Nano Calcium

Article pubs.acs.org/IECR

Fabrication and Characterization of PLLA/Chitosan/Nano Calcium Phosphate Scaffolds by Freeze-Casting Technique Mahboubeh Jafarkhani,† Alireza Fazlali,† Fathollah Moztarzadeh,‡ Zoha Moztarzadeh,§ and Masoud Mozafari*,‡,∥ †

Department of Chemical Engineering, Faculty of Engineering, Arak University, P.O. Box 38156-8-8349, Arak, Iran Biomaterials Group, Faculty of Biomedical Engineering (Center of Excellence), Amirkabir University of Technology, P.O. Box 15875-4413, Tehran, Iran § Institute of Bioinformatic, Münster University, Münster, Germany ∥ Helmerich Advanced Technology Research Center, School of Material Science and Engineering, Oklahoma State University, Stillwater, Oklahoma 74106, United States ‡

ABSTRACT: In this research, nanocomposite scaffolds of chitosan/PLLA/nano calcium phosphate (average crystallite size of 16.5 nm) have been prepared via the freeze-casting method and then characterized. The effects of nano powder contents on the structure of scaffolds were investigated to provide an appropriate nanocomposite for bone tissue engineering applications. The results showed that the scaffolds had high porosity (up to 98%) with open pores of 80−380 μm in diameter. It was also shown that the porosity increased with decreasing nano powder content. Furthermore, the bioactive nano calcium phosphate was homogenously distributed within the polymeric matrix of scaffolds, which contained up to 40% of nano powder. Microstructure studies showed that the pores were distributed very well throughout the structures. This macropores structure with interconnected pores provides the properties of scaffolds required for bone tissue engineering applications.

1. INTRODUCTION Nowadays, nanocomposites made of biopolymers and bioactive materials have been used for application in tissue engineering.1 A wide range of nanocomposites scaffolds has been studied for use as porous scaffolds.2 The aim of making nanocomposites is achieving a better interaction between the bioactive inorganic phase and the organic phase, creating a tough material. Therefore, new types of biocompatible materials are fabricated and characterized for medical applications. Taking into account that natural bone consists of organic and inorganic materials, significant attention was paid to the polymer/ceramic nanocomposites.3 There are many different methods to prepare porous biodegradable scaffolds for tissue engineering, such as gasforming foam, three-dimensional printing, thermal-induced phase separation, electrospinning, and freezecasting.4 Freeze casting is an attractive method because it is an environmentally friendly and cost-effective technique. Also, complex ceramics with different pore morphologies can be prepared by the freezecasting method. Meanwhile, it is an effective way to avoid drying stress and shrinkage. A wide variety of ceramic materials, such as alumina, hydroxyapatite, tricalcium phosphate, Ni−YSZ, yttriastabilized zirconia, titanium dioxide, and silicon nitride, has already been prepared using this method.5 Poly-L-lactide (PLLA) is a synthetic and biodegradable polymer and has appropriate mechanical properties for biomedical applications. PLLA can be used in bioresorbable composites because of its biocompatibility and biodegradability. Due to the appropriate properties, PLLA has attracted attention from many researchers,6,7 but as a major drawback, it decreases © 2012 American Chemical Society

the local pH as a consequence of its hydrolytic degradation and elicited undesirable inflammatory and allergenic reactions.8−12 The combination of a natural polymer, such as chitosan (CHT), with PLLA can overcome some of the drawbacks of PLLA alone and lead to a material with interesting characteristics which can be used in biomedical engineering. CHT is a cationic linear natural polymer, biodegradable, and biocompatible and usually is obtained from chitin, which is an abundant biopolymer in nature. Also, chitosan has good characterictics such as bioadhesion, nontoxicity, nonimmunogenicity, antibacterial and antifungal effects, bioactivity, etc. In addition, CHT has a similar structure to glycosaminoglycan, one important constituent of ECM, which plays a key role in modulating cell morphology, cell functions, cell differentiation, and so forth.13,14 Besides, CHT is osteocompatible and osteoconductive and can accelerate wound healing as well as bone formation.15 Therefore, the physicochemical and biological properties of CHT make it suitable for preparation of scaffolds for new biomedical applications, and it has also been widely studied as an important tissue engineering scaffold material in recent years. Nevertheless, the poor mechanical strength of CHT limits its applications in the tissue engineering field.16−20 Chen et al.21 obtained a successful precipitation of PLLA− CHT blends from a DMSO:acetic acid solution in acetone and different compositions. The reported data indicated that this mixture would form a promising complex which is appropriate Received: Revised: Accepted: Published: 9241

