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chitosan–alginate composite scaffolds for bone tissue engineering
International Journal of Biological Macromolecules 51 (2012) 1079–1085
Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
In vivo evaluation of porous hydroxyapatite/chitosan–alginate composite scaffolds for bone tissue engineering Hyeong-Ho Jin a , Dong-Hyun Kim a , Tae-Wan Kim a , Keun-Koo Shin b , Jin Sup Jung b , Hong-Chae Park a , Seog-Young Yoon a,∗ a b
School of Materials Science and Engineering, Pusan National University, San 30, Jangjeon-dong, Gumjeong-gu, Busan 609-735, Republic of Korea Department of Physiology, School of Medicine, Pusan National University, Yangsan 626-770, Republic of Korea
a r t i c l e
i n f o
Article history: Received 22 May 2012 Received in revised form 3 August 2012 Accepted 23 August 2012 Available online 30 August 2012 Keywords: Hydroxyapatite Chitosan Alginate Scaffold In vivo
a b s t r a c t Porous hydroxyapatite (HAp)/chitosan–alginate composite scaffolds were prepared through in situ coprecipitation and freeze-drying for bone tissue engineering. The composite scaffolds were highly porous and interconnected with a pore size of around 50–220 m at low concentrations of HAp. As the HAp content increased, the porosity of the scaffolds decreased from 84.98 to 74.54%. An MTT assay indicates that the obtained scaffolds have no cytotoxic effects on MG-63 cells, and that they have good biocompatibility. An implantation experiment in mouse skulls revealed that the composite scaffold provides a strong positive effect on bone formation in vivo in mice. Furthermore, that HAp/chitosan–alginate composite scaffold has been shown to be more effective for new bone generation than chitosan–alginate scaffold. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Recently, tissue engineering has emerged as one such promising approach for bone repair and reconstruction [1]. Tissue engineering involves the expansion of cells from a small biopsy, followed by the culturing of the cells in temporary porous scaffolds to form the new organ or tissue [2–4]. With this approach, the porous scaffold serves an important role in the manipulation of the functions of osteoblasts and a central role in the guidance of new bone formation into desired shapes. Therefore, the scaffold materials must be biocompatible, osteoconductive, and osteointegrative, and have enough mechanical strength to provide structural support. Hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HAp) has been widely used in medicine and dentistry because it is biocompatible, osteoconductive, and has excellent chemical and biological affinity with bony tissue [5]. As a result, HAp is accepted as a bioactive scaffold material for guided bone regeneration. In addition to the requirements for chemical composition of the scaffold material, an interconnected porous structure is necessary to allow cell attachment, proliferation, and differentiation, and to provide pathways for biofluids. Chitosan is a natural cationic polymer that is biologically renewable, biodegradable, biocompatible, non-antigenic,
∗ Corresponding author. Tel.: +82 51 510 2487; fax: +82 51 512 0528. E-mail address: [email protected] (S.-Y. Yoon). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.08.027
non-toxic, and biofunctional. It has been studied as a useful biomaterial in diverse tissue engineering applications because of its hydrophilic surface promoting cell adhesion, proliferation and differentiation, good biocompatibility and good host response, high biochemical significance in hemostasis, angiogenesis and macrophage activation, biodegradability by lysozyme and other enzymes, bactericidal/bacteriostatic activity, and capacity to maintain a predefined shape after cross-linking [3,6–8]. Alginate is a biocompatible, hydrophilic, and biodegradable anionic polymer under normal physiological conditions and is widely used as an instant gel for bone tissue engineering [9–11]. Chitosan–alginate scaffolds have been tested in the regeneration of various tissues and bone with promising results [12,13]. However, many biopolymers are fragile and do not exhibit undoubtedly biocompatible behavior. Some polymer and bioactive ceramics composites have been developed for bone tissue engineering in order to increase the bioactivity and mechanical properties of the materials [14–17]. Hydroxyapatite (HAp)/polymer composites have attracted a great deal of attention because they exhibit osteoconductivity because of the presence of HAp [18,19], which has a similar chemical composition and structure as the mineral phase of human bones and hard tissues. In this study, porous HAp/chitosan–alginate composite scaffolds were prepared through in situ co-precipitation and freezedrying for bone tissue engineering. With the aim of fabricating HAp/chitosan–alginate composite scaffolds and in vivo culture of bone tissue engineered constructs.
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Fig. 1. (a) Photograph of HAp/chitosan–alginate composite scaffolds, and SEM morphology of the composite scaffolds with different HAp contents; (b) 0, (c) 10, (d) 30, (e) 50 and (f) 70 wt%.
