyapatite composite

CaCl2/methanol solvent. Lyophilization technique. β-Chitin hydrogel and nHAp in 0.5 and 1% concentration. Chitosan powder was dissolved in 2% (v/v) acetic acid solution at a concentration of 1% (w/v).

Porosity: 1.2–4.5 μm. Swelling: 1400% after 48 h β-Chitin scaffolds can induce apatite formation in the body The apatite crystals were formed on the surface of β-chitin membranes, and their size and deposition were increased with increasing number of immersion cycles. Presence of apatite layer on surface of β-chitin membranes Swelling ratio: 15–20. Biodegradation: 30–40% on 7th day, maximum swelling after 24 h: 2000.

Squid chitosan-based structures

Carboxymethylation of ulvan and squid chitosan plus glass powder

Bone cement

β-Chitin sponge

β-Chitin (1.0 g) and water were blended in a mixer, swollen β-chitin was placed in 5-mm-diameter dishes and frozen and vacuum-dried for 24 h to form the β-chitin sponge. HA range of 1.00 to 3.00 (%w/w)

Chitosan gelatin/hydroxyapatite

β-Chitin/nanosilver composite

Highly porous β-chitin structure

Nanocomposite scaffold using β-chitin hydrogel with bioactive glass ceramic nanoparticles (nBGC) by lyophilization technique

CaCl2/methanol solvent. 0.001, 0.003 and 0.006% of nanosilver. Lyophilization technique Nonsolvent–solvent exchange-induced phase separation and supercritical CO2 drying processes. Repeating the freeze–thaw cycle of the chitin–formic acid solution 0.5 and 1% nBGC CaCl2·2H2O methanol solvent to prepare 0.5 w/v chitin hydrogel.

considerable hygroscopicity primarily as a result of the loss of crystallinity [5]. In addition, bulk density of the squid pen is much lower than those of crab and shrimp shells [16]. Crab and shrimp shells chitosan have been widely used for the fabrication of scaffolds. However, the structure–functional relationship and biological responses of scaffolds made by squid pens chitosan have not been addressed. In this study, we hypothesize that squid pen derived chitosan can be a useful material for the generation of biocomposite. This study describes biocomposites composed of squid pen chitosan and HA/β-TCP which have been obtained from waste mussel shells [17,18]. The objectives of the present study were: (1) to evaluate the microstructure, mechanical and functional properties of biocomposites prepared with squid pen chitosan and HA/β-TCP and (2) to examine the physiochemical properties of the scaffolds. 2. Materials and methods 2.1. Materials Chitosan, with a 75% degree of deacetylation (DD), was obtained from the arrow squid pen (Nototodarus sloanii) (obtained from Independent Fisheries Co., Christchurch, New Zealand) following the method described by Chaussard and Domard [29]. Nano-hydroxyapatite (Ca10(PO4)6 (OH)2) and β-TCP (Ca3(PO4)2) powders were obtained from waste mussel shells as previously described [17,18]. Acetic acid (Univar, USA), ethanol (Ajax Finechem, USA), glycerol, tripolyphosphate (TPP) and NaOH (Sigma, St. Louis, USA) were of analytical grade. 2.2. Preparation of the scaffold The process of the chitosan synthesis and scaffold fabrication is shown in Fig. 1. Due to the high hygroscopicity of squid pen chitosan,

Degradation b10% after 60 days soaking in SBF, pore size up to 67 μm. Compression modulus ~2

[20]

[21]

[22]

MPa. Similar structural stability and morphological features compared to commercial chitosan Low porosity (10%) and a mean pore size of 67 μm [23] Limited water uptake (230%) and weight loss (7%); non-cytotoxic behavior. Pore size ranged from 100 to 200 μm. [24] β-Chitin sponge could absorb 200–230 ml of water. [25] Pore size varied from 50 to 350 μm. Biodegradability up to 56% after 7 days and porosity and swelling about 90%. Wound healing. Bactericidal against Escherichia coli [26] and Staphylococcus aureus 5–25 nm. Drug delivery and dye or heavy-metal ion [27] removal technologies.

[28] Lose 20–25% of their weight after 28 days of incubation with lysozyme. Porosity about 60%. Pore size 100–300 μm. Swelling ratio of 12

the solution was very viscous at 1% chitosan. To prepare a higher concentration, a 1% solution of chitosan dissolved in 1% acetic acid was subjected to microwave irradiation to remove excess water achieving the desired concentration of 2%. After the gel cools, the solution was mixed with an overhead mixer (IKA RW 20 digital) for 10 min. The HA and β-TCP powders were mixed together based on the value of the ratio of HA to β-TCP shown in Table 2. The HA and β-TCP powders were weighed and a paste was formed using ethanol (10% w/v). The paste was added to the chitosan solution and homogenized. The chitosan solution was then sonicated (Elmasonic S40(H)) for 1 h to remove any air bubbles. The air bubble free mixture was transferred to 15 ml Poly-Cons® plastic containers and frozen at −80 °C. Then the samples were freeze-dried for 48 h (Labconco FreeZone 12 Plus) to form the composites. The dried HA/β-TCP/Chi composites were soaked in TPP aqueous solution (2.5%) at 4 °C for 2 h [30]. Then, the HA/β-TCP/Chi composites were rinsed with deionized water for 12 h at 4 °C to remove residual TPP and were freeze dried for 24 h at −40 °C (Fig. 1). 2.3. Characterization of the scaffold The distribution of the crystal HA/β-TCP structure in the chitosan matrix was analyzed using an X-Ray Diffractometer (XRD; PANnalytical X'Pert PRO MPD System) in the range 0° b 2θ b 60° with Cu Kα radiation (k = 0.15418 nm) with a scan speed of 2.63 s [31]. The functional groups of the samples were identified using Fourier transform infrared spectroscopy (FT-IR; Perkin-Elmer #100) in the region 400–4000 cm−1 with 4 cm− 1 spectral resolution using the KBr pellet technique [31]. Thermogravimetric analysis of the composites was carried out using a TGA instrument (TGA; Q 500) up to 1000 °C at a 10 °C/min heating rate under a nitrogen flow. Scanning electron microscopy (SEM) coupled with X-ray analysis (EDS) (JEOL 6700F FESEM JEOL Ltd.,

A. Shavandi et al. / Materials Science and Engineering C 55 (2015) 373–383

375

Waste squid pen Waste mussel shells 5% NaOH

Deproteinization

β-chitin

45% NaOH

Acetic acid

HA &β-TCP

Glycerol

Deacetylation

Chitosan

Add Ethanol

Paste

Mixing

Sonication Casting Freezing Lyophilisation

Cross linking

Freezing

Lyophilisation

Samples

Fig. 1. Graphical presentation for the fabrication process of HA/β-TCP/Chi scaffolds.