January 19, 2012 June 2, 2012 June 15, 2012 June 15, 2012 dx.doi.org/10.1021/ie300173j | Ind. Eng. Chem. Res. 2012, 51, 9241−9249

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for tissue engineering. In another study, Temtem et al.22 reported precipitation of CHT membranes from an aqueous solution of dilute acetic acid. Also, the feasibility of processing CHT from organic solutions has been described, and the effect of different organic solvents on the morphology of the scaffolds was accessed.23 Calcium phosphates have been used as implant materials due to their chemical composition, which is similar to the inorganic component of bone which helps bone repair and regeneration. Because the chemical structure of calcium phosphate is similar to mineral bone, it can promote formation of bone-like apatite on its surface. It is a bioactive material, and its osteoconductivity is suitable for bone tissue repair or substitution applications.24 However, the brittle and rigid nature of calcium phosphate limits its role in biomedical applications. It should be noted that successful design of a bone substitute material requires an appreciation of the structure of bone composed of organic and inorganic materials. Nano calcium phosphate (Nano-CP) is highly promising for a wide range of orthopedic applications because of its excellent biocompatibility and bone replacement capability.25,26 Thus, porous composites made from CHT, PLLA, and calcium phosphate seem to mimic the morphology and properties of natural bone.27 Since the mechanical properties of porous scaffolds depend on the pore structure, a variety of methods of manufacturing strong porous materials have been reported. These include the replication of polymer foams by ceramic dip coating,28,29 foaming of aqueous ceramic powder suspensions,30,31 pyrolysis of preceramic precursors,32 and firing of ceramic powder compacts with pore-forming fugitive phases.33,34 However, none of these methods can completely satisfy all the necessary requirements, a controlled level of interconnected porosity combined with adequate mechanical properties. An example is the foam replication method, which can achieve very high volumes of porosity and excellent interconnectivity levels but characteristically results in poor mechanical properties due to defects generated during pyrolysis of the polymer foam template.29 An alternative approach is the freeze-casting method.35 This has proven to be an attractive method as it allows construction of reticulated porous structures. This avoids some of the inherent problems currently associated with other methods. The process consists of freezing a slurry or blend, which is usually aqueous based, in a mold at low temperatures, followed by demolding and vehicle removal by sublimation.35 Among different methods of fabricating porous materials freeze casting has gained much attention. This method usually produces good results with regard to the mechanical performance of the scaffolds. The freeze-casting method is quite simple and offers the possibility to produce many parts at once. This method has great potential, as it can be employed for fabrication of various kinds of porous materials. In addition, a variety of structures can be produced for a fixed material composition by adjusting the process parameters, such as the freezing temperature and time, freezing direction, freezing vehicle, etc.36 By varying the processing conditions, wide variations in the mechanical properties of materials prepared through the freeze-casting method can be achieved.37,38 Freeze casting consists in freezing a liquid slurry composed of a particular powder, solvent, and organic additive.39 During the freezing, the water slowly solidifies in a dendritic manner at a specific temperature, just below its solidification temperature. It was demonstrated recently that highly aligned porous structures can

provide increased compressive strength.40,42 To fabricate an aligned porous scaffold structure, an anisotropic thermal gradient is usually imposed through the use of a container with a relatively coldfinger at the bottom, which induces directional solidification of the solvent. In the case of the freeze-casting method using ice crystals as templates, unidirectional processing was commonly used as the fabrication method for aligned porous structures. Lots of research groups are now working on this method to improve it as an effective method for the future. They are trying to use different materials as crystal template or using a doubleside cooling procedure.43,44 In this study, 3D porous nanocomposite scaffolds made of nano-CP powders, CHT and PLLA, were developed by the freeze-casting method for bone tissue engineering applications and the effects of nano-CP powder contents on the properties of scaffolds were investigated.

