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Preparation of highly interconnected porous hydroxyapatite scaffolds by chitin gel-casting
Materials Science and Engineering C 31 (2011) 697–701
Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Preparation of highly interconnected porous hydroxyapatite scaffolds by chitin gel-casting J. Zhao a,b, K. Duan b, J.W. Zhang b, L.Y. Guo b, J. Weng b,⁎ a b
School of Mechanical Engineering, Southwest Jiaotong University, Chengdu, Sichuan 610031, China Key Laboratory of Advanced Technologies of Materials (Ministry of Education), Southwest Jiaotong University, Chengdu, Sichuan 610031, China
a r t i c l e
i n f o
Article history: Received 13 March 2010 Received in revised form 15 October 2010 Accepted 29 December 2010 Available online 8 January 2011 Keywords: Hydroxyapatite Chitin Gel-casting Porosity Interconnectivity Compressive strength
a b s t r a c t In this study, a simple and effective method for producing highly interconnected porous hydroxyapatite (HA) scaffolds was developed by combining gel-casting, particle-leaching and extrusion techniques. Chitin (CT) sol was used to disperse HA particles and wax spheres were introduced as porogens for their excellent deformability. In extrusion process, the accumulated wax spheres in point-to-point contact can transform into surface-to-surface contact by means of the extrusion pressure. Thus, the obtained porous HA scaffolds exhibited an interconnected channel network after leaching out of the porogens. The results showed that the scaffolds prepared by different size of wax spheres exhibited nearly the same volumetric porosity of about 86%, while the compressive strengths decreased as the pore size increased. Therefore, the method developed can be used to effectively tailor the pore size of HA scaffolds while maintaining a high porosity. The highly porous HA scaffolds with excellent interconnectivity are expected to be a promising bone substitute in clinical practice. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Porous hydroxyapatite (HA) scaffolds have gained considerable attention as bone substitutes for the reconstruction of bone defects for their excellent osteoconductivity and biocompatibility. The architectures of these scaffolds, particularly pore interconnectivity, play important roles in determining the rate and degree of bone ingrowth by affecting metabolism, nutrient diffusion and neovascularization [1,2]. A survey of the literature shows that interconnected pores larger than 50 μm favored cellular and vascular penetration and ensured bone ingrowth, and a pore diameter of 200–500 μm is optimal for the osteoconductivity [1,3,4]. Therefore, effective control of pore size and interconnectivity is important for accelerating bone regeneration associated with porous scaffolds. Many methods have been developed to fabricate porous scaffolds with well-defined porosity and pore size, such as polymer impregnation, particle leaching, suspension foaming, and three dimensional (3D) printing [5–8]. Among these methods, particle leaching technique is probably the most popular method to produce porous scaffolds. The biggest advantage of this technique is the ease to control the porosity and pore size of biomaterial scaffolds, whereas the major ⁎ Corresponding author. Key Laboratory of Advanced Technologies of Materials (Ministry of Education), Southwest Jiaotong University, Chengdu, 610031 China. Tel./ fax: + 86 28 87601371. E-mail addresses: [email protected] (J. Zhao), [email protected] (J. Weng). 0928-4931/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.12.011
disadvantage is the difficulty to overcome the presence of closed pores, which is adverse to neovascularization and tissue ingrowth [9–12]. The existence of closed pores depends largely on whether the adjacent porogen particles are in contact. Currently, the commonly used porogens are water-soluble particles such as sodium chloride (NaCl) particles, which can be easily removed by water after gelation. However, it is really difficult for these hard cubic particles to form surface-to-surface contact and result in a poor pore interconnectivity [13,14]. In contrast, relatively softer particles can easily deform to achieve effective overlapping contacts under compression during extrusion process. Some softer porogens, such as wax spheres and gelatin spheres, have been introduced to prepare porous scaffolds with better pore interconnectivity than by NaCl particles [15–18]. Gel-casting is a wet ceramic forming technique that involves the polymerization of a monomer in an aqueous or non-aqueous solvent forming a rigid, ceramic-loaded body that can be machined in the green bodies or formed directly in a complex mold [19,20]. After gel formation, gel-cast green samples can be easily demolded and then are dried in controlled conditions [21]. The gel-casting technique was firstly established for preparing dense components, and more recently modified to produce also porous ceramics, by combining with particle leaching methods, or foaming techniques, or even sponge methods [22–24]. Different monomers were employed for gel-casting, such as polysaccharides [25,26], protein [26,27], and polymers [28]. As an abundant natural polymer, chitin (CT) has been widely used as biomaterials owing to its biocompatibility, biodegradability and