Tokyo, Japan) was used to examine the microscopic details of the surface and to identify the elemental composition of the samples. 2.4. Mechanical testing The mechanical properties of the HA/β-TCP/Chi composite scaffolds were tested according to the guidelines of ASTM D5024-95a [32]. The Table 2 Composition of HA/β-TCP/Chi composites. Compound

HA β-TCP Squid pen chitosan

(%)

compression mechanical properties of the scaffolds were determined using a TA.XTPlus, Texture Analyzer (Texture Technologies Corp., Stable Micro Systems, UK). The analysis was carried out on cylindrical samples with dimensions of 25 mm diameter × 12 mm height. A 250 N load cell was operated at a rate of 0.5 mm min− 1 until the sample was compressed to 50% of the original height at room temperature. The compression stress–strain curves were recorded, and Young's modulus was calculated using the Exponent software (version V6.1.5.0) using 5 replicates per each of the composite samples.

2.5. Micro-architectural analysis of scaffold

B1

B2

B3

20 10 70

30 20 50

40 30 30

X-ray microtomographic (μ-CT) systems have been widely used as a non-destructive technique to study or characterize microstructural morphology of various biomaterials [33,34]. To quantify the 3-D

376

A. Shavandi et al. / Materials Science and Engineering C 55 (2015) 373–383

microstructure of the scaffold samples, μ-CT images of the HA/β-TCP/ Chi scaffolds were obtained using a Skyscan 1172 system (BrukerMicro CT, Kontuch, Belgium). The used Skyscan 1172 system had an X-ray source with a focal spot size of b5 μm and was operated at 20–100 kV with 11 Mp detector. The maximum size of the samples was 27 mm. The samples were located between the X-ray source and the detector. The 2-D images generated by X-ray were recorded from a slice to another slice with a rotation of 0.4° by rotating the sample 180°. The scanned images were acquired at a pixel size of 11.4 μm. Total acquisition time was approximately 20 min and more than 900 2-D images were recorded for each sample. The images of the 3-D microstructure were binarized to discriminate voxel structure (the white spots) from empty voxels (the black spots). The image threshold was optimized to distinguish the chitosan polymer from the pore space. The pore size, porosity, total volume, and density of the scaffolds were measured. The porosity analysis of the scaffolds was carried out using the Ctan software (Bruker-microCT, Kontich, Belgium). Regions with white pixels are considered as solid portions and regions with black pixels, which are surrounded by white pixels, are pores. All data was reported as a percentage.

3. Microstructural characteristics 3.1. Pore size determination The pore structure was examined using SEM micrographs and the average pore size was extracted using ImageJ [37]. The mean pore diameter was estimated by measuring about 100 different pores for each scaffold. For this, the scale bar was set and then the image was thresholded and binarized. The threshold was adjusted in a way to be an accurate representation of pore distribution. The ‘Analyse Particles’ command function in imageJ was executed to analyze Ferret's diameter, the longest distance between any two parallel lines tangent to the particle edge. In these samples, the pore diameter cannot be determined using the area of each pore, since the pores were not uniform and were not circular in shape. A single measurement is not an accurate depiction of the pore, since the calculated area is reduced because of the projection of solid material from planes above and below the pore. The Ferret's diameter is therefore a fair approximation since it will measure the longest diameter. 3.2. Cell culture and proliferation

2.6. Water uptake and retention abilities Water uptake and retention ability of the scaffolds were measured using the method described by Thein and Misra [13]. A sample with a known weight (Wd) was immersed in distilled water for 24 h. Then, the sample was gently removed and placed on a wire rack for 1 min. The sample was weighed (Ww) to determine the water uptake. To measure the retention ability of water, the wet scaffold sample was transferred into a falcon tube with a filter paper at the bottom of the tube and centrifuged using a Beckman GPR centrifuge at 500 RPM for 3 min. Then the sample was weighed (W′w). The weight of the sample was averaged from triplicate measurements for each group of samples. The water uptake and retention percentage of the scaffolds were calculated using the following equation [7,13]: EA ¼ ½
ðW d Þ=W h
W −W  d   i100 0 ER ¼ ½ W W −W d =W d  100

where EA is the water uptake and ER is the water retention. 2.7. In vitro degradation of scaffolds Alpha-lactate and lactic acid-buffered stimulated body fluid (LacSBF) with an ionic concentration similar to human blood plasma was prepared as described by Cuneyt Tas [35]. The degradation ability of the composites was studied by incubating dry and weighed (W0) samples in SBF (pH 7.4 at 37 °C) for up to 28 days. Approximately 25 mg samples (n = 3 for each group) were placed in 10 ml of SBF solution and then incubated at 37 °C. The samples were removed at time intervals (1, 7, 14, and 28 days), washed five times with distilled water, and then dried overnight at 60 °C. Changes in the pH of Lac-SBF solution were also measured over the testing time using a pH meter (HI 2211, HANNA instruments, Rhode Island, USA) [36]. The degradation was monitored in terms of changes in the weight of the samples over the time period. The degradation ratio (D) was calculated using the following equation: D ¼ 100−½ðW 0 −W t Þ=W 0 Þ  100

where D is the degradation rate of scaffolds, W0 represents the original weight and Wt is the weight of the sample at time t.