2. EXPERIMENTAL PROCEDURE 2.1. Materials. PLLA (Mw = 1.0 × 105), chloroform, nano calcium phosphate (average particle size 100 nm), acetic acid, and chitosan (Mw = 2.5 × 105, degree of deacetylation = 90%) were obtained from Aldrich Sigma Co. Phosphate-buffered saline (PBS, 0.1 M, pH 7.4) was analytical grade and used as received. 2.2. Preparation of Nanocomposite Scaffolds. CHT solution with a concentration of 1.0 wt % was first prepared by dissolution of CHT in 2.0% (v/v) acetic acid aqueous solution. Then, 5.0% (v/v) PLLA solution in chloroform was slowly introduced into the solution, followed by adding drop by drop with additional stirring. The mixture was stirred at 50 °C for 1 h. The nano-CP/polymers mass ratios of 0, 10, 20, 30, and 40 (wt %) were selected, and an appropriate amount of nano-CP was added to the polymeric solution to make a homogeneous mixture. The newly obtained mixture was cast onto a mold with 6 mm height and 3 mm diameter and then placed in liquid nitrogen to solidify the mixture. The solidified mixture was transferred into a freeze dryer at a preset temperature of −50 °C. The samples were freeze dried at 0.5 mmHg for at least 3 days to completely remove the solvent. Finally, to cross-link CHT polymeric chains and enhance the biomechanical properties of the scaffolds for tissue repair, samples were soaked in a glutaraldehyde solution of 1% (w/v) for 24 h, and after soaking in a glutaraldehyde solution samples were carefully washed. The preparation process of these scaffolds is shown in Figure 1. 3. CHARACTERIZATION 3.1. SEM Analysis. The cross-section morphology of nanocomposite scaffolds and its pore size were evaluated with scanning electron microscopy (SEM). The scaffolds were coated with gold observed with SEM (EMITECH K450X, England) at an accelerating voltage of 15 kV. 3.2. Scaffolds Density Measurement. The apparent density of the samples (ρa) was measured by mercury pycnometry.45 A sample of weight Ws was placed in a pycnometer, which was completely filled with mercury and weighted to obtain Wsl. ρa was calculated according to eq 1 ρa =

W × ρHg W1 − Ws1 + Ws

(1)

where Wl is the weight of the pycnometer filled with mercury and ρHg is the density of mercury (13.5 g/cm3). 9242

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vacuum at 40 °C before their weights were measured. The weight loss (Wloss) of the scaffolds was calculated via following formula Wloss = [(Winit − Wdeg)/Winit] × 100%

where Winit is the initial weight of the sample before degradation and Wdeg the weight of the specimen after different periods of degradation. 3.6. FTIR Analysis. Scaffolds were analyzed by FTIR with a Bomem MB 100 spectrometer. For IR analysis, 1 mg of the scraped samples was mixed with 300 mg of KBr (infrared grade) and pelletized under vacuum condition. Then the pellets were analyzed in the range of 500−4000 cm−1 with 4 cm−1 resolution averaging 120 scans. 3.7. XRD Analysis. The resulting samples were analyzed by XRD with a Rigaku-Dmax 2500 diffractometer. This instrument works with voltage and current settings of 40 kV and 200 mA, respectively, and uses Cu Kα radiation (1.5405 Å). For qualitative analysis, XRD diagrams were recorded in the interval 20° ≤ 2θ ≤ 70° at a scan speed of 2°/min. 3.8. Mechanical Behavior. The mechanical behavior of the prepared nanocomposites scaffold was studied using an instrument (Roel−Amstel) following the ASTM F451-86 guideline. Cylindrical samples were cut to an appropriate size (3 mm in diameter and 5 mm in thickness), and the cross-section of the scaffolds was conducted with a drawing rate of 1 mm/min (load cell, 25 kN; resolution, 1 N). Each test has been repeated four times, and the average amount and standard deviation (SD) of related parameters such as E (Young's modulus) was measured. 3.9. Statistical Analysis. At least four specimens were tested in all experiments. Results are presented as a mean value with its standard error (SE).

Figure 1. Manufacturing process of hybrid nanocomposite scaffolds.