698
J. Zhao et al. / Materials Science and Engineering C 31 (2011) 697–701
favorable effects on wound healing [29–31]. Moreover, the strong shrinkage of CT is reported to enhance the mechanical strength of porous ceramic scaffold [32,33]. In this study, a method was developed to prepare highly interconnected porous HA scaffolds by combining gel-casting, particle-leaching and extrusion techniques. The feasibility of the technique was demonstrated by providing an excellent control over pore size, porosity and pore interconnectivity in one step. The porosity, pore size, cross-sectional shrinkage and compressive strength of porous HA scaffold were systematically investigated. 2. Experimental 2.1. Materials The starting HA powders (mean particle size: 2.1 μm) were synthesized in our laboratory by a wet chemical method according to the literature [34]. CT and polyvinyl alcohol (PVA, average molecular weight: 75,000) were purchased from Zhongren Chemical Ltd. (Chengdu, China). Lithium chloride (LiCl), dimethylacetamide (DMAc), silver nitrate, ethanol and hexane were all of analytical grade and purchased from Chengdu Chemical Ltd. (Chengdu, China). 2.2. Preparation of wax spheres Wax spheres were prepared by an emulsion process [35]. PVA was dissolved at a concentration of 2% (w/v) in distilled water at about 100 °C. Wax was melted at 65 °C and added into the PVA solution under mechanical stirring at 350 rpm. After 20 min, while keeping stirring, cold water was poured into the wax-PVA suspension to rapidly solidify the melted wax into spheres. Finally, these wax spheres were collected by filtration, rinsed with deionized water, dried at room temperature overnight and stored in a vacuum desiccator. 2.3. Preparation of HA/CT suspension LiCl was dissolved in DMAc at a concentration of 5% (w/v), and then CT powders (0.5 w/v %) were added to this LiCl/DMAc solution under stirring until a uniform CT sol was formed. HA powders were mixed with this CT sol at a concentration of 15% (w/v) under intensive stirring to form a homogeneous HA/CT suspension. After stirring for 24 h, the HA/CT suspension was sealed and kept at room temperature for 48 h.
process, the interstices between the wax spheres were filled with the HA/CT suspension by compression under extrusion and the excess HA/CT suspension was extruded through these interstices while maintaining surface-to-surface contact between the adjacent wax spheres. After standing for 30 min at room temperature, the HA/CT samples were rapidly gelated by dropwise addition of ethanol, removed from the mold, and then soaked in ethanol for 12 h to reach complete gelation. Subsequently, the gelated HA/CT samples were immersed in hexane at 50 °C for 5 times to leach out the wax spheres, and then immersed in distilled water for several times to completely remove the remaining LiCl. Finally, the porous HA/CT gel samples were dried at 80 °C overnight, heated at 320 °C for 2 h to burn out CT, and sintered at 1200 °C for 2 h. Three size ranges of wax spheres (small, medium and large) were used as porogens. The porous HA scaffolds prepared with small, medium and large wax spheres are referred to as HA-S, HA-M and HA-L, respectively. 2.5. Characterization Cross-sectional shrinkage (CSS) is a critical factor that is closely related to the final dimension of scaffold as well as the pore size. Therefore, to calculate the CSS during the preparation of scaffolds can provide guidelines for the selection of wax spheres and the determination of the pore size of the sintered HA scaffolds. The CSS was obtained according to Eq. (1):
CSS ¼
πr12 −πr22 r12 −r22 × 100%¼ × 100% πr12 r12
ð1Þ
where r1 represents the radius of the original cylindrical sample after complete gelation; and r2 represents the radius of the porous sample after a process such as wax leaching or sintering. The total porosity of HA scaffold was measured by a gravimetric method [1]. The morphology, porous structure and phase structure of wax spheres and HA scaffolds were studied by optical microscopy (OM), scanning electron microscopy (SEM) and X-ray diffraction (XRD), respectively. The compressive strength was tested using an Instron 5567 universal testing machine equipped with a 30 kN load cell at a crosshead speed of 0.5 mm/min. Five replicate cylindrical samples (Φ 8 × 16 mm) were tested for each type of scaffolds. 3. Results and discussion 3.1. Wax spheres
2.4. Preparation of porous HA scaffolds Porous HA scaffolds were fabricated by combining gel-casting, wax leaching and extrusion techniques, as illustrated in Fig. 1. In this
Fig. 1. Schematics of the gel-casting molding of HA/CT samples (a) and (b) the process of extrusion across wax spheres.