Human osteoblast-like cells (Saos-2) and mouse fibroblastic-like cells (L929) (European Collection of Cell Cultures (ECACC; Salisbury, UK) were prepared for cell culture by the addition of 10% fetal bovine serum (FBS) and penicillin–streptomycin antibiotics into Modified Essential Medium (MEM; Invitrogen). Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. Briefly, the scaffolds were handsectioned with a razor blade (40 mg), placed in 48 well plates and UV sterilized before being immersed in 70% ethanol and allowed to dry. Scaffolds were equilibrated in 1 ml of media and incubated overnight at 37 °C in a humidified atmosphere of 5% CO2. The media were removed and cells were seeded onto each scaffold at a density of 6 × 103 cells/scaffold/well allowing the cells to attach to the scaffolds for 1 h in the incubator before more media were added to cover the scaffolds. Three replicates were used for each scaffold type and in the control cultures the cells were placed directly into the well at the same density as placed onto the scaffolds. Media were replaced every 48 h up to the cultivation period. The cytocompatibility of synthesized scaffolds was determined using the MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-caebozymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay using the CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI). Colorimetric measurement of the samples was performed on a spectrophotometer (Synergy 2 Multi-Mode Microplate Reader) at 490 nm. 3.3. Statistical analysis Results were obtained from triplicate individual experiments. Twoway analysis of variance (ANOVA) was performed to examine the effects of composite's composition and treatment time on the measured properties. Differences among the means were considered statistically significant at P b 0.05 by Tukey's post-tests using the GraphPad Prism software (version 6.00 for Windows, GraphPad Software, California USA, www.graphpad.com). 4. Results and discussion 4.1. X-ray diffraction The phase of the composite was confirmed by XRD. The XRD patterns of the composites with different HA/β-TCP weight percentages are shown in Fig. 2A. The wide peak at 2θ = 20° in all patterns was assigned to chitosan and shows its degree of crystallinity [6]. The sharp peak at around 2θ = 32° corresponds to the HA/β-TCP in the composite [17,18] and as shown by increasing the ratio of HA/β-TCP to

A. Shavandi et al. / Materials Science and Engineering C 55 (2015) 373–383

377

Fig. 2. A) X-ray diffractometer patterns, B) FTIR spectra and C) thermo-gravimetric graph of composites with different ratios of HA/β-TCP/Chi; B1) 30% HA/β-TCP, B2) 50% HA/β-TCP, B3) 70% HA/β-TCP.

chitosan (from B1 to B3) the peak at 2θ = 32° becomes sharper and amplified. Similarly, peaks at 2θ = 33, 34, 40, 47, 48° and 50–54° are also assigned to HA/β-TCP and are narrower and sharper in the composite when the content of HA/β-TCP is higher. These results were in

agreement with previously reported findings [38]. Increasing the HA/ β-TCP content caused the crystallinity of the chitosan to decrease and inferring that the HA/β-TCP weakens the intramolecular interaction of the chitosan chain [6]. The crystallinity of the HA/β-TCP compounds

378

A. Shavandi et al. / Materials Science and Engineering C 55 (2015) 373–383

B1

B2

B3

Fig. 3. The effect of differing weight ratios of HA/β-TCP on the scaffolds. Scanning electron microscopy (SEM) images of scaffolds B1) 30% HA/β-TCP, B2) 50% HA/β-TCP, and B3) 70% HA/β-TCP.

was lower than their corresponding pure HA or β-TCP compounds, due to the presence of the chitosan matrix. However, in natural bone these peaks are low and are overlapped [39]. 4.2. Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy (FTIR) was used to investigate the interaction between chitosan and HA/β-TCP. The IR spectra of the composites are shown in Fig. 2B. The peaks at 1647 and 1560 cm− 1 are assigned to amides Ι and II, respectively [13,40]. The

peak at around 3260 cm−1 represents the stretching vibration of N–H of the chitosan, which became lower by increasing the HA/β-TCP ratio. The sharp peak at 1415 cm−1 is devoted to symmetrical deformation mode of CH3. The peaks at 1029 and 1100 cm− 1 are assigned to the C–O stretching vibrations. The FTIR spectra of all the three composites indicated that characteristic bands of both CaP and chitosan were present in the composites. This observation suggests that the chitosan structure provided a matrix for the HA/β-TCP particles and also binds them together in the composite [13]. The strong peaks in spectra at 1060 cm−1 and 539 cm−1 belong to stretching and bending modes of

A. Shavandi et al. / Materials Science and Engineering C 55 (2015) 373–383

phosphate respectively, and the peak at 1415 cm−1 was due to calcium phosphate [21,41]. As shown by increasing HA/β-TCP content, the typical peak of PO4 at 500–700 cm−1 became stronger [6].

Table 3 Micro-architectural parameters for composites produced with various HA/β-TCP concentrations.

4.3. Thermal analysis Thermal degradation of the composites was studied using thermogravimetric analysis (TGA). Fig. 2C shows the TGA curves of the three composites. It is seen that in the case of B1 (20/10/70, HA/β-TCP/β-Ch, wt.%), which has the lowest HA/β-TCP%, the sample weight decreased very fast by increasing the temperature. The weight loss begins at 50 °C and the sharpest decrease was observed at 200–300 °C, which may be due to water loss and chitosan decomposition [42]. In the case of B2 and B3, which have higher HA/β-TCP%, the thermal decomposition of the chitosan was lower. This heat resistant behavior of chitosan can be assigned to bonds that were formed between hydroxyl and amino groups [42]. A weight loss was initiated at around 550 °C, which indicated the beginning of HA/β-TCP decomposition [42]. After the curve became stable at temperature N550 °C, there were about 33%, 56% and 74% of the weight remaining for B1, B2 and B3 respectively, which was parallel to HA/β-TCP%. 4.4. Scaffold morphology The porous chitosan composite with different HA/β-TCP contents and 1% glycerol was fabricated by lyophilization and cross-linked using 2.5% TPP solution. The SEM was used to examine the microstructure of the scaffolds (Fig. 8), and it can be seen that the pores of the composites were elongated in shape and were irregularly interconnected. Increasing the HA/β-TCP wt.% caused the pores to become more irregular in shape, and the structure walls to be covered with calcium phosphate (CaP) particles. In the case of B3 that had the highest HA/βTCP%, the microstructure of the samples was compact and a thick layer of HA/β-TCP covered the chitosan walls. The EDS chemical analysis confirmed the presence of CaP (Fig. 3). The EDS results showed the presence of HA/β-TCP crystals with CaP ratio ranging from 1.68 to 1.9 in all the three composites. Increasing HA/β-TCP% caused a reduction in the EDS carbon peak which represents chitosan, whereas peaks of Ca and P increased. The obtained CaP ratios correspond to that of HA/β-TCP [43]. 4.5. Porosity and density The porosity of the scaffold plays various important roles. The porosity facilitates cell migration, blood circulation and vascularization. It was reported that composites made via the lyophilization technique have a

A 100

379

Type of scaffold

Volume fraction (%)

Degree of anisotropy

Density (gr/cm3)

Porosity (%)