2. Experimental 2.1. Preparation of HAp/chitosan–alginate composite scaffolds The HAp/chitosan–alginate composite scaffolds were synthesized through in situ co-precipitation. A chitosan aqueous solution was prepared by dissolving 3.84 g of the chitosan powder (viscosity > 200 cP:1 wt% solution in the 1 wt% acetic acid solution in Brookfield, Sigma–Aldrich) in 64 ml 1 M acetic acid. To prepare the alginate solution, 3.84 g of the sodium alginate powder (viscosity 200–400 cP for 1 wt% solution at 20 ◦ C, Sigma–Aldrich) was dissolved in 96 ml of 1 M NaOH. H3 PO4 and Ca(OH)2 were added to the chitosan aqueous and alginate solutions, respectively, to form the HAp/chitosan–alginate composite scaffolds. The ratios of chitosan to H3 PO4 and alginate to Ca(OH)2 were adjusted so that the final HAp/chitosan–alginate weight ratios were 10/90 and 70/30, respectively. The resulting suspension was mixed under constant stirring in a blender for 1 h. Acetic acid was gradually added
Fig. 2. Pore diameter of HAp/chitosan–alginate composite scaffolds with different HAp contents.
drop-wise to the suspension until a pH of 7.4 was obtained. The slurry was placed into 24-well cell culture plates and stored in a freezer at −15 ◦ C until frozen. Subsequently, the samples were lyophilized in a freeze dryer at −80 ◦ C for 24 h. The dried samples were cross-linked with a 1% (w/v) CaCl2 solution for 15 min and then immersed in distilled water for 24 h to remove any residual sodium acetate and unbound CaCl2 [12]. After immersion, the samples were washed three times and then freeze-dried at −80 ◦ C for 24 h to obtain the HAp/chitosan–alginate composite scaffolds. The morphologies, pore configuration, and pore size of the HAp/chitosan–alginate composite scaffolds were investigated using scanning electron microscopy (SEM, S-4300, Hitachi, Japan). The porosity and density were measured by the liquid displacement method [18]. 2.2. Cell culture The cytotoxicity of the HAp/chitosan–alginate composite scaffolds was assessed using the MTT (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide) assay [20]. MG-63 cells
Fig. 3. Density and porosity of HAp/chitosan–alginate composite scaffolds with different HAp contents.
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Fig. 4. (a) Cell viability (MTT assay) of HAp/chitosan–alginate composite scaffolds with different HAp contents. (b) SEM micrographs of the surface and cross-section of the 30 wt% HAp/chitosan–alginate composite scaffold after MTT test.
seeded in 24-well plates with the composite scaffolds at a density of 1 × 105 cells/well, and the plates was incubated at 37 in 5% CO2 . The MG-63 cells were treated with chitosan–alginate scaffold as control [12] and the composite scaffolds for 1, 3, 5, and 7 days. MTT solution was added to each well (final concentration ∼50 mg/ml). The cells were incubated at 37 ◦ C for 3 h and dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance was read at 570 nm in an ELISA reader (E-MAX Molecular Devices) against a reference wavelength of 490 nm. 2.3. In vivo test of the composite scaffolds The HAp/chitosan–alginate composite scaffolds were evaluated qualitatively in vivo using critical-sized calvarial bone defects in adult (6-week-old) severe combined immunodeficient (SCID) mice. The surgical procedures were performed in aseptic conditions under general anesthesia. Briefly, a linear incision (1 cm long) was made on the left side of the skull and the scalp was dissected to expose the calvaria. The periosteum was carefully peeled off and 2 lateral 4-mm-wide calvarial bone defects were performed in each animal using a 3-mm-diameter trephine bur using a slow-speed dental drill. To avoid tissue damage due to overheating, 0.9% saline was dripped onto the contact point between the bur and bone
and great care was taken to avoid dura mater injury. One defect was then used for implanting with HAp/chitosan–alginate composite scaffolds while the contralateral site was implanted with chitosan–alginate scaffolds as a control [12]. The animals were euthanized after 4 and 8 weeks by exposure to hyperbaric carbon dioxide. At each time point, the skulls were harvested and fixed in 4% paraformaldehyde for 12 h. Representative skulls stained with 1 mg/ml alizarin red for 12 h to view bone formation. Calvaria were X-rayed using a volumetric computed tomography (CT) scanner (NFR-MXSCAN-G90: NanoFocusRay, Iksan, Korea) at 50 kVp, 65 A, and 470-ms per frame and then decalcified overnight with decalcifying solution (10% EDTA). Samples were then trimmed, processed, and embedded in paraffin wax. A micro-CT image of mouse calvaria was taken using the CT scanner without having to move the animal’s head position. Paraffin-embedded samples were sectioned at 10-mm thickness with a microtome. Sections were floated in a water bath at 40 ◦ C, placed on poly-l-lysine-coated polysine microscope slides and baked at 37 ◦ C overnight. For hematoxylin and eosin (H&E) staining, sections were dewaxed in xylene and rehydrated in ethanol baths. The sections slides were stained with H&E viewed under an optical microscope for histological observation.
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Fig. 5. Micro-CT images of the mouse skull after transplantation for (a) 4 and (c) 8 weeks. Photographs of mice skulls stained with alizarin red after transplantation for (b) 4 and (d) 8 weeks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3. Results and discussion
3.2. The cytotoxicity of the scaffolds
3.1. Structure of the composite scaffolds
Fig. 4 shows the results of the MTT test and the SEM micrographs of the HAp/chitosan–alginate composite scaffolds. In Fig. 4(a), the density on control and the HAp/chitosan–alginate composite scaffolds increased with increasing time. The density on the composite scaffolds was higher than that on control. These results show that the composite scaffolds exhibited no cytotoxic effects on the MG63 cells, and have good biocompatibility. After the MTT test, the surface and cross-section of the composite scaffolds with 30 wt% HAp are shown in Fig. 4(b). The cells were observed at both surface and cross-section of the composite scaffolds, indicating that the structure of the HAp/chitosan–alginate composite scaffold was favorable for cell attachment and new bone tissue growth. Therefore, 30 wt% HAp/chitosan–alginate composite scaffold could have good biocompatibility and osteoconductivity for bone tissue engineering as the results in Figs. 1–4.