Considering the shrinkage of HA samples in drying and sintering processes, the size selection of wax spheres is larger than that of the asprepared HA scaffold. Fig. 2 shows the appearances of the prepared wax spheres. Here the mean diameters for the small, medium and large wax spheres are 800 μm, 1400 μm and 2000 μm, respectively. All wax spheres
Fig. 2. The appearance of wax spheres with different diameter ranges.
J. Zhao et al. / Materials Science and Engineering C 31 (2011) 697–701
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showed excellent sphericity and very smooth surfaces, both of which were in favor of forming surface-to-surface contact by compression under extrusion and thus ensuring the pore interconnectivity. 3.2. Phase structure Fig. 3 shows the XRD patterns of the starting HA powders and the sintered HA scaffold. All diffraction peaks recorded from the HA powders and the HA scaffold matched the standard pattern of HA (JCPDS09-0432) without additional peaks from other phases being observed, indicating that no chemical reactions occurred during the sintering process. 3.3. Porosity The porosities of the HA scaffolds prepared with the three types of wax spheres were 87.2 ± 2.1% in HA-S, 86.8 ± 2.8% in HA-M, and 86.4 ± 2.6% in HA-L, all of which met the clinical requirements of 50– 90% for nutrient diffusion, metabolite elimination, cell migration and bone ingrowth [1]. Moreover, it is worthwhile to note that these three types of scaffolds with different pore sizes exhibited almost the same total porosities of about 86%, indicating that the porosities of scaffolds prepared by this technique are nearly independent of the particle size of porogen. Therefore, as compared with other studies, the combination of gel-casting, wax leaching and extrusion techniques provided a more convenient technique for preparing highly porous HA scaffolds of different size ranges without requirement of careful measurement and control of the ratio of porogens to scaffold materials [36,37]. 3.4. Porous structure of HA scaffold The three types of HA scaffolds all showed well-defined macroporosity, as shown in Fig. 4. The mean macropore diameter was 300 μm in HA-S, 600 μm in HA-M, and 900 μm in HA-L, as determined by image analyses (Image Pro 6.0). These as-prepared cylindrical HA scaffolds showed a reticular structure with fully interconnected spherical pores similar to a trabecular bone [38]. As expected, under compression during extrusion, the wax spheres easily deformed to form surface-to-surface contact with each other. After wax leaching, the space occupied by the wax spheres was converted into interconnected pores [18]. Therefore, the technique in this study successfully overcame the main disadvantage of poor or non-uniform pore interconnectivity associated with the use of NaCl or sucrose particles as porogens [9,10]. Fig. 5 shows the typical pore morphology and surface topography of the HA-M scaffold produced with medium wax spheres. The scaffold had interconnected spherical pores with diameters ranging from 50 to 200 μm (Fig. 5a and b). These interconnected pores were
Fig. 4. The reticular structures of porous HA-S (a), HA-M (b) and HA-L (c) showing the interconnected channels with different pore sizes.
reported to benefit the circulation of body fluid and the supply of nutrients and exchange of ions [39]. Two types of HA grains were observed in Fig. 5c and d. The smaller grains were uniformly distributed on the larger grains. This structure might improve the mechanical properties of HA scaffolds. In addition, some micropores existed on the wall of macropores, which can further increase the surface roughness of the scaffolds and thus benefit protein absorption and cell adhesion [40]. 3.5. Cross sectional shrinkage (CSS)
Fig. 3. XRD patterns of the starting HA powder and the sintered HA scaffold.
