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chitosan composite scaffolds for bone tissue engineering
Materials Science and Engineering C 54 (2015) 20–25
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Preparation and characterization of nano-sized hydroxyapatite/alginate/chitosan composite scaffolds for bone tissue engineering Hye-Lee Kim a, Gil-Yong Jung b, Jun-Ho Yoon a, Jung-Suk Han b,c, Yoon-Jeong Park b,d, Do-Gyoon Kim e, Miqin Zhang f, Dae-Joon Kim a,⁎ a
Department of Advanced Materials Engineering, Sejong University, 209 Neungdong-ro, Gwangjin-gu, Seoul 143-747, South Korea Interdisciplinary Program for Bioengineering, Seoul National University, Seoul 152-742, South Korea c Department of Prosthodontics and Dental Research Center, School of Dentistry, Seoul National University, 101 Daehak-no, Jongno-gu, Seoul 110-749, South Korea d Department of Craniomaxillofacial Reconstructive Science and Dental Research Institute, School of Dentistry, Seoul National University, 101 Daehak-no, Jongno-gu, Seoul 110-749, South Korea e Division of Orthodontics, College of Dentistry, The Ohio State University, Columbus, OH 43210, USA f Department of Materials Science & Engineering, University of Washington, Seattle, 302 L Roberts Hall, WA 98195-2120, USA b
a r t i c l e
i n f o
Article history: Received 29 July 2014 Received in revised form 17 February 2015 Accepted 21 April 2015 Available online 22 April 2015 Keywords: Hydroxyapatite Chitosan Alginate Porous scaffold Bone tissue engineering
a b s t r a c t The aim of this study was to develop chitosan composite scaffolds with high strength and controlled pore structures by homogenously dispersed nano-sized hydroxyapatite (nano-HAp) powders. In the fabrication of composite scaffolds, nano-HAp powders distributed in an alginate (AG) solution with a pH higher than 10 were mixed with a chitosan (CS) solution and then freeze dried. While the HAp content increased up to 70 wt.%, the compressive strength and the elastic modulus of the composite scaffolds significantly increased from 0.27 MPa and 4.42 MPa to 0.68 MPa and 13.35 MPa, respectively. Higher content of the HAp also helped develop more differentiation and mineralization of the MC3T3-E1 cells on the composite scaffolds. The uniform pore structure and the excellent mechanical properties of the HAp/CS composite scaffolds likely resulted from the use of the AG solution at pH 10 as a dispersant for the nano-HAp powders. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Scaffolds for bone tissue engineering, which are a promising approach for the treatment of defective and lost bone, are required to have osteoconductivity and biodegradability in addition to their three dimensional (3D) interconnected porous networks [1]. It has been indicated that a scaffold pore size in the range of 100–400 μm is favorable for cell colonization, proliferation and penetration [2,3]. Chitosan (CS), a deacetylated derivative of chitin, has long been considered one of the most attractive natural biopolymer matrices for tissue engineering because of its biocompatibility, biodegradability and structural similarity to glycosaminoglycans [4–6]. Nevertheless, the use of CS has been limited to non-load-bearing applications due to its poor mechanical properties [7]. Unlike numerous other reinforcing materials, hydroxyapatite (Ca10(PO4)6(OH)2: HAp) significantly improves the mechanical properties of CS scaffolds because it has a crystal structure similar to that of the inorganic component of natural bone [8–10]. In particular, nano-sized HAp (nano-HAp) morphologically resembles
⁎ Corresponding author. E-mail address: [email protected] (D.-J. Kim).
http://dx.doi.org/10.1016/j.msec.2015.04.033 0928-4931/© 2015 Elsevier B.V. All rights reserved.
biological apatite [11] such that scaffolds composed of HAp and CS (HAp/CS composite scaffolds) have enhanced osteoconductive, osteoinductive and osteogenic properties [10]. HAp/CS composite scaffolds have been fabricated by several methods, such as the mechanical mixing of HAp and CS [12,13], the in situ co-precipitation of HAp [9,14] and the in situ precipitation of HAp into CS [15]. The HAp in the HAp/CS composite scaffolds that were prepared by mechanical mixing of constituents were aggregated and formed large clusters in the scaffolds because of the partial dissolution of nano-HAp particles in the acidic CS solution [12]. In contrast, the preparation of HAp/CS composites by the in situ precipitation of calcium phosphate led to an uncontrollable Ca/P ratio and crystallinity of HAp [16]. It has also been reported that the pore structure of HAp/CS composite scaffolds collapsed as the HAp content increased higher than 30 wt.% due to the agglomeration of HAp particles [13]. When HAp particles in the HAp/CS composite scaffolds were prepared by mechanical mixing, a more extreme heterogeneity of pore structure and low compressive strength were observed because the homogeneous dispersion of nano-HAp within the scaffolds was difficult to accomplish due to particle agglomeration [5]. Thus, uniform dispersion of nano-HAp in the composite scaffolds is a prerequisite to produce controlled pore structure and strength of scaffolds.
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It has been reported that alginate (AG), which is derived primarily from brown seaweed and bacteria, forms stable hydrogels through the ionic interaction between the carboxyl group of AG and Ca2 + [17]. Teng et al. reported that AG promoted the preferential synthesis of HAp oriented in the c direction when composite scaffolds were fabricated by the co-precipitation method [18]. The results indicated a strong ionic interaction between AG and Ca2+, leading to the formation of a specific stereo-chemical arrangement and charge distribution [18]. Furthermore, the combination of AG and CS forms a polyelectrolyte complex (PEC) between the carboxyl groups of AG and protonated amines of CS [19,20]. The formation of a PEC alleviates the shortcomings of CS, such as its poor mechanical properties and structural instability after transplantation [19,20]. Thus, it is expected that AG interacts with both HAp and CS in HAp/CS composites. In this context, AG may be utilized as a dispersant of nano-HAp particles in CS solution since biocompatibility of commercial dispersants for inorganic powders have not been ensured. To the best knowledge of the present authors, there has been no attempt to apply AG as a dispersant for optimization of the pore structure and the mechanical properties of HAp/CS composites. In this study, nano-HAp/AG/CS composite scaffolds were fabricated utilizing AG as a dispersing agent for nano-HAp particles and freezedrying the solution mixtures of HAp, AG and CS. The pore morphologies and mechanical properties of the composite scaffolds were determined, and osteoblast differentiation and mineralization on the composite scaffolds were investigated to evaluate the bioactivity and osteoconductivity of the composite scaffolds.