The SEM micrographs and the pore size of the HAp/chitosan–alginate composite scaffolds with different HAp contents were shown in Figs. 1 and 2. The chitosan–alginate polyelectrolyte complex was highly porous and interconnected with a pore size of around 50–220 m. At low HAp contents, the pore structure of the composite scaffolds (Fig. 1(b) and (c)) was similar to the chitosan–alginate polyelectrolyte complex (Fig. 1(a)). However, the pore size decreased with increasing HAp content above 30 wt%, and eventually the pore structure locally collapsed and appeared to be agglomerated (Fig. 1(d) and (e)). Characterization of the composite scaffolds was described in our previous report [21]. Chitosan and alginate, the polymer matrix of the composite scaffolds, were cross-linked, and the synthesized HAp was low crystallized. The density and porosity of the composite scaffolds with different HAp contents were shown in Fig. 3. Porosity is based on the presence of open pores which are related to properties such as permeability and surface area of the porous structure. High porosity usually means a high surface area/volume ratio, and thus favors cell adhesion to the scaffold and promotes bone tissue regeneration [22]. As the HAp content increased, the porosity of the composite scaffolds decreased, and the density increased. These phenomena would be attributed that the pores became interconnected with more dense and thicker pore walls with increasing of the HAp content. It is worthwhile mentioning that the density of a scaffold decreases with the level of porosity at constant HAp content. The porosity of the composite scaffolds ranged between 84.98 and 74.54%.
3.3. In vivo tissue compatibility Bone defects of the skull were implanted with two types of scaffolds. One site was implanted with chitosan–alginate scaffold as a control. 30 wt% HAp/chitosan–alginate composite scaffold showing the good cell affinity as the result in Fig. 4, was inserted on the opposite site. During the in vivo experiment, mice remained in good health and did not show any wound complications. At explanation, no inflammatory signs or adverse tissue reactions were seen. It indicates that the composite scaffold could be suitable for cell attachment and proliferation as bone substitute materials. Fig. 5 shows micro-CT images and alizarin red analysis of a mouse skull after 4, 8 weeks. After 4 weeks in vivo, low-density mineralization in the area of the bone defect was produced in the site implanted with
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Fig. 6. Histological analysis of (a) the mouse skull with H&E staining after 4 weeks, (b) the site implanted with control sample, (d) high magnification of (b), (c) the site implanted with HAp/chitosan–alginate composite scaffold, (e) high magnification of (c) (HBT: host bone tissue, NBT: new bone tissue, Ob: osteoblast).
the composite scaffold, whereas mineralization was not detected in the control site (Fig. 5(a)). It indicates that the site implanted with the composite scaffold had started to heal with new generation bone. These features were more obvious after 8 weeks of implantation as shown in Fig. 5(c). After 8 weeks of implantation, the site implanted with the composite scaffold had formed sufficient bone to span most of the bone defect, even though the control site had just observed low-density mineralization. Alizarin red analysis of the mouse skull after in vivo for 4 and 8 weeks was shown in Fig. 5(b) and (d). Alizarin red is used in study new bone generation because it stains free calcium and certain calcium compounds a red or light purple color [23]. After 4 weeks in vivo, the light red color can be seen in both implanted sites in Fig. 5(b), however the site implanted with the composite scaffold was observed dark red color after 8 weeks in vivo as shown in Fig. 5(d). Alizarin red staining localized bone formation to areas of mineralization onto the site implanted with the composite scaffold. Figs. 6 and 7 show histological analysis of a mouse skull with H&E staining after 4 and 8 weeks in vivo. H&E staining
showed characteristic bone morphology on the implanted sites. After 4 weeks, the appearance and shape of both implanted sites showed no obvious change, although slight connection between the material and surrounding tissue with osteoblast was detected as shown in Fig. 6. However, in Fig. 7, newly formed bone tissue which filled the area of the bone defect was observed in the site implanted with the composite scaffold after 8 weeks in vivo, whereas bone tissue was not observed in the control site. The new bone tissue in the site implanted with the composite scaffold had not only osteoblast but also osteoclast and Howship’s lacuna. In general, bone regeneration is accompanied by bone reabsorption and bone formation with osteoblast and osteoclast [24]. Therefore, the new bone tissue in the site implanted with the composite scaffold was actively generated, suggesting that degradation of HAp promoted osteoid production [25,26]. In summary, implantation experiments in mouse skulls in the present study have revealed that HAp/chitosan–alginate composite scaffold is more effective for new bone generation than chitosan–alginate scaffold.
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Fig. 7. Histological analysis of (a) the mouse skull with H&E staining after 8 weeks, (b) the site implanted with control sample, (d) high magnification of (b), (c) the site implanted with HAp/chitosan–alginate composite scaffold, (e) high magnification of (c) (HBT: host bone tissue, NBT: new bone tissue, Ob: osteoblast, Oc: osteoclast, B: bone, Hl: Howship’s lacuna).