B1 B2 B3

11.00 35.17 43.61

1.28 ± 0.21 1.09 ± 0.08 1.32 ± 0.24

0.11 ± 0.02 0.15 ± 0.03 0.13 ± 0.01

88.72 ± 0.90 66.40 ± 5.70 56.39 ± 2.41

better pore size compared to the sol–gel and the precipitation method [44]. The porosity and pore morphology are important factors for the evaluation of the scaffolds. In addition to the SEM images, more insights into the microstructure of the scaffold were obtained using μ-CT analysis, which provides simultaneous analysis of the microstructure and pore properties in a 3 dimensional scale. Two dimensional crosssection slices of sequential scanning of the chitosan scaffolds are shown in Fig. 4b. The μ-CT images and data were in good agreement with SEM images (Fig. 3) and show a highly porous structure. The composites have a porosity range from 56 to 88% (Table 3 and Fig. 4). By increasing the HA/β-TCP% in the scaffold, the density increases and the porosity of the composite decreased [45]. The total porosity of the composites was also explored using liquid displacement method, and the results are summarized in Table 3. The scaffold porosity decreased significantly (P b 0.05), as the HA/β-TCP% increased to 70% in the B3 sample [46]. The addition of HA/β-TCP increased the density of the composite (Table 3) and confirmed the observed reduction in the porosity with an increase in the % of HA/β-TCP [45]. As shown in Table 3, increasing the HA/β-TCP% leads to an increase in the volume fraction of the composites. This could be explained by the increase in thickness of pore walls due to the precipitation of HA/ β-TCP on the walls (Fig. 8). Moreover, among the three composites, the composite with the lowest HA/β-TCP% had the highest porous structure, which may be useful to enhance cell adhesion and proliferation and thus promote tissue growth and replacement. Pore size The scaffold morphology is an important feature of the scaffolds for bone tissue engineering. The structure should have sufficiently large pores that allow blood vascularization and cell proliferation. Wide ranges of pore size, from 30 to 1000 μm, have been reported in the literature for adequate bone tissue engineering [47,48]. It was reported that pores greater than 300 μm is essential for vascularization of scaffolds and bone ingrowth [49–52], however, the degree of bone ingrowth depends on the biomaterial and the geometry of the pores. The average

B

a b

P o r o s ity (% )

80

c 60

40

20

0 B1

B2

B3

T y p e o f s c a ffo ld s

Fig. 4. MicroCT analysis of the composites. A) Porosity of composites B1–3. There is a significant decrease (P b 0.05) in porosity with increase in the content of HA/β-TCP in all three composites. B) 2D cross section image of composite B2.

380

A. Shavandi et al. / Materials Science and Engineering C 55 (2015) 373–383

Fig. 5. Pore size of composites with different HA/β-TCP concentrations. B1) 30% HA/β-TCP, B2) 50% HA/β-TCP, B3) 70% HA/β-TCP.

diameter of the pores was 424.6 μm for B1 scaffold, 456.2 μm for B2 scaffold and 225.6 μm for B3 scaffold. In this study, it was revealed that as the HA/β-TCP% increased, the pore structure of the scaffolds became less homogeneous and pore size distribution became more inhomogeneous (Fig. 5). As seen from the SEM images (Fig. 3), the pore walls became thicker due to the deposition of CaP on the wall surface, leading to a reduced pore size. The results of our study were in accordance with previously reported pore size for chitosan/hydroxyapatite scaffolds [53,54].

proper structure for bone regeneration. Therefore, a balanced process which produces both suitable mechanical properties and porosity is essential. It is worth noting that with an increase in the ratio of HA/β-TCP the shrinkage of the membrane decreased, and the least shrinkage was observed in scaffold group B3 with highest HA/β-TCP ratio (Fig. 6B) [55]. Additionally Reys et al. [22], in a recent study, concluded that membranes produced from squid chitosan have higher mechanical properties than those prepared with crab chitosan. 4.7. Water uptake and water retention test

4.6. Mechanical properties of scaffolds The mechanical behavior of the scaffolds is a critical factor for their biomedical application and bone healing. In most polymer matrixes, the incorporation of CaP enhances the mechanical properties of the scaffold. The mechanical properties of the composites are shown in Fig. 6. It is seen that by increasing the HA/β-TCP% up to 50% the compression mechanical properties are increased (Fig. 6A), and the elasticity of the scaffold is improved. However, the intramolecular properties of the chitosan molecules appear to be negatively affected by excess addition of HA/β-TCP. Considering the brittle nature of chitosan, incorporation of higher HA/β-TCP% (70%) increased brittleness of the chitosan composites and led to reduction in the tensile properties of the scaffolds with early failure [6]. Furthermore, cross-linking of chitosan may cause microstructural changes that lead to an even more brittle structure. In addition to this, the high concentration of HA/β-TCP (70%) could result in composites with low porosity (Table 3), generating a scaffold without

The water retention ability of the scaffold is a critical factor for its suitability for bone grafting, and it has been reported that water uptake ability of the composite can significantly affect cell proliferation and differentiation [56]. Results for the water uptake and retentions of the composites in this study are shown in Fig. 7. Increasing the HA/βTCP% in the composites decreased the water uptake and retention ability of composites. The HA/β-TCP particles act as fillers making the polymer chains intact and thus reduce the composite porosity. All the composite scaffolds started uptaking rapidly on the first day, indicating good characteristic swelling; similar trends are reported in the literature [13]. On day 14, maximum water uptake capacity has been attained and thereafter the uptake percentage was found to decrease. This was due to the rupture of the polymer chains at a higher stage of swelling. The chitosan scaffold can take up and hold water more than its own weight (values are higher than 100%). A maximum water uptake recorded in this study was 1600%, which is lower than the 2500% reported for

A b

400

C o m p re s s io n m o d u lu s

U ltim a te te n s ile s tre s s 12

b

Y e ild s tre n g th

ab

b

kPa

kPa

300

200

a

8

a

a

4

100

a

a

0

0

B1

B1

B2

B2

B3

B3

T y p e s o f S c a ffo ld s

T y p e s o f S c a ffo ld s

B

Fig. 6. Mechanical properties of the HA/β-TCP/Chi composites. A) There is a significant increase in mechanical properties with increasing the content of HA/β-TCP up to 50%. a–cBars with different letters are significantly different at P b 0.05 (n = 7). B) The morphological observation of scaffolds.