3.3. Porosity and Pore Size Measurement. The porosity was estimated from density measurements by the following equation46 ρ porosity % = 1 − scaffold ρcomponent (2) where ρscaffold and ρcompact are the densities of the prepared scaffold and of the bulk material .The densities of PLLA, CHT, and Nano-CP are ρ = 1.2, 0.3, and 1.55, respectively. The density of the scaffold was obtained from mass and volume measurements of the scaffolds. Using SEM, the pore size of the crosssection and transverse section and the average pore diameter of the samples were determined. 3.4. Swelling Index. Dry scaffolds were accurately weighted and placed into 15 mL tubes containing 10 mL of PBS solution at 37 °C. PBS solution with pH 7.4 was prepared by dissolving phosphate-buffered saline powders in deionized water. At predetermined time intervals (1, 3, 7, 14, and 21 day), the swollen scaffolds were wiped with soft paper tissue and weighed again. The degree of swelling for all samples at each time was calculated by the following equation SI = [(Wst − Wd)/Wd] × 100%

(4)

4. RESULT AND DISCUSSIONS The present work aimed to design and develop a nanocomposite scaffold fabricated from Nano-CP, natural polymer CHT, and synthetic polymer PLLA using the freeze-casting method. The nanocomposite scaffold would serve as a potential candidate for bone tissue engineering applications. The morphology and pore architecture of the scaffolds were evaluated by scanning electronic microscopy (SEM), and the blend scaffolds were also investigated for their miscibility using IR spectra. In vitro swelling and degradation study was performed in the phosphate buffer solution (PBS, pH 7.2) at a temperature of 37 °C. In order to evaluate the mechanical strength of the scaffolds a compression test was carried out, and the elastic modulus (E) and compressive modulus (σ) of nanocomposite scaffolds were determined. 4.1. Swelling Index of Scaffolds. In tissue engineering, fluid uptake is an important parameter which influences the chemical and physical characteristics of the scaffolds after and prior to cell seeding. Herein, swelling experiments were performed after cross-linking of the scaffolds. To study changes in hydrophilicity, the swelling index of scaffolds was measured. It was found that the swelling index was decreased by the weight percentage of Nano-CP. The dependence of swelling index of samples on the weight percentage of Nano-CP is presented in Figure 2. In addition, Figure 3 shows the effect of the polymeric component ratio on the swelling index of the scaffolds. It can be seen that when CHT increased the swelling index increased. Generally, CHT is swelled in PBS medium alone because it is a quite hydrophilic polymer due to its hydroxyl and amino groups

(3)

where Wd and Ws are the measured masses of dry and swollen scaffolds, respectively. For each reported value at least three replicate measurements were averaged. 3.5. Degradation in PBS Solutions. The scaffolds were immersed into the PBS solutions for degradation assessment by monitoring weight loss of scaffolds. The scaffolds were precisely weighed first and then immersed in the PBS solutions and incubated at 37 °C for various periods up to 21 days without refreshing the media. Four samples with identical masses were used for each specimen. After being incubated for various time durations, the scaffolds were taken out from the PBS media, washed with deionized water repeatedly, and then immersed in deionized water to remove the traces of water-soluble inorganic ions. Subsequently, they were frozen and dried again under 9243



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Figure 2. Dependence of Nano-CP weight percentage on the swelling behavior of scaffolds.

Figure 3. Dependence of the ratio of polymers on the swelling behavior of scaffolds.

on the backbone.47 Therefore, the reason may be that when the content of CHT is lower PLLA chains warped CHT chains up and when the proportion of CHT is higher the situation is reversed. Then, the second case certainly would render the scaffolds with a large swelling index. 4.2. In Vitro Degradation Behavior of Scaffolds. The degradation behavior of porous scaffolds plays a key role in the engineering process of a new tissue. The degradation rate of porous scaffolds affects cell vitality, cell growth, and even host response. The degradation behavior of prepared scaffolds is shown in Figures 4 and 5. In tissue engineering it is expected that the degradation rate of porous scaffolds is tunable in the repair or

regeneration process of tissue, which is achieved by adjusting the composition of the first and second phases. Therefore, in this paper, five porous scaffolds with different Nano-CP contents were characterized for in vitro degradation. It was observed that the scaffolds with a lower amount of Nano-CP have a higher weight loss rate within the period of the degradation time. It can be seen that degradation is decreased by the weight percentage of Nano-CP. From Figure 5, it can be observed that the degradation rate of the samples decreased when CHT:PLLA increased. Because CHT is generally degraded through lysozyme-mediated systems by randomly cleaving glucosamine and N-acetyl-glucosamine 9244



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Figure 4. dependence Nano-CP weight percentage on the degradation rate of scaffolds.