2. Materials and methods Nano-HAp was hydrothermally synthesized using 1 M Ca(NO3)2·4H2O (Sigma-Aldrich, St. Louis, MO, USA) and 1 M H3PO4 (Sigma-Aldrich). The solutions were mixed to achieve the Ca/P ratio of 1.67. Then, NH3 (30%, Sigma-Aldrich) was added into the solution mixture to precipitate the hydroxide containing Ca2+ and PO3− 4 . The coprecipitates were aged for 24 h at 80 °C and were then placed in an autoclave for 2 h at 180 °C. The hydrothermally synthesized powders were washed three times with deionized water to remove any unreacted substances. A 4 wt.% CS (practical grade, N 75% deacetylated, MW = 190 kDa– 375 kDa, Sigma-Aldrich) solution was prepared by dissolving the CS in deionized water with 1 wt.% acetic acid and the pH of CS solution was 5.2. In addition, 2 and 3.75 wt.% AG (alginic acid from brown seaweed, MW = 80 kDa–120 kDa, Sigma-Aldrich) solutions were prepared for the dispersion of nano-HAp particles and to form a PEC with CS, respectively, by dissolving the AG in deionized water. Prior to the dispersion of the nano-HAp powder, the pH of the 2 wt.% AG solution was adjusted to 9.5–10.0 by adding 5 wt.% NH4OH solution. During the dispersion of the nano-HAp powder in the 2 wt.% AG solution the pH of the slurry was maintained at about 9.0 using the NH4OH solution. The dispersion was facilitated by a magnetic stirrer for 1 h and a subsequent sonication for 2 h. The dispersed nano-HAp slurry was mixed with the 3.75 wt.% AG solution using a planetary mixer (ARM-300, Thinky, Laguna Hills, CA, USA) at 2000 rpm for 5 min. HAp/AG/CS composite scaffolds were prepared by mixing the HAp/AG mixture with the 4 wt.% CS solution using the planetary mixer. The nano-HAp content of the composite scaffolds ranged from 10 to 70 wt.%. The pH of HAp/AG/CS solutions was 6.3. Following mixing, the mixture was cast in a 24-well plate and refrigerated at 4 °C for 12 h. During the refrigeration, the plate was covered with paraffin film to prevent water from evaporating. The plate was frozen at − 20 °C overnight and then lyophilized for 24 h with a freeze drier (Labconco FreeZone 6Plus Freeze Drier, Labconco, Kansas, MO, USA) under vacuum with the collector temperature set at −89 °C to sublimate the ice from the composite scaffolds. Each composite scaffold was crosslinked by 0.2 M CaCl2 solution for 15 min and was then washed three times with deionized water and immersed in deionized
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Fig. 1. Transmission electron microscopic images of the nano-HAp particles.
water to remove unbound CaCl2. Wet composite scaffolds were refrozen and lyophilized following the procedure described above. The prepared nano-HAp, their morphology and particle size were examined using transmission electron microscopy (TEM). This was performed by a JEOL 2010 (Tokyo, Japan), using an accelerating voltage of 200 kV and a copper electron microscopic grid supported by a porous carbon (mesh size 300) film. The zeta potentials of nano-HAp particles suspended in deionized water were measured using electrophoretic light scattering (Otsuka Electronics, Hirakata, Osaka, Japan). The infrared spectra of the composite scaffolds were obtained by Fourier transform infrared (FT-IR) attenuated total reflection spectroscopy (Thermo Fisher Scientific Inc., Franklin, MA, USA). The porosities of the composite scaffolds were measured using the liquid displacement method [21] and calculated by: Porosityð%Þ ¼
ðW 1 −W 0 Þ 100 ρ V0
where W1, W0, ρ and V0 are the weight of the composite scaffolds saturated with ethanol, the dry weight of the scaffolds, the density of the ethanol and the initial volume of the composite scaffolds, respectively. Morphometric analysis of composite scaffolds was carried out using microcomputed tomography (micro-CT, Skyscan, Aartselaar, Belgium). The microstructures of cross-sections of the composite scaffolds were observed by scanning electron microscopy (SEM, JEOL, Tokyo, Japan). The mechanical properties of composite scaffolds were determined for circular disk specimens (12 mmØ × 10 mm, n = 5) under compression using a universal testing machine (Instron, Norwood, MA, USA) at a crosshead speed of 1 mm/min until the composite scaffolds reached a 30% reduction
Fig. 2. Effect of pH on the zeta potentials of the nano-HAp particles.
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Fig. 3. FT-IR spectra of (a) alginate, (b) nano-HAp particles and (c) alginate containing 50 wt.% nano-HAp particles.
in height. The elastic modulus was measured using the linear slope of stress–strain curve. To evaluate the cell behaviors on composite scaffolds, newborn mouse calvaria-derived MC3T3-E1 (subclone 4) preosteoblastic cells (American Type Culture Collection, Manassas, VA, USA) were used. MC3T3-E1 cells were cultured in Alpha Minimum Essential Medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) as well as 100 U/ml penicillin and streptomycin (Invitrogen). Media were further supplemented with 50 μg/ml ascorbic acid (Invitrogen) and 5 mM β-glycerophosphate (Invitrogen) for differentiation. MC3T3-E1 cells were seeded onto each scaffold at a density of 1.0 × 105 cells. Composite scaffolds (12 mmØ × 3 mm) were sanitized in ethanol 70% and then washed with deionized water sterilized by autoclaving at 121 °C. Cell differentiation and calcium-rich deposits by cells on composite scaffolds were determined by alkaline phosphatase (ALP) expression using a QuantiChrom ALP kit (BioAssay Systems, Hayward, CA, USA) and 0.04 M alizarin red S (Sigma-Aldrich, pH 4.2). Data were evaluated for statistical significance using one-way analysis of variance, and results were expressed as means ± standard deviations. Experiments were repeated three times unless otherwise specified. In all analyses, comparisons among means were determined using Tukey's post hoc test, and a p-value of less than 0.05 was considered statistically significant. 3. Results and discussion The morphology of the hydrothermally prepared nano-HAp particles is shown in Fig. 1. The particle size was 57 ± 13 nm and a
Fig. 5. FT-IR spectra of (a) alginate containing 50 wt.% nano-HAp particles (b) chitosan containing 85 wt.% nano-HAp particles and (c) composite of 70 wt.% nano-HAp/18 wt.% alginate/12 wt.% chitosan.