4. Conclusions Porous HAp/chitosan–alginate composite scaffolds were prepared through in situ co-precipitation and freeze-drying for bone tissue engineering. At low HAp concentrations, the HAp/chitosan–alginate composite scaffolds were highly porous and interconnected with a pore size of around 50–220 m. As the HAp content increased, the porosity of the scaffolds decreased from 84.98 to 74.54%, whereas the density increased from 0.12 to 0.24 g/cm3 . An MTT assay indicates that the obtained scaffolds have no cytotoxic effects on MG-63 cells, and that they have good biocompatibility. 30 wt% HAp/chitosan–alginate composite scaffold could have good biocompatibility and osteoconductivity for bone tissue engineering. The cells were observed at both the surface and cross-section of the composite scaffolds after MTT test. After 8 weeks of implantation, the site implanted with the composite scaffold had formed sufficient bone to span most of the bone defect, even though the control site had just observed low-density mineralization. Alizarin red staining localized bone formation to areas of mineralization onto the site implanted with the composite
scaffold. Newly formed bone tissue which filled the area of the bone defect was observed in the site implanted with the composite scaffold after 8 weeks in vivo, whereas bone tissue was not observed in the control site. Acknowledgment This work was supported by National Research Foundation of Korea Grant funded by the Korean Government Ministry of Education, Science and Technology (NRF-2011-355-D00021). References [1] [2] [3] [4] [5] [6] [7]
R.F. Service, Science 289 (2000) 1498–1500. S. Bose, S. Tarafder, Acta Biomater. 8 (2012) 1401–1421. R.A.A. Muzzarelli, Carbohydr. Polym. 83 (4) (2011) 1433–1445. J.R. Jones, J. Eur. Ceram. Soc. 29 (2009) 1275–1281. L.L. Hench, J. Am. Ceram. Soc. 74 (1991) 1487–1510. E. Khor, L.Y. Lim, Biomaterials 24 (2003) 2339–2349. R.A.A. Muzzarelli, V. Ramos, V. Stanic, B. Dubini, M. Mattioli-Belmonte, G. Tosi, R. Giardino, Carbohydr. Polym. 36 (4) (1998) 267–276.
H.-H. Jin et al. / International Journal of Biological Macromolecules 51 (2012) 1079–1085 [8] M. Mattioli-Belmonte, G. Biagini, R.A.A. Muzzarelli, C. Castaldini, M.G. Gandolfi, A. Krajewski, A. Ravaglioli, M. Fini, R. Giardino, J. Bioact. Compat. Polym. 10 (1995) 249–257. [9] S. Zmora, R. Glicklis, S. Cohen, Biomaterials 23 (2002) 4087–4094. [10] S. Sakai, H. Masuhara, Y. Yamada, T. Ono, H. Ijima, K. Kawakami, J. Biosci. Bioeng. 100 (2005) 127–129. [11] W.M. Parks, Y.B. Guo, Mater. Sci. Eng. C 28 (2008) 1435–1440. [12] Z. Li, H.R. Ramay, K.D. Hauch, D. Xial, M. Zhang, Biomaterials 26 (2005) 3919–3928. [13] F.M. Kievit, S.J. Florczyk, M.C. Leung, O. Veiseh, J.O. Park, M.L. Disis, M. Zhang, Biomaterials 31 (2010) 5903–5910. [14] L. Kong, Y. Gao, W. Cao, Y. Gong, N. Zhao, X. Zhang, J. Biomed. Mater. Res. A 75 (2005) 275–282. [15] K. Rezwan, Q.Z. Chen, J.J. Blaker, A.R. Boccaccini, Biomaterials 27 (2006) 3413–3431. [16] J. Han, Z. Zhou, R. Yin, D. Yang, J. Nie, Int. J. Biol. Macromol. 46 (2010) 199–205.
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[17] G. Wei, P.X. Ma, Biomaterials 25 (2004) 4749–4757. [18] Y. Zhang, M. Zhang, J. Biomed. Mater. Res. A 62 (2002) 378–386. [19] L. Jiang, Y. Li, X. Wang, L. Zhang, J. Wen, M. Gong, Carbohydr. Polym. 74 (2008) 680–684. [20] G. Mickisch, S. Fajta, G. Keilhauer, E. Schlick, R. Tschada, P. Alken, Urol. Res. 18 (1990) 131–136. [21] H.H. Jin, C.H. Lee, W.K. Lee, J.K. Lee, H.C. Park, S.Y. Yoon, Mater. Lett. 62 (2008) 1630–1633. [22] T.J. Flatley, K.L. Lynch, M. Benson, Clin. Orthop. 179 (1983) 246–252. [23] H. Puchtler, S.N. Meloan, M.S. Terry, J. Histochem. Cytochem. 17 (1969) 110–124. [24] G. Vaes, Basic Sci. Pathol. 231 (1988) 239–271. [25] N. Masahisa, N. Takashi, K. Tadashi, T. Masami, O. Masaki, Biomaterials 17 (1996) 1771–1777. [26] J.S. Sun, Y.H. Tsuang, C.J. Liao, H.C. Liu, Y.S. Hang, F.H. Lin, J. Biomed. Mater. Res. A 37 (1997) 324–334.
Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
In vivo evaluation of porous hydroxyapatite/chitosan–alginate composite scaffolds for bone tissue engineering Hyeong-Ho Jin a , Dong-Hyun Kim a , Tae-Wan Kim a , Keun-Koo Shin b , Jin Sup Jung b , Hong-Chae Park a , Seog-Young Yoon a,∗ a b
School of Materials Science and Engineering, Pusan National University, San 30, Jangjeon-dong, Gumjeong-gu, Busan 609-735, Republic of Korea Department of Physiology, School of Medicine, Pusan National University, Yangsan 626-770, Republic of Korea
a r t i c l e
i n f o
Article history: Received 22 May 2012 Received in revised form 3 August 2012 Accepted 23 August 2012 Available online 30 August 2012 Keywords: Hydroxyapatite Chitosan Alginate Scaffold In vivo
a b s t r a c t Porous hydroxyapatite (HAp)/chitosan–alginate composite scaffolds were prepared through in situ coprecipitation and freeze-drying for bone tissue engineering. The composite scaffolds were highly porous and interconnected with a pore size of around 50–220 m at low concentrations of HAp. As the HAp content increased, the porosity of the scaffolds decreased from 84.98 to 74.54%. An MTT assay indicates that the obtained scaffolds have no cytotoxic effects on MG-63 cells, and that they have good biocompatibility. An implantation experiment in mouse skulls revealed that the composite scaffold provides a strong positive effect on bone formation in vivo in mice. Furthermore, that HAp/chitosan–alginate composite scaffold has been shown to be more effective for new bone generation than chitosan–alginate scaffold. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Recently, tissue engineering has emerged as one such promising approach for bone repair and reconstruction [1]. Tissue engineering involves the expansion of cells from a small biopsy, followed by the culturing of the cells in temporary porous scaffolds to form the new organ or tissue [2–4]. With this approach, the porous scaffold serves an important role in the manipulation of the functions of osteoblasts and a central role in the guidance of new bone formation into desired shapes. Therefore, the scaffold materials must be biocompatible, osteoconductive, and osteointegrative, and have enough mechanical strength to provide structural support. Hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HAp) has been widely used in medicine and dentistry because it is biocompatible, osteoconductive, and has excellent chemical and biological affinity with bony tissue [5]. As a result, HAp is accepted as a bioactive scaffold material for guided bone regeneration. In addition to the requirements for chemical composition of the scaffold material, an interconnected porous structure is necessary to allow cell attachment, proliferation, and differentiation, and to provide pathways for biofluids. Chitosan is a natural cationic polymer that is biologically renewable, biodegradable, biocompatible, non-antigenic,
∗ Corresponding author. Tel.: +82 51 510 2487; fax: +82 51 512 0528. E-mail address: [email protected] (S.-Y. Yoon). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.08.027
non-toxic, and biofunctional. It has been studied as a useful biomaterial in diverse tissue engineering applications because of its hydrophilic surface promoting cell adhesion, proliferation and differentiation, good biocompatibility and good host response, high biochemical significance in hemostasis, angiogenesis and macrophage activation, biodegradability by lysozyme and other enzymes, bactericidal/bacteriostatic activity, and capacity to maintain a predefined shape after cross-linking [3,6–8]. Alginate is a biocompatible, hydrophilic, and biodegradable anionic polymer under normal physiological conditions and is widely used as an instant gel for bone tissue engineering [9–11]. Chitosan–alginate scaffolds have been tested in the regeneration of various tissues and bone with promising results [12,13]. However, many biopolymers are fragile and do not exhibit undoubtedly biocompatible behavior. Some polymer and bioactive ceramics composites have been developed for bone tissue engineering in order to increase the bioactivity and mechanical properties of the materials [14–17]. Hydroxyapatite (HAp)/polymer composites have attracted a great deal of attention because they exhibit osteoconductivity because of the presence of HAp [18,19], which has a similar chemical composition and structure as the mineral phase of human bones and hard tissues. In this study, porous HAp/chitosan–alginate composite scaffolds were prepared through in situ co-precipitation and freezedrying for bone tissue engineering. With the aim of fabricating HAp/chitosan–alginate composite scaffolds and in vivo culture of bone tissue engineered constructs.
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Fig. 1. (a) Photograph of HAp/chitosan–alginate composite scaffolds, and SEM morphology of the composite scaffolds with different HAp contents; (b) 0, (c) 10, (d) 30, (e) 50 and (f) 70 wt%.