A. Shavandi et al. / Materials Science and Engineering C 55 (2015) 373–383

A

2000

B B3 a

1500

b

b b

b

B3

b

B2

B1

a

1000

a

B2

a

a

ab

800

b b

600

W /W (% )

W /W (% )

381

b

500

bc

B1 cd

cd

cd cd

400

cd cd

c

c

c

200

0

0

1

7

14

28

1

T im e (d a y s )

7

14

28

T im e (d a y s )

Fig. 7. Water uptake (A) and water retention (B) ability of HA/β-TCP/Chi composites after 28 days of soaking in SBF solution. a–dBars with different letters are significantly different at P b 0.05.

chitosan/CaP [13,45]. The lower water uptake found in this study may be due to the cross-linking and shrinkage of the scaffold which reduces its water uptake ability. Water uptake of composite B1, was significantly (P b 0.05) higher than composites B2 and B3 over the test period. On the basis of a previous finding, β-chitin/chitosan, has higher affinity for solvents and therefore, has higher water uptake and retention compared to α-chitin/ chitosan. This may be due to the weak hydrogen bonding and intramolecular force of β-chitin/chitosan, which make β-chitin/chitosan more reactive compared to α-chitin/chitosan [57–59].

4.9. Change in pH of physiological solution

4.8. Degradation The biodegradation properties of the scaffolds are an important factor on the long term functionality of the bone graft. The backbone of the chitosan as a natural polymer is hydrolytically unstable [60] which enhances its degradation. In a study by Kumar et al., after 28 day incubation, 30% and 45% degradation was reported for β-chitin composites with HA (0.5%) and without HA (1%) [21]. However, after 60 days of incubation less than 10% of degradation was observed by Reys et al., who used squid pen chitosan without incorporating CaP compounds [22]. These differences may be due to the different sources of chitin and different isolation processes and degrees of deacetylation where degradation rate increases at a higher degree of deacetylation [22]. It is suggested that the degradation rate is higher when chitosan molecular weight, crystallinity index and degree of deacetylation are low [61,62]. Part of the degradation products is chitooligosaccharides which have a positive role for the bioactive functionality of scaffolds [21,63]. Therefore, a chitosan with low DD and molecular weight could be a good

100

D e g r a d a t io n ( % )

a B1

80

B2 B3

b

60

bc bc 40

candidate for tissue engineering applications [62]. The degradation profile of the synthesized chitosan composites in SBF over 28 days is presented in Fig. 8. After 28 days of in vitro degradation, composites B1 and B2 were degraded by about 31 and 37%, respectively. In the case of composite B3, which had the highest HA/β-TCP%, the degradation rate was the lowest until day 14 while B3 lost around 83% of its weight after 28 days of soaking in SBF at 37 °C. As shown in Fig. 8, the weight loss gradually increased with time. The degradation rate of scaffold was higher in high HA/β-TCP% samples.

cd

cde def f

20

def

bc

ef

f

0 1

7

14

28

T im e (d a y s )

Fig. 8. Degradation rate of HA/β-TCP/Chi composites after 28 days of soaking in SBF solution. There are no significant differences in biodegradation between B1, B2 and B3 composites. a–fBars with different letters are significantly different at P b 0.05.

Chemical stability of the scaffold is of great importance. Solubility of the scaffold compartments can lead to variation in pH and ion conductivity of the environment and may negatively affect the cell response [64]. The rate of scaffold degradation was found to be closely related to ions released from the scaffold. Ionic conductivity measurement can be used to estimate the changes in ion concentration of the scaffold environment [36]. The pH variation of the SBF solution of the composites was monitored over a four-week period (Fig. 9). During the first week of incubation in SBF, the pH value of all the composite groups was increased slightly in the first week and then decreased over the following week (Fig. 9). This increase might be due to the alkalescent nature of chitosan, the presence of alkaline groups, and the generation of alkaline ions from the degradation of CaP compounds [65,66]. The decrease in pH in the second week was significant (P b 0.05) and can be due to deposition of SBF calcium and phosphate ions on the composite surface [67]. Then the value of the pH gradually increased up to 28 days. The pH variation pattern of the SBF solution might be related to the dissolution of HA/β-TCP and to the degradation profile of chitosan. As shown in Fig. 9, at all the tested time points, composite B1, which had the lowest HA/β-TCP weight ratio, experienced a sharper pH change compared to B2 and B3 composites. This may be related to a higher dissolution rate of chitosan in B1. HA and β-TCP particles slow down the degradation of chitosan and thus the pH change is lower at the higher HA/β-TCP% [68]. The gradual pH increase from the second week onward could be due to faster degradation of composites, which was inversely related to pH [61]. In a study, by Czechowska et al., β-TCP/chitosan composites were tested in SBF solution, and pH of the solution changed slightly from 7.46 to 7. 28. However, in this study, the pH of SBF solution deviated from pH 7.45 and fluctuated between 7.23 and 7.95. This could be due to different degradation behavior, and composition of the composites tested in this study [36]. The ion conductivity measurement results are displayed in Fig. 9. The greatest changes occurred in the first and last weeks (P b 0.05). The ion release process from the scaffolds is non-linear and in the first week, a slight decrease from 13 to 10 (mS/cm) was observed for B1 and B2 samples. In consecutive examined days, a slight

A. Shavandi et al. / Materials Science and Engineering C 55 (2015) 373–383

A

B

10

B3 B2

9

B1

pH

8

7

6

5 1

7

14

Io n ic c o n d u c tiv ity ( m S /c m )

382

14

B3

12

B2 B1

10 8 6 4 2 0

28

1

7

T im e (d a y s )

14

28

T im e (d a y s )

Fig. 9. pH and ion conductivity vs. time of incubation in SBF. The pH (A) and ion conductivity (B) values of the SBF solution in which the samples were incubated.

6 x10

5

4 x10

C o n tro l

cd cd de 2 x10

5

ef

ef f

5

8 .0 x 1 0

4

6 .0 x 1 0

4

4 .0 x 1 0

4

2 .0 x 1 0

4

B

f

f

B2 ab

B3

ab

C o n tro l

b b

b b b

b

0

B1

a

B1 bcd b c

5

1 .0 x 1 0

B2

ab

C e ll n u m b e r

C e ll n u m b e r

B3

a

A

b

b

b

0 1

3

7

1

T im e (d a y s )

3

7

T im e (d a y s )

Fig. 10. Viability level of L929 (A) and Saos-2 (B) cells on the scaffolds as a function of time, assessed by MTS assay, which shows active mitochondrial activity of living cells. The number of cells on the all composites increased from days 1 to 7. a–bBars with different letters are significantly different at P b 0.05.

decrease in ion conductivity was recorded until it reached a steady state of about 8 mS/cm on day 28.

HA/β-TCP-Chitosan composite as a potential scaffold material for biomedical field, however; further research is needed on its possible application for tissue engineering.

