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collagen composite
Materials Science and Engineering C 31 (2011) 683–687
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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
Biomimetic properties of an injectable chitosan/nano-hydroxyapatite/collagen composite Zhi Huang a, Qingling Feng a,⁎, Bo Yu b, Songjian Li b a b
Laboratory of Advanced Materials, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084, China Department of Orthopedics, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China
a r t i c l e
i n f o
Article history: Received 25 May 2010 Received in revised form 2 November 2010 Accepted 29 December 2010 Available online 8 January 2011 Keywords: Biomimetic Injectable Hydrogel In situ Bone
a b s t r a c t To meet the challenges of designing an injectable scaffold and regenerating bone with complex threedimensional (3D) structures, a biomimetic and injectable hydrogel scaffold based on nano-hydroxyapatite (HA), collagen (Col) and chitosan (Chi) is synthesized. The chitosan/nano-hydroxyapatite/collagen (Chi/HA/ Col) solution rapidly forms a stable gel at body temperature. It shows some features of natural bone both in main composition and microstructure. The Chi/HA/Col system can be expected as a candidate for workable systemic minimally invasive scaffolds with surface properties similar to physiological bone based on scanning electron microscopic (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) results. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Materials that enhance bone regeneration have a wealth of potential clinical applications from the treatment of nonunion fractures to spinal fusion [1]. Injectable materials, particularly those delivered in an aqueous solution, are considered ideal delivery vehicles for cells and bioactive factors and can also be delivered through minimally invasive methods and fill complex 3D shapes [2]. The thermosensitive approach can be advantageous for particular applications as it does not require organic solvents, co-polymerization agents, or an externally applied trigger for gelation [3]. Chitosan/β-glycerophosphate (C/GP) formulations undergo thermosensitive sol–gel transition at body temperature [4]. These formulations possess a physiological pH and can be held liquid below room temperature for encapsulating living cells [5–13]. Recent studies have demonstrated the application of the C/GP hydrogel as an injectable scaffold for tissue engineering [10,14–16]. Extracellular matrix (ECM) in natural tissues supports cell attachment, proliferation, and differentiation. Ideally the scaffold should mimic natural ECM as much as possible [17]. The design of scaffolds with surface properties similar to physiological bone would undoubtedly aid in the formation of new bone at the tissue/ biomaterial interface and, therefore, improve orthopedic/dental implant efficacy [18–20]. Bone is mainly composed of nanohydroxyapatite and collagen fibers, in which the c-axes of the HA ⁎ Corresponding author. Department of Materials Science and Engineering, Tsinghua University, Beijing 100084, China. Tel.: +86 10 62782770; fax: +86 10 62771160. E-mail address: [email protected] (Q. Feng). 0928-4931/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.12.014
are regularly aligned along the collagen fibers [21]. While considerable effort has gone into determining the relationship between collagen structure and mineral orientation, synthetic re-creation of this most fundamental level of bone structure has eluded the materials engineer seeking to fabricate bone-like composites. It would be desirable to mimic both the composition and structure of bone for synthetic bone graft substitutes [22,23]. Biomimetic bone materials can be used in conjunction with natural bone, to induce new bone tissue formation and promote bone remodeling. At present this is the most promising route for the repair of defects in natural bone [24–27]. The use of in situ gel-forming scaffolds from bioceramic and polymer components to support bone cell and tissue growth is a longstanding area of interest [1]. In our previous study, the feasibility of developing a thermosensitive and injectable chitosan solution in the presence of HA/Col (nHAC) was demonstrated [8]. It is likely that biofunctionalization strategies will approach to better integrate micron- and nanoscale features into designed scaffolds [1,28,29]. Little is known about the bone-like feature of the thermosensitive and injectable chitosan solution in the presence of HA/Col. In this paper, the main composition and microstructure were investigated. 2. Materials and methods 2.1. Materials Medical grade type I collagen was purchased from YierKang Company (China). Medical grade chitosan was provided by Shandong AK Biotech Ltd. (China). The degree of deacetylation of chitosan was