2. Experimental 2.1. Preparation of HAp/chitosan–alginate composite scaffolds The HAp/chitosan–alginate composite scaffolds were synthesized through in situ co-precipitation. A chitosan aqueous solution was prepared by dissolving 3.84 g of the chitosan powder (viscosity > 200 cP:1 wt% solution in the 1 wt% acetic acid solution in Brookfield, Sigma–Aldrich) in 64 ml 1 M acetic acid. To prepare the alginate solution, 3.84 g of the sodium alginate powder (viscosity 200–400 cP for 1 wt% solution at 20 ◦ C, Sigma–Aldrich) was dissolved in 96 ml of 1 M NaOH. H3 PO4 and Ca(OH)2 were added to the chitosan aqueous and alginate solutions, respectively, to form the HAp/chitosan–alginate composite scaffolds. The ratios of chitosan to H3 PO4 and alginate to Ca(OH)2 were adjusted so that the final HAp/chitosan–alginate weight ratios were 10/90 and 70/30, respectively. The resulting suspension was mixed under constant stirring in a blender for 1 h. Acetic acid was gradually added
Fig. 2. Pore diameter of HAp/chitosan–alginate composite scaffolds with different HAp contents.
drop-wise to the suspension until a pH of 7.4 was obtained. The slurry was placed into 24-well cell culture plates and stored in a freezer at −15 ◦ C until frozen. Subsequently, the samples were lyophilized in a freeze dryer at −80 ◦ C for 24 h. The dried samples were cross-linked with a 1% (w/v) CaCl2 solution for 15 min and then immersed in distilled water for 24 h to remove any residual sodium acetate and unbound CaCl2 [12]. After immersion, the samples were washed three times and then freeze-dried at −80 ◦ C for 24 h to obtain the HAp/chitosan–alginate composite scaffolds. The morphologies, pore configuration, and pore size of the HAp/chitosan–alginate composite scaffolds were investigated using scanning electron microscopy (SEM, S-4300, Hitachi, Japan). The porosity and density were measured by the liquid displacement method [18]. 2.2. Cell culture The cytotoxicity of the HAp/chitosan–alginate composite scaffolds was assessed using the MTT (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide) assay [20]. MG-63 cells
Fig. 3. Density and porosity of HAp/chitosan–alginate composite scaffolds with different HAp contents.
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Fig. 4. (a) Cell viability (MTT assay) of HAp/chitosan–alginate composite scaffolds with different HAp contents. (b) SEM micrographs of the surface and cross-section of the 30 wt% HAp/chitosan–alginate composite scaffold after MTT test.
seeded in 24-well plates with the composite scaffolds at a density of 1 × 105 cells/well, and the plates was incubated at 37 in 5% CO2 . The MG-63 cells were treated with chitosan–alginate scaffold as control [12] and the composite scaffolds for 1, 3, 5, and 7 days. MTT solution was added to each well (final concentration ∼50 mg/ml). The cells were incubated at 37 ◦ C for 3 h and dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance was read at 570 nm in an ELISA reader (E-MAX Molecular Devices) against a reference wavelength of 490 nm. 2.3. In vivo test of the composite scaffolds The HAp/chitosan–alginate composite scaffolds were evaluated qualitatively in vivo using critical-sized calvarial bone defects in adult (6-week-old) severe combined immunodeficient (SCID) mice. The surgical procedures were performed in aseptic conditions under general anesthesia. Briefly, a linear incision (1 cm long) was made on the left side of the skull and the scalp was dissected to expose the calvaria. The periosteum was carefully peeled off and 2 lateral 4-mm-wide calvarial bone defects were performed in each animal using a 3-mm-diameter trephine bur using a slow-speed dental drill. To avoid tissue damage due to overheating, 0.9% saline was dripped onto the contact point between the bur and bone
and great care was taken to avoid dura mater injury. One defect was then used for implanting with HAp/chitosan–alginate composite scaffolds while the contralateral site was implanted with chitosan–alginate scaffolds as a control [12]. The animals were euthanized after 4 and 8 weeks by exposure to hyperbaric carbon dioxide. At each time point, the skulls were harvested and fixed in 4% paraformaldehyde for 12 h. Representative skulls stained with 1 mg/ml alizarin red for 12 h to view bone formation. Calvaria were X-rayed using a volumetric computed tomography (CT) scanner (NFR-MXSCAN-G90: NanoFocusRay, Iksan, Korea) at 50 kVp, 65 A, and 470-ms per frame and then decalcified overnight with decalcifying solution (10% EDTA). Samples were then trimmed, processed, and embedded in paraffin wax. A micro-CT image of mouse calvaria was taken using the CT scanner without having to move the animal’s head position. Paraffin-embedded samples were sectioned at 10-mm thickness with a microtome. Sections were floated in a water bath at 40 ◦ C, placed on poly-l-lysine-coated polysine microscope slides and baked at 37 ◦ C overnight. For hematoxylin and eosin (H&E) staining, sections were dewaxed in xylene and rehydrated in ethanol baths. The sections slides were stained with H&E viewed under an optical microscope for histological observation.
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Fig. 5. Micro-CT images of the mouse skull after transplantation for (a) 4 and (c) 8 weeks. Photographs of mice skulls stained with alizarin red after transplantation for (b) 4 and (d) 8 weeks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3. Results and discussion
3.2. The cytotoxicity of the scaffolds
3.1. Structure of the composite scaffolds
Fig. 4 shows the results of the MTT test and the SEM micrographs of the HAp/chitosan–alginate composite scaffolds. In Fig. 4(a), the density on control and the HAp/chitosan–alginate composite scaffolds increased with increasing time. The density on the composite scaffolds was higher than that on control. These results show that the composite scaffolds exhibited no cytotoxic effects on the MG63 cells, and have good biocompatibility. After the MTT test, the surface and cross-section of the composite scaffolds with 30 wt% HAp are shown in Fig. 4(b). The cells were observed at both surface and cross-section of the composite scaffolds, indicating that the structure of the HAp/chitosan–alginate composite scaffold was favorable for cell attachment and new bone tissue growth. Therefore, 30 wt% HAp/chitosan–alginate composite scaffold could have good biocompatibility and osteoconductivity for bone tissue engineering as the results in Figs. 1–4.