4.10. Cytocompatibility of scaffolds Acknowledgments Cytocompatibility of a tissue is another crucial factor that needs to be considered in a material targeted for tissue engineering. For this purpose, the cytotoxicity of the composites was investigated using the MTS assay. The results shown in Fig. 10 indicate that the cell's density was increased over the testing period, and the scaffold composition materials were biocompatible with the L929 and Saos-2 cells. This shows that both HA/β-TCP and chitosan do not exert any significant toxic effect on the cells. Furthermore, the cell density on the scaffolds was much higher than the control sample. Additionally, it can be seen that at a higher concentration of HA/β-TCP, the cell density also increased indicating the positive effect of HA/TCP concentration on the growth of these cells. These results are in accordance the observations reported by Reys et al. [22] who also compared chitosan composites from different sources (squid and crab) and concluded that the noncytotoxic behavior of the composites is independent of the chitosan type and the degree of deacetylation [22,69]. In another study, βchitin/HA composite was fabricated and enhanced cell growth obtained on composites with HA, they suggested that HA causes adsorption of more protein and consequently results in better cell attachment to the scaffold [21]. 5. General conclusions A highly porous HA/β-TCP-Chitosan composite fabricated by freeze drying was characterized using a variety of mechanical and physiochemical methods. We have shown that cross-linking greatly influenced the properties of the composites. Scaffolds with the highest concentration of HA/β-TCP exhibited greater mechanical properties but slower degradation, increased thermal stability, and lower water swelling and retention. HA/β-TCP/CH composites exhibited significant interactions between the HA/β-TCP and chitosan that were homogeneously distributed within the chitosan structure. This study shows

The authors acknowledge the facilities as well as the scientific and technical assistance from staff at the Otago Centre for Electron Microscopy (OCEM) at University of Otago. We would also like to thank Mr Damian Wallas for his help and support to use XRD. The first author acknowledges the PhD scholarship awarded by University of Otago, New Zealand. References [1] P. Jongwattanapisan, N. Charoenphandhu, N. Krishnamra, J. Thongbunchoo, I.M. Tang, R. Hoonsawat, et al., In vitro study of the SBF and osteoblast-like cells on hydroxyapatite/chitosan–silica nanocomposite, Mater. Sci. Eng. C 31 (2) (2011) 290–299. [2] A.F. Khan, M. Saleem, A. Afzal, A. Ali, A. Khan, A.R. Khan, Bioactive behavior of silicon substituted calcium phosphate based bioceramics for bone regeneration, Mater. Sci. Eng. C 35 (2014) 245–252. [3] C. Tsioptsias, I. Tsivintzelis, L. Papadopoulou, C. Panayiotou, A novel method for producing tissue engineering scaffolds from chitin, chitin–hydroxyapatite, and cellulose, Mater. Sci. Eng. C 29 (1) (2009) 159–164. [4] S.V. Dorozhkin, Biocomposites and hybrid biomaterials based on calcium orthophosphates, Biomaterials 1 (1) (2011) 3–56. [5] K. Kurita, K. Tomita, T. Tada, S. Ishii, S.-I. Nishimura, K. Shimoda, Squid chitin as a potential alternative chitin source: deacetylation behavior and characteristic properties, J. Polym. Sci. A Polym. Chem. 31 (2) (1993) 485–491. [6] S.H. Teng, E.J. Lee, B.H. Yoon, D.S. Shin, H.E. Kim, J.S. Oh, Chitosan/ nanohydroxyapatite composite membranes via dynamic filtration for guided bone regeneration, J. Biomed. Mater. Res. Part A 88 (3) (2009) 569–580. [7] M.V. Zugravu, R.A. Smith, B.T. Reves, J.A. Jennings, J.O. Cooper, W.O. Haggard, et al., Physical properties and in vitro evaluation of collagen-chitosan-calcium phosphate microparticle-based scaffolds for bone tissue regeneration, J. Biomater. Appl. 28 (4) (2013) 566–579. [8] W.W. Thein-Han, R.D. Misra, Biomimetic chitosan–nanohydroxyapatite composite scaffolds for bone tissue engineering, Acta Biomater. 5 (4) (2009) 1182–1197. [9] E. Khor, L.Y. Lim, Implantable applications of chitin and chitosan, Biomaterials 24 (13) (2003) 2339–2349. [10] P. Wang, L. Zhao, J. Liu, M.D. Weir, X. Zhou, H.H.K. Xu, Bone tissue engineering via nanostructured calcium phosphate biomaterials and stem cells, Bone Res. 2 (2014) 1–13.