Fig. 6 shows the CSS values of HA scaffolds after wax leaching and sintering. The three types of HA scaffolds had nearly the same CSS values of about 66% after wax leaching and about 81% after sintering. Therefore, scaffolds with a tailored pore size can be easily prepared by selecting the wax spheres with suitable size ranges. In the wax leaching process, the significant CSS of porous HA/CT gel samples resulted from the motion of CT chains during drying was mainly
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Fig. 5. SEM images of the typical pore structure (a) and a pore (b) and surface topography at different (c, d) magnifications of the HA-M scaffold.
In Fig. 7, the compressive strengths of the three types of HA scaffolds, which were produced with the three types of wax spheres and presented nearly the same porosity of about 86%, decreased with
increasing pore size. The relationship between compressive strength and pore size was consistent with previous studies [43]. Liu et al. demonstrated that at a same porosity the compressive strength of porous HA scaffolds decreased roughly linearly with increasing macropore size [44], because in the same cross-section area the scaffolds with smaller pore size had more struts than those with a larger pore size and thus could better distribute the load [45]. Furthermore, the present as-prepared HA scaffolds in this study exhibited higher compressive strengths than HA scaffolds with similar porosity and pore size reported in other studies [46–48], indicating that the high shrinkage of CT gel might be helpful in improving the mechanical properties of porous scaffolds.
Fig. 6. Cross-sectional shrinkage of porous HA scaffolds prepared with the three types of wax spheres after wax leaching and sintering processes.
Fig. 7. Compressive strengths of HA scaffold produced with the three types of wax spheres (mean ± standard deviation, n = 5).
dependent on the concentration of CT [41,42]. While in the sintering process, the CSS was probably attributed to the decomposition of CT, the loss of crystallization water in HA grains and the realignment of HA grains. The presence of shrinkage during wax leaching and sintering processes would facilitate the densification of scaffolds and thus improve their mechanical properties accordingly. 3.6. Compressive strength
J. Zhao et al. / Materials Science and Engineering C 31 (2011) 697–701
4. Conclusions Highly interconnected porous HA scaffolds were successfully fabricated by combining gel-casting, particle-leaching and extrusion techniques in one step. The porosities of these porous HA scaffolds were approximately 86% regardless of the size of the porogens, and the pore size can be tailored independent of porosity by this technique. The compressive strength was closely related to the pore size. With increasing pore size, the compressive strength decreased significantly. This kind of porous HA scaffolds with excellent porosity and interconnectivity is expected to be a promising bone substitute in the future clinical practice of tissue engineering. Acknowledgement This study was financially supported by National Basic Research Program (No. 2011CB707604), National Natural Science Foundation of China (No. 30870630), Ph.D. Programs Foundation of MEC (No. 20070613062) and Fundamental Research Fund of Sichuan (No. 2008JY0062). References [1] V. Karageorgiou, D. Kaplan, Biomaterials 26 (2005) 5474. [2] F.C. Fierz, F. Beckmann, M. Huser, S.H. Irsen, B. Leukers, F. Witte, O. Degistirici, A. Andronache, M. Thie, B. Müller, Biomaterials 29 (2008) 3799. [3] E. Saiz, L. Gremillard, G. Menendez, P. Miranda, K. Gryn, A.P. Tomsia, Mater. Sci. Eng. C. 27 (2007) 546. [4] O. Gauthier, J.M. Bouler, E. Aguado, P. Pilet, G. Daculsi, Biomaterials 19 (1998) 133. [5] Y. Zhang, M. Zhang, J. Biomed. Mater. Res. 61 (2002) 1. [6] C. Tsioptsias, I. Tsivintzelis, L. Papadopoulou, C. Panayiotou, Mater. Sci. Eng. C. 29 (2009) 159. [7] X. Mao, S. Wang, S. Shimai, Ceram. Int. 34 (2008) 107. [8] A. Park, B. Wu, L.G. Griffith, J. Biomat. Sci. polym. E. 9 (1998) 89. [9] E. Chevalier, D. Chulia, C. Pouget, M. Viana, J. Pharm. Sci. 97 (2008) 1135. [10] K. Rezwan, Q.Z. Chen, J.J. Blaker, A.R. Boccaccini, Biomaterials 27 (2006) 3413. [11] K. Prabhakaran, A. Melkeri, N.M. Gokhale, S.C. Sharma, Ceram. Int. 33 (2007) 77. [12] A.R. Studart, U.T. Gonzenbach, E. Tervoort, L.J. Gauckler, J. Am. Ceram. Soc. 89 (2006) 1771.