considerable number of particles were entangled to form agglomerates. It was difficult for the nanoparticles to be dispersed homogeneously due to their extremely high surface areas [22]. Thus, they were weakly agglomerated in order to reduce surface energy. The tendency of the agglomeration to occur in solution depended on the pH and was characterized by zeta potential [23]. The zeta potentials of the nano-HAp particles dispersed in deionized water are shown in Fig. 2. The point of zero charge of the nano-HAp particles was a pH of about 6.3 and the negative zeta potential increased with the increase in pH, which was beneficial to deagglomeration. This result indicates that the negatively charged nano-HAp particles were electrostatically repulsive in an alkaline solution, leading to the dispersion of the nano-HAp particles. The dispersion of the nano-HAp particles can be further improved by mechanical mixing with an AG solution that is soluble at high pH [18], since the negatively charged carboxylates of the AG are likely to form a complex with Ca2+ in the HAp to provide steric hindrance for the stabilization. Indeed, the nano-HAp particles, mixed with the AG solution at pH 10 were well dispersed, and a probable reaction between the HAp particles and AG was observed by FT-IR, as shown in Fig. 3, where HAp and AG are in the ratio of 1:1. The AG spectra show the characteristic absorption bands of COO− (1597 cm− 1, 1408 cm− 1) and C–O–C stretching (1022 cm− 1) [24,25]. The nano-HAp spectra show a strong absorption band at 1019 cm−1 corresponding to the v3 −1 band of PO3− and 1087 cm−1, which re4 and the shoulders at 962 cm flect the v1 band of P–O stretching [25,26]. The AG scaffold containing 70 wt.% nano-HAp particles exhibits a prominent band around 1009 cm−1 that may be caused by a shift of either the C–O–C in AG or the PO3− in HAp. It has been reported that C–O–C in AG immersed in 4 CaCl2 shifts towards lower wave numbers as Ca2 + content increases [25]. The shift to lower frequencies indicates weakening in C–O–C bonds, most likely due to these bonds being shared with Ca2+. Thus, the bond at 1009 cm−1 may result from a shift of the C–O–C in AG due to strong ionic interactions between C–O–C and Ca2+ in the nanoHAp. Furthermore, the HAp/gelatin nanocomposites, fabricated by coTable 1 Properties of alginate–chitosan composite scaffolds containing various nanohydroxyapatites.
Fig. 4. FT-IR spectra of (a) chitosan, (b) nano-HAp particles and (c) chitosan mixed with 85 wt.% nano-HAp particles.
HAp contents in wt.% in AG/CS composite scaffolds
Characteristics of the composite scaffolds Porosity (%)
Scaffold surface/volume ratio (mm−1)
0 10 30 50 70
78 ± 4.64 84 ± 5.45 81 ± 4.56 79 ± 4.42 81 ± 5.15
164.71 ± 23.64 187.18 ± 21.14 171.62 ± 30.41 181.29 ± 28.26 176.68 ± 26.84
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Fig. 6. Micro-CT images (a, b) and 3D reconstructions (c, d) of the alginate/chitosan composite scaffolds (a, c) and the alginate/chitosan composite scaffolds containing 70 wt.% nano-HAp (b, d).
precipitation in the presence of AG, demonstrated a strong ionic interaction between HAp and AG to form a specific stereo-chemical arrangement [18]. The interaction has been rationalized by the “egg-box model”, where Ca2+ in nano-HAp is located in the electronegative cavities of guluronic acids of AG. Thus, the dispersion of nano-HAp in AG solution at pH 10 is likely achieved both by the electrostatic repulsion and by the formation of ionic complexes between nano-HAp and AG. The characteristic FT-IR spectra of CS appeared at 1645, 1556, 1410, 1375 and 1022 cm−1, corresponding to amide I, amide II, CH2 bending,
CH3 symmetrical deformation and C–O–C, respectively, as seen in Fig. 4 [1,26]. In the CS composite scaffolds containing 70 wt.% nano-HAp particles, the bands for amide I, amide II and CH2 bending shifted to lower wave numbers of 1637, 1548 and 1403 cm−1, respectively, indicating chemical interaction between either the amino groups in CS and the Ca2 + in nano-HAp or between the amino groups and PO34 − [5]. Comparison of the FT-IR spectra for the composite scaffolds of 85 wt.% HAp/15 wt.% CS, 50 wt.% HAp/50 wt.% AG and 70 wt.% HAp/ 18 wt.% AG/12 wt.% CS in Fig. 5 strongly supports that the interaction
Fig. 7. Microstructures of cross-sectional images of alginate/chitosan composite scaffolds containing various HAp contents as observed by SEM. The nano-HAp contents in the composite scaffolds were: (a) 0 wt.%, (b) 10 wt.%, (c) 30 wt.%, (d) 50 wt.% and (e) 70 wt.%. Original magnifications were 50×.