Figure 5. dependence of the ratio of polymers on the degradation rate of scaffolds.

onto the pore’s surface of the scaffolds, which is consistent with the higher roughness and lower porosity. The effect of the polymeric components ratio on the morphology of scaffolds is shown in Figure.7. Note that in this figure the interconnectivity of the scaffold with the lowest content of CHT is not compromised by the scaffold with the highest content of it. With increasing CHT concentration the volume of the pores was occupied with CHT network because of its low density. In addition, using SEM, the average pore size of the scaffolds was observed. Table 1 presents the changes of the porosity and the average pore size of the samples with the concentration of the components. It can be observed that the porosity and average

oligomers, degradation of CHT hardly occurs in PBS medium alone. Then the scaffolds with a lower content of CHT had a higher degradation rate in the buffer solution.48 4.3. SEM Observation. The microstructures of the crosssection of scaffolds were observed with SEM, as shown in Figure 4. The PLLA/CHT scaffolds showed a well-developed porous structure, consisting of open interconnected pores. It can be also inferred from Figure 6 that the interconnectivity of the initial scaffolds was not compromised by the presence of Nano-CP particles. With increasing Nano-CP mass percentage the volume of the pores was occupied and the average pore size decreased. The value of Nano-CP has a significant effect on the pore structure of the hybrid scaffolds. Nano-CP particles deposited 9245



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Figure 6. SEM micrographs of nanocomposite scaffolds: (a) without Nano-CP, (b) with 10% wt Nano-CP, (c) with 20% wt Nano-CP, and (d) with 40% wt Nano-CP.

Figure 7. SEM micrographs of the scaffolds without Nano-CP: (a) CHT:PLLA = 20:80, (b) CHT:PLLA = 50:50, and (c) CHT:PLLA = 80:20.

Table 1. Effect of the Concentration of the Components on Pore Parameters of Scaffolds CHT:PLLA 20:80

CHT:PLLA 50:50

CHT:PLLA 80:20

Nano-CP (wt %)

porosity (%)

pore size

porosity (%)

pore size

porosity (%)

pore size

0 10 20 30 40

98.0 ± 2.4 97.8 ± 3.1 97.3 ± 2.6 96.1 ± 2.3 95.8 ± 3.1

387 ± 8.7 337 ± 8.4 246 ± 6.8 210 ± 7.4 148 ± 8.1

97.0 ± 3.2 96.7 ± 2.6 96.0 ± 2.3 95.8 ± 2.7 95.0 ± 2.7

261 ± 7.6 216 ± 6.9 168 ± 6.6 122 ± 7.3 45 ± 7.1

95.0 ± 2.2 94.4 ± 2.5 93.9 ± 2.1 93.1 ± 2.2 92.8 ± 2.3

221 ± 6.5 181 ± 6.7 121 ± 6.8 80 ± 6.3 38 ± 6.7

several characteristic bands of PLLA were located at 795 (CH bend, crystalline-sensitive band), 1265 (dCsO stretch), and 3504−3631 cm−1 (OH stretch, end group). The main bands in the spectrum of CHT can be seen as follows: a broad and strong overlapped band at around 3455 cm−1 (OH and NH stretch); two weak bands at 2913 cm−1 (CH stretch); two bands at 1675 and 1589 cm−1 (amide I and amide II); bands at 1380, 1326, and 1255 cm−1 (deformation of CsCH3 and amide III); bands at 1153 and 1071 cm−1 (saccharide structure). It can be also observed

pore size of scaffolds decreased when the Nano-CP and CHT content increased. It is worth noting that the same trend was also reported by Duarte et al.49 Here, the porosity and pore size were determined to be approximately 92% −0.98% and 80−387 μm, respectively. 4.4. FTIR Analysis. FTIR spectroscopy is a common method for investigating the intermolecular and intramolecular interactions in polymers. Figure 8 represents FTIR spectra of PLLA, CHT, and Nano-CP and hybrid scaffolds. It can be seen that 9246



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Figure 8. FTIR spectra of PLLA, CHT, Nano-CP, and nanocomposite scaffold with 30 wt % Nano-CP.