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Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Preparation of highly interconnected porous hydroxyapatite scaffolds by chitin gel-casting J. Zhao a,b, K. Duan b, J.W. Zhang b, L.Y. Guo b, J. Weng b,⁎ a b
School of Mechanical Engineering, Southwest Jiaotong University, Chengdu, Sichuan 610031, China Key Laboratory of Advanced Technologies of Materials (Ministry of Education), Southwest Jiaotong University, Chengdu, Sichuan 610031, China
a r t i c l e
i n f o
Article history: Received 13 March 2010 Received in revised form 15 October 2010 Accepted 29 December 2010 Available online 8 January 2011 Keywords: Hydroxyapatite Chitin Gel-casting Porosity Interconnectivity Compressive strength
a b s t r a c t In this study, a simple and effective method for producing highly interconnected porous hydroxyapatite (HA) scaffolds was developed by combining gel-casting, particle-leaching and extrusion techniques. Chitin (CT) sol was used to disperse HA particles and wax spheres were introduced as porogens for their excellent deformability. In extrusion process, the accumulated wax spheres in point-to-point contact can transform into surface-to-surface contact by means of the extrusion pressure. Thus, the obtained porous HA scaffolds exhibited an interconnected channel network after leaching out of the porogens. The results showed that the scaffolds prepared by different size of wax spheres exhibited nearly the same volumetric porosity of about 86%, while the compressive strengths decreased as the pore size increased. Therefore, the method developed can be used to effectively tailor the pore size of HA scaffolds while maintaining a high porosity. The highly porous HA scaffolds with excellent interconnectivity are expected to be a promising bone substitute in clinical practice. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Porous hydroxyapatite (HA) scaffolds have gained considerable attention as bone substitutes for the reconstruction of bone defects for their excellent osteoconductivity and biocompatibility. The architectures of these scaffolds, particularly pore interconnectivity, play important roles in determining the rate and degree of bone ingrowth by affecting metabolism, nutrient diffusion and neovascularization [1,2]. A survey of the literature shows that interconnected pores larger than 50 μm favored cellular and vascular penetration and ensured bone ingrowth, and a pore diameter of 200–500 μm is optimal for the osteoconductivity [1,3,4]. Therefore, effective control of pore size and interconnectivity is important for accelerating bone regeneration associated with porous scaffolds. Many methods have been developed to fabricate porous scaffolds with well-defined porosity and pore size, such as polymer impregnation, particle leaching, suspension foaming, and three dimensional (3D) printing [5–8]. Among these methods, particle leaching technique is probably the most popular method to produce porous scaffolds. The biggest advantage of this technique is the ease to control the porosity and pore size of biomaterial scaffolds, whereas the major ⁎ Corresponding author. Key Laboratory of Advanced Technologies of Materials (Ministry of Education), Southwest Jiaotong University, Chengdu, 610031 China. Tel./ fax: + 86 28 87601371. E-mail addresses: [email protected] (J. Zhao), [email protected] (J. Weng). 0928-4931/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.12.011
disadvantage is the difficulty to overcome the presence of closed pores, which is adverse to neovascularization and tissue ingrowth [9–12]. The existence of closed pores depends largely on whether the adjacent porogen particles are in contact. Currently, the commonly used porogens are water-soluble particles such as sodium chloride (NaCl) particles, which can be easily removed by water after gelation. However, it is really difficult for these hard cubic particles to form surface-to-surface contact and result in a poor pore interconnectivity [13,14]. In contrast, relatively softer particles can easily deform to achieve effective overlapping contacts under compression during extrusion process. Some softer porogens, such as wax spheres and gelatin spheres, have been introduced to prepare porous scaffolds with better pore interconnectivity than by NaCl particles [15–18]. Gel-casting is a wet ceramic forming technique that involves the polymerization of a monomer in an aqueous or non-aqueous solvent forming a rigid, ceramic-loaded body that can be machined in the green bodies or formed directly in a complex mold [19,20]. After gel formation, gel-cast green samples can be easily demolded and then are dried in controlled conditions [21]. The gel-casting technique was firstly established for preparing dense components, and more recently modified to produce also porous ceramics, by combining with particle leaching methods, or foaming techniques, or even sponge methods [22–24]. Different monomers were employed for gel-casting, such as polysaccharides [25,26], protein [26,27], and polymers [28]. As an abundant natural polymer, chitin (CT) has been widely used as biomaterials owing to its biocompatibility, biodegradability and