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between AG and nano-HAp is dominant in the ternary composite and that the interaction contributes to the dispersion of nano-HAp in the ternary composite. Scaffolds used in tissue engineering should have a high porosity structure to provide the sites for cell attachment, proliferation and differentiation [1–3]. The porosity and other data on the pore structure of CS/AG composite scaffolds containing various amounts of nano-HAp are listed in Table 1. The porosity of the composite scaffolds ranged from 78% to 84% and was independent of nano-HAp content; no significant differences in the surface to volume ratio were observed. Scaffolds for tissue engineering require an interconnected pore structure in order to facilitate the migration of invading cells into scaffolds [1]. Micro-CT images demonstrated that the AG/CS composite scaffolds containing various amounts of nano-HAp had internally interconnected porous structures, as shown in Fig. 6. The ternary composite scaffolds exhibited anisotropic porous networks with pore sizes ranging from 100 to 400 μm. The high surface area to volume ratios of the composite scaffolds, calculated using the CTAn program embedded in the micro-CT system, range from 164.71 mm−1 to 187.18 mm−1, may be favorable for cell adhesion to scaffolds and bone tissue regeneration [2,3]. The ternary composite scaffolds showed no significant differences in interconnected pore structure depending on the nano-HAp content. The pore structure of the composite scaffolds was sufficient for tissue engineering, since the scaffolds require pores larger than 100 μm in size and porosity of about 80%, considering cell size, migration requirements, and transport. The microstructure of the composite scaffolds was observed by SEM and the results are shown in Fig. 7. The ternary composite scaffold shows homogeneously distributed pores and continuous pore channels.
Fig. 8. Comparison of (a) compressive strengths and (b) elastic modulus of alginate/chitosan composite scaffolds containing various nano-HAp contents (n = 5).
The pore structures of the composite scaffolds were not influenced by the content of the nano-HAp. It has been reported that high amounts of HAp in polymer solution often result in a decrease in porosity, irregularly sized pores and the collapse of pore structure [13,27,28]. This is probably because the rheological behavior of polymer solutions containing HAp is inhomogeneous due to the agglomerated and nonuniform nature of the HAp distributed in suspension. In the fabrication of scaffolds by freeze-drying, the high viscosity of the suspension prevents water migration for the growth of ice crystals, and thus reduces the pore size of the scaffolds [15,29]. Hence, the uniform pore structure of the ternary composite scaffolds in Fig. 7 demonstrates that the nano-HAp particles, dispersed in AG solution at pH 10 prior to being mixed with CS, did not alter significantly the rheological behavior of the solution mixtures. The compressive strengths and elastic modulus of the composite scaffolds containing various amounts of nano-HAp are shown in Fig. 8. The compressive strength increased from 0.27 MPa to 0.68 MPa and the elastic modulus improved from 4.4 MPa to 13.4 MPa with increasing nano-HAp content in the composite scaffolds up to 70 wt.% nano-HAp. The improved mechanical properties in CS scaffolds containing HAp are thought to result from chemical interactions between the amino groups in CS and the Ca2+ in the nano-HAp or between the amino groups and PO3− 4 [5]. The interactions between CS and nano-HAp were observed in the ternary composite scaffolds as shown in Figs. 3–5. Thus, the mechanical properties in the ternary composite scaffolds were improved mainly by the strong interaction between CS and nano-HAp, which was facilitated by the uniform distribution of nano-HAp through the ionic
Fig. 9. Biocompatibility of alginate/chitosan composite scaffolds containing various nanoHAp contents. (a) ALP assays for cell differentiation and (b) alizarin Red-S staining for mineralization were conducted after MC3T3-E1 cells were incubated for the designated times on each composite scaffold. Data are presented as means ± standard deviations of three separate experiments. *p b 0.05; **p b 0.01.
H.-L. Kim et al. / Materials Science and Engineering C 54 (2015) 20–25
interaction between AG and nano-HAp. In addition, the uniform pore structure with thick pore walls contributed to the improved compressive strengths and elastic modulus. As demonstrated by Parminik et al., the increase in HAp/CS composite scaffold mechanical properties ended at a HAp content of 40 wt.% [12] when composites were prepared by mechanical mixing of the constituents. This is probably due to insufficient dispersion of HAp particles in the composite scaffolds, which resulted in nonuniform pore morphology. Osteoblasts secrete and mineralize the bone matrix during differentiation, and the differentiation phase of osteoblasts is determined using ALP activity and mineralization. ALP activity was measured over 14 and 21 days of culture as a marker of the matrix maturation phase during the differentiation of osteoblasts on the ternary composite scaffolds (Fig. 9(a)). On the ternary composite scaffolds, APL activity increased significantly after 14 and 21 days of culture as compared to the activity on AG/CS scaffolds. The composite scaffolds containing 70% nano-HAp showed a 1.45-fold increase in enzyme activity after 21 days of cultivation in comparison with that of the AG/CS scaffolds. Similar to the ALP activity results, the mineralization of osteoblasts on the composite scaffolds was enhanced on the ternary scaffolds as compared to the AG/CS scaffolds (Fig. 9(b)). Since the increase in extracellular Ca2+ improved osteoblast differentiation, the Ca2+ released from HAp contributed to the increase in osteoblast differentiation due to the increase in osteopontin and bone sialoprotein expression through L-type Ca channels, which triggered the Ca2+/calmodulin-dependent protein kinase 2 pathway [30]. 4. Conclusions An alginate solution at pH 10 can be utilized as a bio-inspired dispersant for nano-sized HAp particles as result of both electrostatic repulsion and steric hindrance. The nano-HAp/chitosan composite scaffolds containing alginate demonstrated uniform pore structure even with a nano-HAp content of 70 wt.%. The even pore morphology contributed to an increase in the compressive strength and elastic modulus of composite scaffolds, which increased as a function of nano-HAp content. The composite scaffolds containing nano-HAp, as compared to the chitosan/ alginate composite scaffolds, exhibited improved osteoblastic differentiation for bone regeneration, as determined by ALP activity and the mineralization of alginate/chitosan scaffolds. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2010-0024260). References [1] I. Manjubala, S. Scheler, J. Bössert, K.D. Jandt, Mineralization of chitosan scaffolds with nano-apatite formation by double diffusion technique, Acta Biomater. 2 (2006) 75. [2] A.G. Mikos, G. Sarakinos, M.D. Lyman, D.E. Ingber, J.P. Vacanti, R. Langer, Prevascularization of porous biodegradable polymers, Biotechnol. Bioeng. 42 (1993) 716.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Preparation and characterization of nano-sized hydroxyapatite/alginate/chitosan composite scaffolds for bone tissue engineering Hye-Lee Kim a, Gil-Yong Jung b, Jun-Ho Yoon a, Jung-Suk Han b,c, Yoon-Jeong Park b,d, Do-Gyoon Kim e, Miqin Zhang f, Dae-Joon Kim a,⁎ a