A. Shavandi et al. / Materials Science and Engineering C 55 (2015) 373–383 [11] S. Yamada, D. Heymann, J.M. Bouler, G. Daculsi, Osteoclastic resorption of calcium phosphate ceramics with different hydroxyapatite/β-tricalcium phosphate ratios, Biomaterials 18 (15) (1997) 1037–1041. [12] A. Ogose, T. Hotta, H. Kawashima, N. Kondo, W. Gu, T. Kamura, et al., Comparison of hydroxyapatite and beta tricalcium phosphate as bone substitutes after excision of bone tumors, J. Biomed. Mater. Res. B Appl. Biomater. 72B (1) (2005) 94–101. [13] Khaled R. Mohamed, Biocomposite Materials, Composites and Their Applications, in: Prof. Ning Hu (Ed.), 2012 ISBN: 978-953-51-0706-4, InTech, http://dx.doi.org/10. 5772/48302. Available from:/ http://www.intechopen.com/books/composites-andtheir-applications/biocomposite-materials. [14] R. Minke, J. Blackwell, The structure of α-chitin, J. Mol. Biol. 120 (2) (1978) 167–181. [15] N.E. Dweltz, The structure of β-chitin, Biochim. Biophys. Acta 51 (2) (1961) 283–294. [16] Y. Fan, T. Saito, A. Isogai, Preparation of chitin nanofibers from squid pen β-chitin by simple mechanical treatment under acid conditions, Biomacromolecules 9 (7) (2008) 1919–1923. [17] A. Shavandi, A.E.-D.A. Bekhit, A. Ali, Z. Sun, Synthesis of nano-hydroxyapatite (nHA) from waste mussel shells using a rapid microwave method, Mater. Chem. Phys. 149–150 (2015) 607–616. [18] A. Shavandi, A. Bekhit AE-D, A. Ali, Z. Sun, J.T. Ratnayake, Microwave-assisted synthesis of high purity β-tricalcium phosphate crystalline powder from the waste of Green mussel shells (Perna canaliculus), Powder Technol. 273 (2015) 33–39. [19] Y. Maeda, R. Jayakumar, H. Nagahama, T. Furuike, H. Tamura, Synthesis, characterization and bioactivity studies of novel beta-chitin scaffolds for tissue-engineering applications, Int. J. Biol. Macromol. 42 (5) (2008) 463–467. [20] K. Madhumathi, N.S. Binulal, H. Nagahama, H. Tamura, K.T. Shalumon, N. Selvamurugan, et al., Preparation and characterization of novel β-chitin–hydroxyapatite composite membranes for tissue engineering applications, Int. J. Biol. Macromol. 44 (1) (2009) 1–5. [21] P.T. Sudheesh Kumar, S. Srinivasan, V.-K. Lakshmanan, H. Tamura, S.V. Nair, R. Jayakumar, β-Chitin hydrogel/nano hydroxyapatite composite scaffolds for tissue engineering applications, Carbohydr. Polym. 85 (3) (2011) 584–591. [22] L.L. Reys, S.S. Silva, J.M. Oliveira, S.G. Caridade, J.F. Mano, T.H. Silva, et al., Revealing the potential of squid chitosan-based structures for biomedical applications, Biomed. Mater. 8 (4) (2013) 1–11. [23] A.A.A. Barros, A. Alves, C. Nunes, M.A. Coimbra, R.A. Pires, R.L. Reis, Carboxymethylation of ulvan and chitosan and their use as polymeric components of bone cements, Acta Biomater. 9 (11) (2013) 9086–9097. [24] M. Abe, M. Takahashi, S. Tokura, H. Tamura, A. Nagano, Cartilage-scaffold Composites Produced by Bioresorbable β-chitin Sponge with Cultured Rabbit Chondrocytes, 10 (3–4)2004. 585–594. [25] Characterization of porous scaffold from chitosan–gelatin/hydroxyapatite, in: W. Wattanutchariya, W. Changkowchai (Eds.), for Bone Grafting Proceedings of the International MultiConference of Engineers and Computer Scientists IMECS 2014, The International Association of Engineers (IAENG), Hong Kong, March 12–14 2014. [26] P.T.S. Kumar, S. Abhilash, K. Manzoor, S.V. Nair, H. Tamura, R. Jayakumar, Preparation and characterization of novel β-chitin/nanosilver composite scaffolds for wound dressing applications, Carbohydr. Polym. 80 (3) (2010) 761–767. [27] P. Ratanajiajaroen, M. Ohshima, Preparation of highly porous β-chitin structure through nonsolvent–solvent exchange-induced phase separation and supercritical CO2 drying, J. Supercrit. Fluids 68 (2012) 31–38. [28] S. Sowmya, P.T.S. Kumar, K.P. Chennazhi, S.V. Nair, H. Tamura, R. Jayakumar, Biocompatible chitin hydrogel/nanobioactive glass ceramic nanocomposite scaffolds for periodontal bone regeneration, Trends Biomater. Artif. Organs 25 (1) (2011) 1–11. [29] G. Chaussard, A. Domard, New aspects of the extraction of chitin from squid pens, Biomacromolecules 5 (2) (2004) 559–564. [30] S.H. Yu, S.J. Wu, J.Y. Wu, C.K. Peng, F.L. Mi, Tripolyphosphate cross-linked macromolecular composites for the growth of shape- and size-controlled apatites, Molecules 18 (1) (2012) 27–40. [31] G.S. Kumar, A. Thamizhavel, E.K. Girija, Microwave conversion of eggshells into flower-like hydroxyapatite nanostructure for biomedical applications, Mater. Lett. 76 (2012) 198–200. [32] H. Lima, F. Lia, S. Ramdayal, Preparation and characterization of chitosan–insulin– tripolyphosphate membrane for controlled drug release: effect of cross linking agent, J. Biomed. Nanotechnol. 5 (2014) 211–219. [33] A.C. Jones, B. Milthorpe, H. Averdunk, A. Limaye, T.J. Senden, A. Sakellariou, et al., Analysis of 3D bone ingrowth into polymer scaffolds via micro-computed tomography imaging, Biomaterials 25 (20) (2004) 4947–4954. [34] A.S. Lin, T.H. Barrows, S.H. Cartmell, R.E. Guldberg, Microarchitectural and mechanical characterization of oriented porous polymer scaffolds, Biomaterials 24 (3) (2003) 481–489. [35] Tas A. Cuneyt, Submicron spheres of amorphous calcium phosphate forming in a stirred SBF solution at 55 °C, J. Non-Cryst. Solids 400 (2014) 27–32. [36] J. Czechowska, A. Zima, Z. Paszkiewicz, J. Lis, A. Ślósarczyk, Physicochemical properties and biomimetic behaviour of α-TCP-chitosan based materials, Ceram. Int. 40 (4) (2014) 5523–5532. [37] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, NIH Image to ImageJ: 25 years of image analysis, Nat. Mater. 9 (7) (2012) 671–675. [38] Q. Hu, B. Li, M. Wang, J. Shen, Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a potential material as internal fixation of bone fracture, Biomaterials 25 (5) (2004) 779–785. [39] C. Meneghini, M.C. Dalconi, S. Nuzzo, S. Mobilio, R.H. Wenk, Rietveld refinement on X-ray diffraction patterns of bioapatite in human fetal bones, Biophys. J. 8 (3) (2003) 2021–2029.