698
J. Zhao et al. / Materials Science and Engineering C 31 (2011) 697–701
favorable effects on wound healing [29–31]. Moreover, the strong shrinkage of CT is reported to enhance the mechanical strength of porous ceramic scaffold [32,33]. In this study, a method was developed to prepare highly interconnected porous HA scaffolds by combining gel-casting, particle-leaching and extrusion techniques. The feasibility of the technique was demonstrated by providing an excellent control over pore size, porosity and pore interconnectivity in one step. The porosity, pore size, cross-sectional shrinkage and compressive strength of porous HA scaffold were systematically investigated. 2. Experimental 2.1. Materials The starting HA powders (mean particle size: 2.1 μm) were synthesized in our laboratory by a wet chemical method according to the literature [34]. CT and polyvinyl alcohol (PVA, average molecular weight: 75,000) were purchased from Zhongren Chemical Ltd. (Chengdu, China). Lithium chloride (LiCl), dimethylacetamide (DMAc), silver nitrate, ethanol and hexane were all of analytical grade and purchased from Chengdu Chemical Ltd. (Chengdu, China). 2.2. Preparation of wax spheres Wax spheres were prepared by an emulsion process [35]. PVA was dissolved at a concentration of 2% (w/v) in distilled water at about 100 °C. Wax was melted at 65 °C and added into the PVA solution under mechanical stirring at 350 rpm. After 20 min, while keeping stirring, cold water was poured into the wax-PVA suspension to rapidly solidify the melted wax into spheres. Finally, these wax spheres were collected by filtration, rinsed with deionized water, dried at room temperature overnight and stored in a vacuum desiccator. 2.3. Preparation of HA/CT suspension LiCl was dissolved in DMAc at a concentration of 5% (w/v), and then CT powders (0.5 w/v %) were added to this LiCl/DMAc solution under stirring until a uniform CT sol was formed. HA powders were mixed with this CT sol at a concentration of 15% (w/v) under intensive stirring to form a homogeneous HA/CT suspension. After stirring for 24 h, the HA/CT suspension was sealed and kept at room temperature for 48 h.
process, the interstices between the wax spheres were filled with the HA/CT suspension by compression under extrusion and the excess HA/CT suspension was extruded through these interstices while maintaining surface-to-surface contact between the adjacent wax spheres. After standing for 30 min at room temperature, the HA/CT samples were rapidly gelated by dropwise addition of ethanol, removed from the mold, and then soaked in ethanol for 12 h to reach complete gelation. Subsequently, the gelated HA/CT samples were immersed in hexane at 50 °C for 5 times to leach out the wax spheres, and then immersed in distilled water for several times to completely remove the remaining LiCl. Finally, the porous HA/CT gel samples were dried at 80 °C overnight, heated at 320 °C for 2 h to burn out CT, and sintered at 1200 °C for 2 h. Three size ranges of wax spheres (small, medium and large) were used as porogens. The porous HA scaffolds prepared with small, medium and large wax spheres are referred to as HA-S, HA-M and HA-L, respectively. 2.5. Characterization Cross-sectional shrinkage (CSS) is a critical factor that is closely related to the final dimension of scaffold as well as the pore size. Therefore, to calculate the CSS during the preparation of scaffolds can provide guidelines for the selection of wax spheres and the determination of the pore size of the sintered HA scaffolds. The CSS was obtained according to Eq. (1):
CSS ¼
πr12 −πr22 r12 −r22 × 100%¼ × 100% πr12 r12
ð1Þ
where r1 represents the radius of the original cylindrical sample after complete gelation; and r2 represents the radius of the porous sample after a process such as wax leaching or sintering. The total porosity of HA scaffold was measured by a gravimetric method [1]. The morphology, porous structure and phase structure of wax spheres and HA scaffolds were studied by optical microscopy (OM), scanning electron microscopy (SEM) and X-ray diffraction (XRD), respectively. The compressive strength was tested using an Instron 5567 universal testing machine equipped with a 30 kN load cell at a crosshead speed of 0.5 mm/min. Five replicate cylindrical samples (Φ 8 × 16 mm) were tested for each type of scaffolds. 3. Results and discussion 3.1. Wax spheres
2.4. Preparation of porous HA scaffolds Porous HA scaffolds were fabricated by combining gel-casting, wax leaching and extrusion techniques, as illustrated in Fig. 1. In this
Fig. 1. Schematics of the gel-casting molding of HA/CT samples (a) and (b) the process of extrusion across wax spheres.