Department of Advanced Materials Engineering, Sejong University, 209 Neungdong-ro, Gwangjin-gu, Seoul 143-747, South Korea Interdisciplinary Program for Bioengineering, Seoul National University, Seoul 152-742, South Korea c Department of Prosthodontics and Dental Research Center, School of Dentistry, Seoul National University, 101 Daehak-no, Jongno-gu, Seoul 110-749, South Korea d Department of Craniomaxillofacial Reconstructive Science and Dental Research Institute, School of Dentistry, Seoul National University, 101 Daehak-no, Jongno-gu, Seoul 110-749, South Korea e Division of Orthodontics, College of Dentistry, The Ohio State University, Columbus, OH 43210, USA f Department of Materials Science & Engineering, University of Washington, Seattle, 302 L Roberts Hall, WA 98195-2120, USA b
a r t i c l e
i n f o
Article history: Received 29 July 2014 Received in revised form 17 February 2015 Accepted 21 April 2015 Available online 22 April 2015 Keywords: Hydroxyapatite Chitosan Alginate Porous scaffold Bone tissue engineering
a b s t r a c t The aim of this study was to develop chitosan composite scaffolds with high strength and controlled pore structures by homogenously dispersed nano-sized hydroxyapatite (nano-HAp) powders. In the fabrication of composite scaffolds, nano-HAp powders distributed in an alginate (AG) solution with a pH higher than 10 were mixed with a chitosan (CS) solution and then freeze dried. While the HAp content increased up to 70 wt.%, the compressive strength and the elastic modulus of the composite scaffolds significantly increased from 0.27 MPa and 4.42 MPa to 0.68 MPa and 13.35 MPa, respectively. Higher content of the HAp also helped develop more differentiation and mineralization of the MC3T3-E1 cells on the composite scaffolds. The uniform pore structure and the excellent mechanical properties of the HAp/CS composite scaffolds likely resulted from the use of the AG solution at pH 10 as a dispersant for the nano-HAp powders. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Scaffolds for bone tissue engineering, which are a promising approach for the treatment of defective and lost bone, are required to have osteoconductivity and biodegradability in addition to their three dimensional (3D) interconnected porous networks [1]. It has been indicated that a scaffold pore size in the range of 100–400 μm is favorable for cell colonization, proliferation and penetration [2,3]. Chitosan (CS), a deacetylated derivative of chitin, has long been considered one of the most attractive natural biopolymer matrices for tissue engineering because of its biocompatibility, biodegradability and structural similarity to glycosaminoglycans [4–6]. Nevertheless, the use of CS has been limited to non-load-bearing applications due to its poor mechanical properties [7]. Unlike numerous other reinforcing materials, hydroxyapatite (Ca10(PO4)6(OH)2: HAp) significantly improves the mechanical properties of CS scaffolds because it has a crystal structure similar to that of the inorganic component of natural bone [8–10]. In particular, nano-sized HAp (nano-HAp) morphologically resembles
⁎ Corresponding author. E-mail address: [email protected] (D.-J. Kim).
http://dx.doi.org/10.1016/j.msec.2015.04.033 0928-4931/© 2015 Elsevier B.V. All rights reserved.
biological apatite [11] such that scaffolds composed of HAp and CS (HAp/CS composite scaffolds) have enhanced osteoconductive, osteoinductive and osteogenic properties [10]. HAp/CS composite scaffolds have been fabricated by several methods, such as the mechanical mixing of HAp and CS [12,13], the in situ co-precipitation of HAp [9,14] and the in situ precipitation of HAp into CS [15]. The HAp in the HAp/CS composite scaffolds that were prepared by mechanical mixing of constituents were aggregated and formed large clusters in the scaffolds because of the partial dissolution of nano-HAp particles in the acidic CS solution [12]. In contrast, the preparation of HAp/CS composites by the in situ precipitation of calcium phosphate led to an uncontrollable Ca/P ratio and crystallinity of HAp [16]. It has also been reported that the pore structure of HAp/CS composite scaffolds collapsed as the HAp content increased higher than 30 wt.% due to the agglomeration of HAp particles [13]. When HAp particles in the HAp/CS composite scaffolds were prepared by mechanical mixing, a more extreme heterogeneity of pore structure and low compressive strength were observed because the homogeneous dispersion of nano-HAp within the scaffolds was difficult to accomplish due to particle agglomeration [5]. Thus, uniform dispersion of nano-HAp in the composite scaffolds is a prerequisite to produce controlled pore structure and strength of scaffolds.
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It has been reported that alginate (AG), which is derived primarily from brown seaweed and bacteria, forms stable hydrogels through the ionic interaction between the carboxyl group of AG and Ca2 + [17]. Teng et al. reported that AG promoted the preferential synthesis of HAp oriented in the c direction when composite scaffolds were fabricated by the co-precipitation method [18]. The results indicated a strong ionic interaction between AG and Ca2+, leading to the formation of a specific stereo-chemical arrangement and charge distribution [18]. Furthermore, the combination of AG and CS forms a polyelectrolyte complex (PEC) between the carboxyl groups of AG and protonated amines of CS [19,20]. The formation of a PEC alleviates the shortcomings of CS, such as its poor mechanical properties and structural instability after transplantation [19,20]. Thus, it is expected that AG interacts with both HAp and CS in HAp/CS composites. In this context, AG may be utilized as a dispersant of nano-HAp particles in CS solution since biocompatibility of commercial dispersants for inorganic powders have not been ensured. To the best knowledge of the present authors, there has been no attempt to apply AG as a dispersant for optimization of the pore structure and the mechanical properties of HAp/CS composites. In this study, nano-HAp/AG/CS composite scaffolds were fabricated utilizing AG as a dispersing agent for nano-HAp particles and freezedrying the solution mixtures of HAp, AG and CS. The pore morphologies and mechanical properties of the composite scaffolds were determined, and osteoblast differentiation and mineralization on the composite scaffolds were investigated to evaluate the bioactivity and osteoconductivity of the composite scaffolds.