that several noticeable changes occurred in the spectrum of scaffolds in comparison with the spectrum of each component. An original strong band of PLLA component at 1751 cm−1 for the ester group and two bands at 750 and 865 cm−1 for the CH bend became significantly weaker. In addition, spectra of nanocomposites revealed the presence of characteristic bands for the Nano-CP with a peak at 565 cm−1 indicative of a P−O band.50,51 The chemical bonding between CP, CHT, and PLLA indicated that the calcium and phosphate ions were homogenously trapped onto the CHT/PLLA matrix at a molecular scale. 4.5. XRD Analysis. The XRD patterns of the scaffolds containing different concentrations are shown in Figure 9. The XRD peaks for three composite samples indicated typical CHT, PLLA, and Nano-CP peaks and showed the increase in Nano-CP weight percentage increased its peak intensities. The average nanocrystallite size of Nano-CP was determined from the halfwidth of diffraction major peaks using the Debye−Scherrer formula in eq 5 D = kλ /β cos θ

(5)

where D is the crystallite diameter, k is a constant (shape factor, about 0.9), λ is the X-ray wavelength (1.5405 Å as mentioned before), β is the full width at half-maximum (fwhm) of the diffraction line, and θ is the diffraction angle. The average crystallite size was estimated at approximately 16.5 nm. 4.6. Compressive Mechanical Test. A compression test is appropriate to evaluate the mechanical strength of tissue

Figure 9. XRD spectra of various scaffolds on the weight percentage of Nano-CP.

engineering scaffolds. Table 2 presents the data obtained from mechanical compressive tests of the samples. According to this 9247



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Table 2. Mechanical Compressive Properties of Nanocomposite Samples CHT:PLLA 20:80

CHT:PLLA 50:50

E (MPa)

σ (MPa)

E (MPa)

σ (MPa)

E (MPa)

σ (MPa)