Considering the shrinkage of HA samples in drying and sintering processes, the size selection of wax spheres is larger than that of the asprepared HA scaffold. Fig. 2 shows the appearances of the prepared wax spheres. Here the mean diameters for the small, medium and large wax spheres are 800 μm, 1400 μm and 2000 μm, respectively. All wax spheres
Fig. 2. The appearance of wax spheres with different diameter ranges.
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showed excellent sphericity and very smooth surfaces, both of which were in favor of forming surface-to-surface contact by compression under extrusion and thus ensuring the pore interconnectivity. 3.2. Phase structure Fig. 3 shows the XRD patterns of the starting HA powders and the sintered HA scaffold. All diffraction peaks recorded from the HA powders and the HA scaffold matched the standard pattern of HA (JCPDS09-0432) without additional peaks from other phases being observed, indicating that no chemical reactions occurred during the sintering process. 3.3. Porosity The porosities of the HA scaffolds prepared with the three types of wax spheres were 87.2 ± 2.1% in HA-S, 86.8 ± 2.8% in HA-M, and 86.4 ± 2.6% in HA-L, all of which met the clinical requirements of 50– 90% for nutrient diffusion, metabolite elimination, cell migration and bone ingrowth [1]. Moreover, it is worthwhile to note that these three types of scaffolds with different pore sizes exhibited almost the same total porosities of about 86%, indicating that the porosities of scaffolds prepared by this technique are nearly independent of the particle size of porogen. Therefore, as compared with other studies, the combination of gel-casting, wax leaching and extrusion techniques provided a more convenient technique for preparing highly porous HA scaffolds of different size ranges without requirement of careful measurement and control of the ratio of porogens to scaffold materials [36,37]. 3.4. Porous structure of HA scaffold The three types of HA scaffolds all showed well-defined macroporosity, as shown in Fig. 4. The mean macropore diameter was 300 μm in HA-S, 600 μm in HA-M, and 900 μm in HA-L, as determined by image analyses (Image Pro 6.0). These as-prepared cylindrical HA scaffolds showed a reticular structure with fully interconnected spherical pores similar to a trabecular bone [38]. As expected, under compression during extrusion, the wax spheres easily deformed to form surface-to-surface contact with each other. After wax leaching, the space occupied by the wax spheres was converted into interconnected pores [18]. Therefore, the technique in this study successfully overcame the main disadvantage of poor or non-uniform pore interconnectivity associated with the use of NaCl or sucrose particles as porogens [9,10]. Fig. 5 shows the typical pore morphology and surface topography of the HA-M scaffold produced with medium wax spheres. The scaffold had interconnected spherical pores with diameters ranging from 50 to 200 μm (Fig. 5a and b). These interconnected pores were
Fig. 4. The reticular structures of porous HA-S (a), HA-M (b) and HA-L (c) showing the interconnected channels with different pore sizes.
reported to benefit the circulation of body fluid and the supply of nutrients and exchange of ions [39]. Two types of HA grains were observed in Fig. 5c and d. The smaller grains were uniformly distributed on the larger grains. This structure might improve the mechanical properties of HA scaffolds. In addition, some micropores existed on the wall of macropores, which can further increase the surface roughness of the scaffolds and thus benefit protein absorption and cell adhesion [40]. 3.5. Cross sectional shrinkage (CSS)
Fig. 3. XRD patterns of the starting HA powder and the sintered HA scaffold.