2. Materials and methods Nano-HAp was hydrothermally synthesized using 1 M Ca(NO3)2·4H2O (Sigma-Aldrich, St. Louis, MO, USA) and 1 M H3PO4 (Sigma-Aldrich). The solutions were mixed to achieve the Ca/P ratio of 1.67. Then, NH3 (30%, Sigma-Aldrich) was added into the solution mixture to precipitate the hydroxide containing Ca2+ and PO3− 4 . The coprecipitates were aged for 24 h at 80 °C and were then placed in an autoclave for 2 h at 180 °C. The hydrothermally synthesized powders were washed three times with deionized water to remove any unreacted substances. A 4 wt.% CS (practical grade, N 75% deacetylated, MW = 190 kDa– 375 kDa, Sigma-Aldrich) solution was prepared by dissolving the CS in deionized water with 1 wt.% acetic acid and the pH of CS solution was 5.2. In addition, 2 and 3.75 wt.% AG (alginic acid from brown seaweed, MW = 80 kDa–120 kDa, Sigma-Aldrich) solutions were prepared for the dispersion of nano-HAp particles and to form a PEC with CS, respectively, by dissolving the AG in deionized water. Prior to the dispersion of the nano-HAp powder, the pH of the 2 wt.% AG solution was adjusted to 9.5–10.0 by adding 5 wt.% NH4OH solution. During the dispersion of the nano-HAp powder in the 2 wt.% AG solution the pH of the slurry was maintained at about 9.0 using the NH4OH solution. The dispersion was facilitated by a magnetic stirrer for 1 h and a subsequent sonication for 2 h. The dispersed nano-HAp slurry was mixed with the 3.75 wt.% AG solution using a planetary mixer (ARM-300, Thinky, Laguna Hills, CA, USA) at 2000 rpm for 5 min. HAp/AG/CS composite scaffolds were prepared by mixing the HAp/AG mixture with the 4 wt.% CS solution using the planetary mixer. The nano-HAp content of the composite scaffolds ranged from 10 to 70 wt.%. The pH of HAp/AG/CS solutions was 6.3. Following mixing, the mixture was cast in a 24-well plate and refrigerated at 4 °C for 12 h. During the refrigeration, the plate was covered with paraffin film to prevent water from evaporating. The plate was frozen at − 20 °C overnight and then lyophilized for 24 h with a freeze drier (Labconco FreeZone 6Plus Freeze Drier, Labconco, Kansas, MO, USA) under vacuum with the collector temperature set at −89 °C to sublimate the ice from the composite scaffolds. Each composite scaffold was crosslinked by 0.2 M CaCl2 solution for 15 min and was then washed three times with deionized water and immersed in deionized
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Fig. 1. Transmission electron microscopic images of the nano-HAp particles.
water to remove unbound CaCl2. Wet composite scaffolds were refrozen and lyophilized following the procedure described above. The prepared nano-HAp, their morphology and particle size were examined using transmission electron microscopy (TEM). This was performed by a JEOL 2010 (Tokyo, Japan), using an accelerating voltage of 200 kV and a copper electron microscopic grid supported by a porous carbon (mesh size 300) film. The zeta potentials of nano-HAp particles suspended in deionized water were measured using electrophoretic light scattering (Otsuka Electronics, Hirakata, Osaka, Japan). The infrared spectra of the composite scaffolds were obtained by Fourier transform infrared (FT-IR) attenuated total reflection spectroscopy (Thermo Fisher Scientific Inc., Franklin, MA, USA). The porosities of the composite scaffolds were measured using the liquid displacement method [21] and calculated by: Porosityð%Þ ¼
ðW 1 −W 0 Þ 100 ρ V0
where W1, W0, ρ and V0 are the weight of the composite scaffolds saturated with ethanol, the dry weight of the scaffolds, the density of the ethanol and the initial volume of the composite scaffolds, respectively. Morphometric analysis of composite scaffolds was carried out using microcomputed tomography (micro-CT, Skyscan, Aartselaar, Belgium). The microstructures of cross-sections of the composite scaffolds were observed by scanning electron microscopy (SEM, JEOL, Tokyo, Japan). The mechanical properties of composite scaffolds were determined for circular disk specimens (12 mmØ × 10 mm, n = 5) under compression using a universal testing machine (Instron, Norwood, MA, USA) at a crosshead speed of 1 mm/min until the composite scaffolds reached a 30% reduction
Fig. 2. Effect of pH on the zeta potentials of the nano-HAp particles.
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Fig. 3. FT-IR spectra of (a) alginate, (b) nano-HAp particles and (c) alginate containing 50 wt.% nano-HAp particles.
in height. The elastic modulus was measured using the linear slope of stress–strain curve. To evaluate the cell behaviors on composite scaffolds, newborn mouse calvaria-derived MC3T3-E1 (subclone 4) preosteoblastic cells (American Type Culture Collection, Manassas, VA, USA) were used. MC3T3-E1 cells were cultured in Alpha Minimum Essential Medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) as well as 100 U/ml penicillin and streptomycin (Invitrogen). Media were further supplemented with 50 μg/ml ascorbic acid (Invitrogen) and 5 mM β-glycerophosphate (Invitrogen) for differentiation. MC3T3-E1 cells were seeded onto each scaffold at a density of 1.0 × 105 cells. Composite scaffolds (12 mmØ × 3 mm) were sanitized in ethanol 70% and then washed with deionized water sterilized by autoclaving at 121 °C. Cell differentiation and calcium-rich deposits by cells on composite scaffolds were determined by alkaline phosphatase (ALP) expression using a QuantiChrom ALP kit (BioAssay Systems, Hayward, CA, USA) and 0.04 M alizarin red S (Sigma-Aldrich, pH 4.2). Data were evaluated for statistical significance using one-way analysis of variance, and results were expressed as means ± standard deviations. Experiments were repeated three times unless otherwise specified. In all analyses, comparisons among means were determined using Tukey's post hoc test, and a p-value of less than 0.05 was considered statistically significant. 3. Results and discussion The morphology of the hydrothermally prepared nano-HAp particles is shown in Fig. 1. The particle size was 57 ± 13 nm and a
Fig. 5. FT-IR spectra of (a) alginate containing 50 wt.% nano-HAp particles (b) chitosan containing 85 wt.% nano-HAp particles and (c) composite of 70 wt.% nano-HAp/18 wt.% alginate/12 wt.% chitosan.