0 10 20 30 40

4.0 ± 1.1 5.7 ± 1.3 9.8 ± 1.6 11.3 ± 2.1 15.6 ± 2.5

0.54 ± 0.13 0.61 ± 0.11 0.65 ± 0.16 0.83 ± 0.12 0.92 ± 0.12

12.2 ± 2.3 14.7 ± 2.2 23 ± 2.7 33.2 ± 2.4 53.1 ± 2.6

0.65 ± 0.10 0.77 ± 0.13 0.94 ± 0.13 1.14 ± 0.17 1.76 ± 0.14

15.5 ± 2.1 24.3 ± 3.2 57 ± 3.3 68.2 ± 3.2 82.6 ± 3.4

0.68 ± 0.15 0.87 ± 0.12 0.91 ± 0.16 0.123 ± 0.15 0.148 ± 0.17

(5) Zhang, Y.; Zuo, K.; Zeng, Y. Effects of gelatin addition on the microstructure of freeze-cast porous hydroxyapatite ceramics. Ceram. Int. 2009, 35, 2151−2154. (6) Martin, O.; Averous, L. New Polylactide/Layered Silicate Nanocomposite: Nanoscale Control over Multiple Properties. Polymer 2001, 42, 6209. (7) Yamane, H.; Sasai, K. Effect of the addition of poly(D-lactic acid) on the thermal property of poly(L-lactic acid). Polymer 2003, 44, 2569. (8) Zhang, X. F.; Hua, H.; Shen, X. Y.; Yang, Q. Invitro degradation and biocompatibility of poly(l-lactic acid)/chitosanfibercomposites. Polymer 2007, 48 (4), 1005−1011. (9) Lu, L.; Peter, S. J.; Lyman, M. D.; Lai, H. L.; Leite, S. M.; Tamada, J. A.; Vacanti, J. P.; Langer, R.; Mikos, A. G. In vitro degradation of porous poly(L-lactic acid) foams. Biomaterials 2000, 21, 1595−1605. (10) Yuan, Y.; Zhang, P.; Yang, Y.; Wang, X.; Gu, X. The interaction of Schwann cells with chitosan membranes and fibers in vitro. Biomaterials 2004, 25, 4273−4278. (11) Seol, Y. J.; Lee, J. Y.; Park, Y. J.; Lee, Y. M.; Ku, Y.; Rhyu, C. I.; Lee, S. J.; Han, S. B.; Chung, C. P. Chitosan sponges as tissue engineering scaffolds for bone formation. Biotechnol. Lett. 2004, 26, 1037−1041. (12) Sundararajan, V. M.; Howard, M. Porous chitosan scaffolds for tissue engineering. Biomaterials 1999, 20, 1133−1142. (13) Nishikawa, H.; Ueno, A.; Nishikawa, S.; Kido, J.; Ohishi, M.; Inoue, H.; Nagata, T. Sulfated glycosaminoglycan synthesis and its regulation by transforming growth factor-b in rat clonal dental pulp cells. J. Endod. 2000, 26, 169−171. (14) Sauced, S.; Watanabe, K.; Yamaguchi, Y. Differentiation/ regeneration of oligodendrocytes entails the assembly of a cellassociated matrix. Int. J. Dev. Neurosci. 2000, 18, 705−720. (15) Wang, J. W.; Hon, H. Sugar-mediated chitosan/poly(ethyleneglycol)-b-dicalcium pyrophospate composite: Mechanical andmicrostructural properties. J. Biomed. Mater. Res. A 2003, 64, 262− 272. (16) Ding, Z.; Chen, J. N.; Gao, S. Y.; Chang, J. B.; Zhang, J. F.; Kang, E. T. Immobilization of chitosan onto poly-L-lactic acid film surface by plasma graft polymerization to control the morphology of fibroblast and liver cells. Biomaterials 2004, 25, 1059−1067. (17) Chen, C.; Dong, L.; Cheung, M. K. Preparation and characterization of biodegradable poly(L-lactide)/chitosan blends. Eur. Polym. J. 2005, 41, 958−966. (18) Madihally, S. V.; Matthew, H. W. Porous chitosan scaffolds for tissue engineering. Biomaterials 1999, 20, 1133−1142. (19) Suh, J. K. F.; Matthew, H. W. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: A review. Biomaterials 2000, 21, 2589−2598. (20) Zhang, Y.; Zhang, M. Cell growth and function on calcium phosphate reinforced chitosan scaffolds. J. Mater. Sci. Mater. Med. 2004, 15, 255−260. (21) Chen, C.; Dong, L.; Cheung, M. K. Preparation and characterization of biodegradable poly(l-lactide)/chitosan blends. Eur. Polym. J. 2005, 41 (5), 958−966. (22) Temtem, M.; Silva, L. M. C.; Andrade, P. Z.; dosSantos, F.; daSilva, C. L.; Cabral, J. M. S.; Abecasis, M. M.; Aguiar-Ricardo, A. Supercritical CO2 generating chitosan devices with controlled morphology. Potential application for drug delivery and mesenchymal stem cell culture. J. Supercrit. Fluids 2009, 48 (3), 269−277. (23) Duarte, A. R. C.; Mano, J. F.; Reis, R. L. Chitosan scaffolds prepared by supercritical assisted phase inversion for tissue engineering

table, the elastic modulus (E) and compressive modulus (σ) of nanocomposite scaffolds enhanced obviously along with increasing the weight percentage of the Nano-CP and CHT content. Ying Wan et al.52 reported that the porosity has a significant influence the mechanical properties of porous scaffolds. In our study, the results represented in Table 2 indicated that E and σ both increased progressively with the increment of the content of Nano-CP and CHT, and consequently, the decrease of porosity of the scaffolds is basically in agreement with published results.53,54 It is worth mentioning that σ and E of the samples were in the range of spongy bone, and this comparison indicated that the properties of the prepared nanocomposites were close to natural spongy bone.2

6. CONCLUSIONS In this study, nanocomposite scaffolds were prepared by the freeze-casting method for tissue engineering applications. The results have shown that the prepared 3D nanocomposite scaffolds were highly porous and the elastic modulus of the scaffolds was comparative to the natural spongy bone. Also, it is found that the swelling ability of the scaffolds was reduced when the Nano-CP and CHT content increased. An increased weight percentage of Nano-CP and CHT decreased the porosity and increased the mechanical strength. The results obtained from a degradation test in PBS showed that increasing the Nano-CP and CHT content inside the scaffolds decreased the degradation of the scaffolds.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +1 918 594 8634. Fax: +1 270 897 1179. E-mail: masoud. [email protected]. Notes

The authors declare no competing financial interest.

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CHT:PLLA 80:20

Nano-CP (wt %)

REFERENCES

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