The SEM micrographs and the pore size of the HAp/chitosan–alginate composite scaffolds with different HAp contents were shown in Figs. 1 and 2. The chitosan–alginate polyelectrolyte complex was highly porous and interconnected with a pore size of around 50–220 m. At low HAp contents, the pore structure of the composite scaffolds (Fig. 1(b) and (c)) was similar to the chitosan–alginate polyelectrolyte complex (Fig. 1(a)). However, the pore size decreased with increasing HAp content above 30 wt%, and eventually the pore structure locally collapsed and appeared to be agglomerated (Fig. 1(d) and (e)). Characterization of the composite scaffolds was described in our previous report [21]. Chitosan and alginate, the polymer matrix of the composite scaffolds, were cross-linked, and the synthesized HAp was low crystallized. The density and porosity of the composite scaffolds with different HAp contents were shown in Fig. 3. Porosity is based on the presence of open pores which are related to properties such as permeability and surface area of the porous structure. High porosity usually means a high surface area/volume ratio, and thus favors cell adhesion to the scaffold and promotes bone tissue regeneration [22]. As the HAp content increased, the porosity of the composite scaffolds decreased, and the density increased. These phenomena would be attributed that the pores became interconnected with more dense and thicker pore walls with increasing of the HAp content. It is worthwhile mentioning that the density of a scaffold decreases with the level of porosity at constant HAp content. The porosity of the composite scaffolds ranged between 84.98 and 74.54%.
3.3. In vivo tissue compatibility Bone defects of the skull were implanted with two types of scaffolds. One site was implanted with chitosan–alginate scaffold as a control. 30 wt% HAp/chitosan–alginate composite scaffold showing the good cell affinity as the result in Fig. 4, was inserted on the opposite site. During the in vivo experiment, mice remained in good health and did not show any wound complications. At explanation, no inflammatory signs or adverse tissue reactions were seen. It indicates that the composite scaffold could be suitable for cell attachment and proliferation as bone substitute materials. Fig. 5 shows micro-CT images and alizarin red analysis of a mouse skull after 4, 8 weeks. After 4 weeks in vivo, low-density mineralization in the area of the bone defect was produced in the site implanted with
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Fig. 6. Histological analysis of (a) the mouse skull with H&E staining after 4 weeks, (b) the site implanted with control sample, (d) high magnification of (b), (c) the site implanted with HAp/chitosan–alginate composite scaffold, (e) high magnification of (c) (HBT: host bone tissue, NBT: new bone tissue, Ob: osteoblast).
the composite scaffold, whereas mineralization was not detected in the control site (Fig. 5(a)). It indicates that the site implanted with the composite scaffold had started to heal with new generation bone. These features were more obvious after 8 weeks of implantation as shown in Fig. 5(c). After 8 weeks of implantation, the site implanted with the composite scaffold had formed sufficient bone to span most of the bone defect, even though the control site had just observed low-density mineralization. Alizarin red analysis of the mouse skull after in vivo for 4 and 8 weeks was shown in Fig. 5(b) and (d). Alizarin red is used in study new bone generation because it stains free calcium and certain calcium compounds a red or light purple color [23]. After 4 weeks in vivo, the light red color can be seen in both implanted sites in Fig. 5(b), however the site implanted with the composite scaffold was observed dark red color after 8 weeks in vivo as shown in Fig. 5(d). Alizarin red staining localized bone formation to areas of mineralization onto the site implanted with the composite scaffold. Figs. 6 and 7 show histological analysis of a mouse skull with H&E staining after 4 and 8 weeks in vivo. H&E staining
showed characteristic bone morphology on the implanted sites. After 4 weeks, the appearance and shape of both implanted sites showed no obvious change, although slight connection between the material and surrounding tissue with osteoblast was detected as shown in Fig. 6. However, in Fig. 7, newly formed bone tissue which filled the area of the bone defect was observed in the site implanted with the composite scaffold after 8 weeks in vivo, whereas bone tissue was not observed in the control site. The new bone tissue in the site implanted with the composite scaffold had not only osteoblast but also osteoclast and Howship’s lacuna. In general, bone regeneration is accompanied by bone reabsorption and bone formation with osteoblast and osteoclast [24]. Therefore, the new bone tissue in the site implanted with the composite scaffold was actively generated, suggesting that degradation of HAp promoted osteoid production [25,26]. In summary, implantation experiments in mouse skulls in the present study have revealed that HAp/chitosan–alginate composite scaffold is more effective for new bone generation than chitosan–alginate scaffold.
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Fig. 7. Histological analysis of (a) the mouse skull with H&E staining after 8 weeks, (b) the site implanted with control sample, (d) high magnification of (b), (c) the site implanted with HAp/chitosan–alginate composite scaffold, (e) high magnification of (c) (HBT: host bone tissue, NBT: new bone tissue, Ob: osteoblast, Oc: osteoclast, B: bone, Hl: Howship’s lacuna).