considerable number of particles were entangled to form agglomerates. It was difficult for the nanoparticles to be dispersed homogeneously due to their extremely high surface areas [22]. Thus, they were weakly agglomerated in order to reduce surface energy. The tendency of the agglomeration to occur in solution depended on the pH and was characterized by zeta potential [23]. The zeta potentials of the nano-HAp particles dispersed in deionized water are shown in Fig. 2. The point of zero charge of the nano-HAp particles was a pH of about 6.3 and the negative zeta potential increased with the increase in pH, which was beneficial to deagglomeration. This result indicates that the negatively charged nano-HAp particles were electrostatically repulsive in an alkaline solution, leading to the dispersion of the nano-HAp particles. The dispersion of the nano-HAp particles can be further improved by mechanical mixing with an AG solution that is soluble at high pH [18], since the negatively charged carboxylates of the AG are likely to form a complex with Ca2+ in the HAp to provide steric hindrance for the stabilization. Indeed, the nano-HAp particles, mixed with the AG solution at pH 10 were well dispersed, and a probable reaction between the HAp particles and AG was observed by FT-IR, as shown in Fig. 3, where HAp and AG are in the ratio of 1:1. The AG spectra show the characteristic absorption bands of COO− (1597 cm− 1, 1408 cm− 1) and C–O–C stretching (1022 cm− 1) [24,25]. The nano-HAp spectra show a strong absorption band at 1019 cm−1 corresponding to the v3 −1 band of PO3− and 1087 cm−1, which re4 and the shoulders at 962 cm flect the v1 band of P–O stretching [25,26]. The AG scaffold containing 70 wt.% nano-HAp particles exhibits a prominent band around 1009 cm−1 that may be caused by a shift of either the C–O–C in AG or the PO3− in HAp. It has been reported that C–O–C in AG immersed in 4 CaCl2 shifts towards lower wave numbers as Ca2 + content increases [25]. The shift to lower frequencies indicates weakening in C–O–C bonds, most likely due to these bonds being shared with Ca2+. Thus, the bond at 1009 cm−1 may result from a shift of the C–O–C in AG due to strong ionic interactions between C–O–C and Ca2+ in the nanoHAp. Furthermore, the HAp/gelatin nanocomposites, fabricated by coTable 1 Properties of alginate–chitosan composite scaffolds containing various nanohydroxyapatites.
Fig. 4. FT-IR spectra of (a) chitosan, (b) nano-HAp particles and (c) chitosan mixed with 85 wt.% nano-HAp particles.
HAp contents in wt.% in AG/CS composite scaffolds
Characteristics of the composite scaffolds Porosity (%)
Scaffold surface/volume ratio (mm−1)
0 10 30 50 70
78 ± 4.64 84 ± 5.45 81 ± 4.56 79 ± 4.42 81 ± 5.15
164.71 ± 23.64 187.18 ± 21.14 171.62 ± 30.41 181.29 ± 28.26 176.68 ± 26.84
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Fig. 6. Micro-CT images (a, b) and 3D reconstructions (c, d) of the alginate/chitosan composite scaffolds (a, c) and the alginate/chitosan composite scaffolds containing 70 wt.% nano-HAp (b, d).
precipitation in the presence of AG, demonstrated a strong ionic interaction between HAp and AG to form a specific stereo-chemical arrangement [18]. The interaction has been rationalized by the “egg-box model”, where Ca2+ in nano-HAp is located in the electronegative cavities of guluronic acids of AG. Thus, the dispersion of nano-HAp in AG solution at pH 10 is likely achieved both by the electrostatic repulsion and by the formation of ionic complexes between nano-HAp and AG. The characteristic FT-IR spectra of CS appeared at 1645, 1556, 1410, 1375 and 1022 cm−1, corresponding to amide I, amide II, CH2 bending,
CH3 symmetrical deformation and C–O–C, respectively, as seen in Fig. 4 [1,26]. In the CS composite scaffolds containing 70 wt.% nano-HAp particles, the bands for amide I, amide II and CH2 bending shifted to lower wave numbers of 1637, 1548 and 1403 cm−1, respectively, indicating chemical interaction between either the amino groups in CS and the Ca2 + in nano-HAp or between the amino groups and PO34 − [5]. Comparison of the FT-IR spectra for the composite scaffolds of 85 wt.% HAp/15 wt.% CS, 50 wt.% HAp/50 wt.% AG and 70 wt.% HAp/ 18 wt.% AG/12 wt.% CS in Fig. 5 strongly supports that the interaction
Fig. 7. Microstructures of cross-sectional images of alginate/chitosan composite scaffolds containing various HAp contents as observed by SEM. The nano-HAp contents in the composite scaffolds were: (a) 0 wt.%, (b) 10 wt.%, (c) 30 wt.%, (d) 50 wt.% and (e) 70 wt.%. Original magnifications were 50×.