383

[40] R. Barabás, M. Czikó, I. Dékány, L. Bizo, E. Bogya, Comparative study of particle size analysis of hydroxyapatite-based nanomaterials, Chem. Pap. 67 (11) (2013) 1414–1423. [41] C. Wang, P. Xiao, J. Zhao, X. Zhao, Y. Liu, Z. Wang, Biomimetic synthesis of hydrophobic calcium carbonate nanoparticles via a carbonation route, Powder Technol. 170 (1) (2006) 31–35. [42] M.C. Sunny, P. Ramesh, H.K. Varma, Microstructured microspheres of hydroxyapatite bioceramic, J. Mater. Sci. Mater. Med. 13 (7) (2002) 623–632. [43] R. Jayakumar, V.V. Divya Rani, K.T. Shalumon, P.T. Kumar, S.V. Nair, T. Furuike, et al., Bioactive and osteoblast cell attachment studies of novel alpha- and beta-chitin membranes for tissue-engineering applications, Int. J. Biol. Macromol. 45 (3) (2009) 260–264. [44] J. Venkatesan, R. Pallela, I. Bhatnagar, S.K. Kim, Chitosan–amylopectin/hydroxyapatite and chitosan–chondroitin sulphate/hydroxyapatite composite scaffolds for bone tissue engineering, Int. J. Biol. Macromol. 51 (5) (2012) 1033–1042. [45] A. Sionkowska, J. Kozlowska, Properties and modification of porous 3-D collagen/hydroxyapatite composites, Int. J. Biol. Macromol. 52 (2013) 250–259. [46] J.P. Gleeson, N.A. Plunkett, F.J. O'Brien, Addition of hydroxyapatite improves stiffness, interconnectivity and osteogenic potential of a highly porous collagen-based scaffold for bone tissue regeneration, Eur. Cell. Mater. 20 (2010) 218–230. [47] K. Tuzlakoglu, C.M. Alves, J.F. Mano, R.L. Reis, Production and characterization of chitosan fibers and 3-D fiber mesh scaffolds for tissue engineering applications, Macromol. Biosci. 4 (8) (2004) 811–819. [48] J.M. Oliveira, M.T. Rodrigues, S.S. Silva, P.B. Malafaya, M.E. Gomes, C.A. Viegas, et al., Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissueengineering applications: scaffold design and its performance when seeded with goat bone marrow stromal cells, Biomaterials 27 (36) (2006) 6123–6137. [49] V. Karageorgiou, D. Kaplan, Porosity of 3D biomaterial scaffolds and osteogenesis, Biomaterials 26 (27) (2005) 5474–5491. [50] Y. Kuboki, Q. Jin, H. Takita, Geometry of Carriers Controlling Phenotypic Expression in BMP-induced Osteogenesis and Chondrogenesis, 2001. (S105-S15 p.). [51] C.M. Murphy, M.G. Haugh, F.J. O'Brien, The effect of mean pore size on cell attachment, proliferation and migration in collagen–glycosaminoglycan scaffolds for bone tissue engineering, Biomaterials 31 (3) (2010) 461–466. [52] H.E. Götz, M. Müller, A. Emmel, U. Holzwarth, R.G. Erben, R. Stangl, Effect of surface finish on the osseointegration of laser-treated titanium alloy implants, Biomaterials 25 (18) (2004) 4057–4064. [53] F. Zhao, Y. Yin, W.W. Lu, J.C. Leong, W. Zhang, J. Zhang, et al., Preparation and histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan–gelatin network composite scaffolds, Biomaterials 23 (15) (2002) 3227–3234. [54] Y. Zhang, M. Zhang, Three-dimensional macroporous calcium phosphate bioceramics with nested chitosan sponges for load-bearing bone implants, J. Biomed. Mater. Res. 61 (1) (2002) 1–8. [55] M. Ito, Y. Hidaka, M. Nakajima, H. Yagasaki, A.H. Kafrawy, Effect of hydroxyapatite content on physical properties and connective tissue reactions to a chitosan–hydroxyapatite composite membrane, J. Biomed. Mater. Res. 45 (3) (1999) 204–208. [56] N. Shanmugasundaram, P. Ravichandran, P.N. Reddy, N. Ramamurty, S. Pal, K.P. Rao, Collagen–chitosan polymeric scaffolds for the in vitro culture of human epidermoid carcinoma cells, Biomaterials 22 (14) (2001) 1943–1951. [57] J. Jung, Y. Zhao, Impact of the structural differences between α- and β-chitosan on their depolymerizing reaction and antibacterial activity, J. Agric. Food Chem. 61 (37) (2013) 8783–8789. [58] M.-K. Jang, B.-G. Kong, Y.-I. Jeong, C.H. Lee, J.-W. Nah, Physicochemical characterization of α-chitin, β-chitin, and γ-chitin separated from natural resources, J. Polym. Sci. A Polym. Chem. 42 (14) (2004) 3423–3432. [59] D.K. Youn, H.K. No, W. Prinyawiwatkul, Preparation and characteristics of squid pen β-chitin prepared under optimal deproteinisation and demineralisation condition, Int. J. Food Sci. Technol. 48 (3) (2013) 571–577. [60] B.D. Ulery, L.S. Nair, C.T. Laurencin, Biomedical applications of biodegradable polymers, J. Polym. Sci. B Polym. Phys. 49 (12) (2011) 832–864. [61] A.M. Martins, R.C. Pereira, I.B. Leonor, H.S. Azevedo, R.L. Reis, Chitosan scaffolds incorporating lysozyme into CaP coatings produced by a biomimetic route: a novel concept for tissue engineering combining a self-regulated degradation system with in situ pore formation, Acta Biomater. 5 (9) (2009) 3328–3336. [62] N. Nwe, T. Furuike, H. Tamura, The mechanical and biological properties of chitosan scaffolds for tissue regeneration templates are significantly enhanced by chitosan from Gongronella butleri, Materials 2 (2) (2009) 374–398. [63] S. Aiba, Studies on chitosan: 4. Lysozymic hydrolysis of partially N-acetylated chitosans, Int. J. Biol. Macromol. 14 (4) (1992) 225–228. [64] J. Gustavsson, M.P. Ginebra, E. Engel, J. Planell, Ion reactivity of calcium-deficient hydroxyapatite in standard cell culture media, Acta Biomater. 7 (12) (2011) 4242–4252. [65] X. Wang, G. Song, T. Lou, W. Peng, Fabrication of nano-fibrous PLLA scaffold reinforced with chitosan fibers, J. Biomater. Sci. 20 (14) (2009) 1995–2002. [66] X.-m. Pu, Q.-q. Yao, Y. Yang, Z.-z. Sun, Q.-q. Zhang, In vitro degradation of threedimensional chitosan/apatite composite rods prepared via in situ precipitation, Int. J. Biol. Macromol. 51 (5) (2012) 868–873. [67] A. Yoshida, T. Miyazaki, E. Ishida, M. Ashizuka, Preparation of bioactive chitosan–hydroxyapatite nanocomposites for bone repair through mechanochemical reaction, Mater. Trans. 45 (4) (2004) 994–998. [68] D. Xu-Liang, S. Gang, Z. Min-Li, C. Guo-Qiang, Y. Xiao-Ping, Poly(L-lactic acid)/hydroxyapatite hybrid nanofibrous scaffolds prepared by electrospinning, J. Biomater. Sci. 18 (1) (2007) 117–130. [69] C. Chatelet, O. Damour, A. Domard, Influence of the degree of acetylation on some biological properties of chitosan films, Biomaterials 22 (3) (2001) 261–268.