4. Conclusions Porous HAp/chitosan–alginate composite scaffolds were prepared through in situ co-precipitation and freeze-drying for bone tissue engineering. At low HAp concentrations, the HAp/chitosan–alginate composite scaffolds were highly porous and interconnected with a pore size of around 50–220 m. As the HAp content increased, the porosity of the scaffolds decreased from 84.98 to 74.54%, whereas the density increased from 0.12 to 0.24 g/cm3 . An MTT assay indicates that the obtained scaffolds have no cytotoxic effects on MG-63 cells, and that they have good biocompatibility. 30 wt% HAp/chitosan–alginate composite scaffold could have good biocompatibility and osteoconductivity for bone tissue engineering. The cells were observed at both the surface and cross-section of the composite scaffolds after MTT test. After 8 weeks of implantation, the site implanted with the composite scaffold had formed sufficient bone to span most of the bone defect, even though the control site had just observed low-density mineralization. Alizarin red staining localized bone formation to areas of mineralization onto the site implanted with the composite
scaffold. Newly formed bone tissue which filled the area of the bone defect was observed in the site implanted with the composite scaffold after 8 weeks in vivo, whereas bone tissue was not observed in the control site. Acknowledgment This work was supported by National Research Foundation of Korea Grant funded by the Korean Government Ministry of Education, Science and Technology (NRF-2011-355-D00021). References [1] [2] [3] [4] [5] [6] [7]
R.F. Service, Science 289 (2000) 1498–1500. S. Bose, S. Tarafder, Acta Biomater. 8 (2012) 1401–1421. R.A.A. Muzzarelli, Carbohydr. Polym. 83 (4) (2011) 1433–1445. J.R. Jones, J. Eur. Ceram. Soc. 29 (2009) 1275–1281. L.L. Hench, J. Am. Ceram. Soc. 74 (1991) 1487–1510. E. Khor, L.Y. Lim, Biomaterials 24 (2003) 2339–2349. R.A.A. Muzzarelli, V. Ramos, V. Stanic, B. Dubini, M. Mattioli-Belmonte, G. Tosi, R. Giardino, Carbohydr. Polym. 36 (4) (1998) 267–276.
H.-H. Jin et al. / International Journal of Biological Macromolecules 51 (2012) 1079–1085 [8] M. Mattioli-Belmonte, G. Biagini, R.A.A. Muzzarelli, C. Castaldini, M.G. Gandolfi, A. Krajewski, A. Ravaglioli, M. Fini, R. Giardino, J. Bioact. Compat. Polym. 10 (1995) 249–257. [9] S. Zmora, R. Glicklis, S. Cohen, Biomaterials 23 (2002) 4087–4094. [10] S. Sakai, H. Masuhara, Y. Yamada, T. Ono, H. Ijima, K. Kawakami, J. Biosci. Bioeng. 100 (2005) 127–129. [11] W.M. Parks, Y.B. Guo, Mater. Sci. Eng. C 28 (2008) 1435–1440. [12] Z. Li, H.R. Ramay, K.D. Hauch, D. Xial, M. Zhang, Biomaterials 26 (2005) 3919–3928. [13] F.M. Kievit, S.J. Florczyk, M.C. Leung, O. Veiseh, J.O. Park, M.L. Disis, M. Zhang, Biomaterials 31 (2010) 5903–5910. [14] L. Kong, Y. Gao, W. Cao, Y. Gong, N. Zhao, X. Zhang, J. Biomed. Mater. Res. A 75 (2005) 275–282. [15] K. Rezwan, Q.Z. Chen, J.J. Blaker, A.R. Boccaccini, Biomaterials 27 (2006) 3413–3431. [16] J. Han, Z. Zhou, R. Yin, D. Yang, J. Nie, Int. J. Biol. Macromol. 46 (2010) 199–205.
1085
[17] G. Wei, P.X. Ma, Biomaterials 25 (2004) 4749–4757. [18] Y. Zhang, M. Zhang, J. Biomed. Mater. Res. A 62 (2002) 378–386. [19] L. Jiang, Y. Li, X. Wang, L. Zhang, J. Wen, M. Gong, Carbohydr. Polym. 74 (2008) 680–684. [20] G. Mickisch, S. Fajta, G. Keilhauer, E. Schlick, R. Tschada, P. Alken, Urol. Res. 18 (1990) 131–136. [21] H.H. Jin, C.H. Lee, W.K. Lee, J.K. Lee, H.C. Park, S.Y. Yoon, Mater. Lett. 62 (2008) 1630–1633. [22] T.J. Flatley, K.L. Lynch, M. Benson, Clin. Orthop. 179 (1983) 246–252. [23] H. Puchtler, S.N. Meloan, M.S. Terry, J. Histochem. Cytochem. 17 (1969) 110–124. [24] G. Vaes, Basic Sci. Pathol. 231 (1988) 239–271. [25] N. Masahisa, N. Takashi, K. Tadashi, T. Masami, O. Masaki, Biomaterials 17 (1996) 1771–1777. [26] J.S. Sun, Y.H. Tsuang, C.J. Liao, H.C. Liu, Y.S. Hang, F.H. Lin, J. Biomed. Mater. Res. A 37 (1997) 324–334.