Fig. 6 shows the CSS values of HA scaffolds after wax leaching and sintering. The three types of HA scaffolds had nearly the same CSS values of about 66% after wax leaching and about 81% after sintering. Therefore, scaffolds with a tailored pore size can be easily prepared by selecting the wax spheres with suitable size ranges. In the wax leaching process, the significant CSS of porous HA/CT gel samples resulted from the motion of CT chains during drying was mainly
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Fig. 5. SEM images of the typical pore structure (a) and a pore (b) and surface topography at different (c, d) magnifications of the HA-M scaffold.
In Fig. 7, the compressive strengths of the three types of HA scaffolds, which were produced with the three types of wax spheres and presented nearly the same porosity of about 86%, decreased with
increasing pore size. The relationship between compressive strength and pore size was consistent with previous studies [43]. Liu et al. demonstrated that at a same porosity the compressive strength of porous HA scaffolds decreased roughly linearly with increasing macropore size [44], because in the same cross-section area the scaffolds with smaller pore size had more struts than those with a larger pore size and thus could better distribute the load [45]. Furthermore, the present as-prepared HA scaffolds in this study exhibited higher compressive strengths than HA scaffolds with similar porosity and pore size reported in other studies [46–48], indicating that the high shrinkage of CT gel might be helpful in improving the mechanical properties of porous scaffolds.
Fig. 6. Cross-sectional shrinkage of porous HA scaffolds prepared with the three types of wax spheres after wax leaching and sintering processes.
Fig. 7. Compressive strengths of HA scaffold produced with the three types of wax spheres (mean ± standard deviation, n = 5).
dependent on the concentration of CT [41,42]. While in the sintering process, the CSS was probably attributed to the decomposition of CT, the loss of crystallization water in HA grains and the realignment of HA grains. The presence of shrinkage during wax leaching and sintering processes would facilitate the densification of scaffolds and thus improve their mechanical properties accordingly. 3.6. Compressive strength
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4. Conclusions Highly interconnected porous HA scaffolds were successfully fabricated by combining gel-casting, particle-leaching and extrusion techniques in one step. The porosities of these porous HA scaffolds were approximately 86% regardless of the size of the porogens, and the pore size can be tailored independent of porosity by this technique. The compressive strength was closely related to the pore size. With increasing pore size, the compressive strength decreased significantly. This kind of porous HA scaffolds with excellent porosity and interconnectivity is expected to be a promising bone substitute in the future clinical practice of tissue engineering. Acknowledgement This study was financially supported by National Basic Research Program (No. 2011CB707604), National Natural Science Foundation of China (No. 30870630), Ph.D. Programs Foundation of MEC (No. 20070613062) and Fundamental Research Fund of Sichuan (No. 2008JY0062). References [1] V. Karageorgiou, D. Kaplan, Biomaterials 26 (2005) 5474. [2] F.C. Fierz, F. Beckmann, M. Huser, S.H. Irsen, B. Leukers, F. Witte, O. Degistirici, A. Andronache, M. Thie, B. Müller, Biomaterials 29 (2008) 3799. [3] E. Saiz, L. Gremillard, G. Menendez, P. Miranda, K. Gryn, A.P. Tomsia, Mater. Sci. Eng. C. 27 (2007) 546. [4] O. Gauthier, J.M. Bouler, E. Aguado, P. Pilet, G. Daculsi, Biomaterials 19 (1998) 133. [5] Y. Zhang, M. Zhang, J. Biomed. Mater. Res. 61 (2002) 1. [6] C. Tsioptsias, I. Tsivintzelis, L. Papadopoulou, C. Panayiotou, Mater. Sci. Eng. C. 29 (2009) 159. [7] X. Mao, S. Wang, S. Shimai, Ceram. Int. 34 (2008) 107. [8] A. Park, B. Wu, L.G. Griffith, J. Biomat. Sci. polym. E. 9 (1998) 89. [9] E. Chevalier, D. Chulia, C. Pouget, M. Viana, J. Pharm. Sci. 97 (2008) 1135. [10] K. Rezwan, Q.Z. Chen, J.J. Blaker, A.R. Boccaccini, Biomaterials 27 (2006) 3413. [11] K. Prabhakaran, A. Melkeri, N.M. Gokhale, S.C. Sharma, Ceram. Int. 33 (2007) 77. [12] A.R. Studart, U.T. Gonzenbach, E. Tervoort, L.J. Gauckler, J. Am. Ceram. Soc. 89 (2006) 1771.
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