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between AG and nano-HAp is dominant in the ternary composite and that the interaction contributes to the dispersion of nano-HAp in the ternary composite. Scaffolds used in tissue engineering should have a high porosity structure to provide the sites for cell attachment, proliferation and differentiation [1–3]. The porosity and other data on the pore structure of CS/AG composite scaffolds containing various amounts of nano-HAp are listed in Table 1. The porosity of the composite scaffolds ranged from 78% to 84% and was independent of nano-HAp content; no significant differences in the surface to volume ratio were observed. Scaffolds for tissue engineering require an interconnected pore structure in order to facilitate the migration of invading cells into scaffolds [1]. Micro-CT images demonstrated that the AG/CS composite scaffolds containing various amounts of nano-HAp had internally interconnected porous structures, as shown in Fig. 6. The ternary composite scaffolds exhibited anisotropic porous networks with pore sizes ranging from 100 to 400 μm. The high surface area to volume ratios of the composite scaffolds, calculated using the CTAn program embedded in the micro-CT system, range from 164.71 mm−1 to 187.18 mm−1, may be favorable for cell adhesion to scaffolds and bone tissue regeneration [2,3]. The ternary composite scaffolds showed no significant differences in interconnected pore structure depending on the nano-HAp content. The pore structure of the composite scaffolds was sufficient for tissue engineering, since the scaffolds require pores larger than 100 μm in size and porosity of about 80%, considering cell size, migration requirements, and transport. The microstructure of the composite scaffolds was observed by SEM and the results are shown in Fig. 7. The ternary composite scaffold shows homogeneously distributed pores and continuous pore channels.
Fig. 8. Comparison of (a) compressive strengths and (b) elastic modulus of alginate/chitosan composite scaffolds containing various nano-HAp contents (n = 5).
The pore structures of the composite scaffolds were not influenced by the content of the nano-HAp. It has been reported that high amounts of HAp in polymer solution often result in a decrease in porosity, irregularly sized pores and the collapse of pore structure [13,27,28]. This is probably because the rheological behavior of polymer solutions containing HAp is inhomogeneous due to the agglomerated and nonuniform nature of the HAp distributed in suspension. In the fabrication of scaffolds by freeze-drying, the high viscosity of the suspension prevents water migration for the growth of ice crystals, and thus reduces the pore size of the scaffolds [15,29]. Hence, the uniform pore structure of the ternary composite scaffolds in Fig. 7 demonstrates that the nano-HAp particles, dispersed in AG solution at pH 10 prior to being mixed with CS, did not alter significantly the rheological behavior of the solution mixtures. The compressive strengths and elastic modulus of the composite scaffolds containing various amounts of nano-HAp are shown in Fig. 8. The compressive strength increased from 0.27 MPa to 0.68 MPa and the elastic modulus improved from 4.4 MPa to 13.4 MPa with increasing nano-HAp content in the composite scaffolds up to 70 wt.% nano-HAp. The improved mechanical properties in CS scaffolds containing HAp are thought to result from chemical interactions between the amino groups in CS and the Ca2+ in the nano-HAp or between the amino groups and PO3− 4 [5]. The interactions between CS and nano-HAp were observed in the ternary composite scaffolds as shown in Figs. 3–5. Thus, the mechanical properties in the ternary composite scaffolds were improved mainly by the strong interaction between CS and nano-HAp, which was facilitated by the uniform distribution of nano-HAp through the ionic
Fig. 9. Biocompatibility of alginate/chitosan composite scaffolds containing various nanoHAp contents. (a) ALP assays for cell differentiation and (b) alizarin Red-S staining for mineralization were conducted after MC3T3-E1 cells were incubated for the designated times on each composite scaffold. Data are presented as means ± standard deviations of three separate experiments. *p b 0.05; **p b 0.01.
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interaction between AG and nano-HAp. In addition, the uniform pore structure with thick pore walls contributed to the improved compressive strengths and elastic modulus. As demonstrated by Parminik et al., the increase in HAp/CS composite scaffold mechanical properties ended at a HAp content of 40 wt.% [12] when composites were prepared by mechanical mixing of the constituents. This is probably due to insufficient dispersion of HAp particles in the composite scaffolds, which resulted in nonuniform pore morphology. Osteoblasts secrete and mineralize the bone matrix during differentiation, and the differentiation phase of osteoblasts is determined using ALP activity and mineralization. ALP activity was measured over 14 and 21 days of culture as a marker of the matrix maturation phase during the differentiation of osteoblasts on the ternary composite scaffolds (Fig. 9(a)). On the ternary composite scaffolds, APL activity increased significantly after 14 and 21 days of culture as compared to the activity on AG/CS scaffolds. The composite scaffolds containing 70% nano-HAp showed a 1.45-fold increase in enzyme activity after 21 days of cultivation in comparison with that of the AG/CS scaffolds. Similar to the ALP activity results, the mineralization of osteoblasts on the composite scaffolds was enhanced on the ternary scaffolds as compared to the AG/CS scaffolds (Fig. 9(b)). Since the increase in extracellular Ca2+ improved osteoblast differentiation, the Ca2+ released from HAp contributed to the increase in osteoblast differentiation due to the increase in osteopontin and bone sialoprotein expression through L-type Ca channels, which triggered the Ca2+/calmodulin-dependent protein kinase 2 pathway [30]. 4. Conclusions An alginate solution at pH 10 can be utilized as a bio-inspired dispersant for nano-sized HAp particles as result of both electrostatic repulsion and steric hindrance. The nano-HAp/chitosan composite scaffolds containing alginate demonstrated uniform pore structure even with a nano-HAp content of 70 wt.%. The even pore morphology contributed to an increase in the compressive strength and elastic modulus of composite scaffolds, which increased as a function of nano-HAp content. The composite scaffolds containing nano-HAp, as compared to the chitosan/ alginate composite scaffolds, exhibited improved osteoblastic differentiation for bone regeneration, as determined by ALP activity and the mineralization of alginate/chitosan scaffolds. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2010-0024260). References [1] I. Manjubala, S. Scheler, J. Bössert, K.D. Jandt, Mineralization of chitosan scaffolds with nano-apatite formation by double diffusion technique, Acta Biomater. 2 (2006) 75. [2] A.G. Mikos, G. Sarakinos, M.D. Lyman, D.E. Ingber, J.P. Vacanti, R. Langer, Prevascularization of porous biodegradable polymers, Biotechnol. Bioeng. 42 (1993) 716.
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