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estimated to be 95.6% by colloidal titration with polyvinyl sulfate potassium. The molecular weight was 2.5 × 105 Da determined by size exclusion chromatography using dextran as standards. Hydrated βglycerophosphate disodium salt (GP), (C 3 H 7 O 3 PO 3 Na 2 5H 2 O; Mw = 306), was from Sigma (USA). The chemicals in analytical pure grade were from Chemical Agents Co. Ltd., Beijing, China. The water used in the experiments was triply distilled. 2.2. Mineralization of collagen fibrils (HA/Col) Synthesis of HA/Col has been reported previously [30]. Collagen was diluted at a concentration of 0.6 mg/ml by 10 mM hydrochloric acid at 4 °C. CaCl2 solution (1.4 ml, 0.1 M) was added into 10 ml of collagen solution and maintained for 10 min after mixture. NaH2PO4 solution (0.84 ml, 0.1 M) was added and the pH was adjusted to 7.0 by 0.1 M NaOH solution. When the pH exceeded about 6.0, the solution became supersaturated and calcium phosphate started to precipitate with collagen. The solution was maintained at pH 7.0 for 1 h, after which the composite was harvested by centrifugation at 5000 rpm and suspended in deionized water to remove the salts. The centrifugation and suspension cycle was repeated 3 times. After the last suspension the sample was freeze-dried. The precipitate was ground into fine powder. 2.3. Synthesis of thermosensitive Chi/HA/Col composite The Chi/HA/Col composite solution was prepared in a clean room (grade 100) in sterile conditions. Firstly, chitosan solution was obtained by dissolving 0.2 g of chitosan in 9 ml of HCl solution (0.1 M). The pH of the chitosan solution was adjusted to 4.0 by adding droplets of NaHCO3 solution (1 M). The mineralized collagen was added at a weight ratio of HA/Col: Chi = 1:1. The solution was then stirred gently at room temperature for 4–6 h and was cooled down to 4 °C. To the resulting solution, the pH of the solution was adjusted to 7.0 by adding droplets of GP solution [30% (w/v), sterilized by filtration] [8]. Thermosensitive chitosan solution was prepared as a control group [8,31]. Briefly, the chitosan solution was obtained by dissolving 0.2 g of chitosan in 9 ml of HCl solution (0.1 M). The pH of the chitosan solution was adjusted to 4.0 by adding droplets of NaHCO3 solution (1 M). The solution was then stirred gently at room temperature for 4–6 h and was cooled down to 4 °C. To the resulting solution, the pH of the solution was adjusted to 7.0 by adding droplets of GP solution [30% (w/v), sterilized by filtration]. 2.4. Characterization of thermosensitivity A simple test tube inverting method [32] was employed to determine the occurrence of sol–gel transition. The sol phase was defined as flowing liquid and the gel phase as nonflowing gel when the hydrogel solution in the test tube was inverted. The sample (10 ml) was added into 20 ml tube to study sol–gel transition characteristics in a water bath of 37 ± 0.5 °C. At a predetermined interval, the tube was taken out and inverted to observe the state of the sample. The gelation point was determined by flow or no-flow criterion over 30 s with the test tube inverted. The sol–gel transition behavior of the Chi/HA/Col composite system was further illustrated by shear viscosity measurement on physica MCR300 Modular Compact Rheometer (Germany) using a cone-plane CC 27 geometry at 37 ± 0.1 °C. Solution aliquots of 18 ml were poured into the concentric cylinders and then covered with mineral oil in order to prevent evaporation during the measurements. Shear viscosity measurements were made at a fixed shear rate of 6.28 rad/s, and the acquisition rate was set up at two points per 1 min.
2.5. Morphology observation The Chi/HA/Col hydrogel was frozen in a refrigerator at −20 °C for 12 h and then lyophilized in a freeze drier. Lyophilized samples were placed on a double-sided tape, sputter-coated with gold and examined with a scanning electron microscope (LEO Gemini 1530 Field Emission Gun SEM, Germany). The accelerating voltage used in this study was 5 kV. 2.6. Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) The Chi/HA/Col hydrogel was frozen in refrigerator at − 20 °C for 12 h and then lyophilized in a freeze drier. Lyophilized samples were characterized using bright-field transmission electron microscopy to assess for mineral crystal morphology. Samples were embedded in epoxy resin and sectioned using the ultracut (Leika ultracut UCT) technique and then transferred onto Formvar-coated [4% (w/v)] copper grids. TEM observation was carried out with a JEOL 2010F instrument operated in a transmission mode at 200 kV. Electron diffraction of the crystals was conducted to identify calcium phosphate phase [30]. 2.7. X-ray diffraction The Chi, Chi/HA/Col, and HA/Col samples were frozen in a refrigerator at − 20 °C for 12 h and then lyophilized in a freeze drier. The samples were placed in a sample holder and the surface of the sample was flattened. The samples were placed in the XRD equipment (Rigaku D/max-RB diffractometer, Rigaku, Tokyo) with Cu anticathode. A diffraction range of 10°–60° (2θ) was selected and the XRD analysis was carried out at 8°/min. 2.8. X-ray photoelectron spectroscopy (XPS) analysis The dried scaffolds of Chi/HA/Col were cut into small slices and fixed on stubs with adhesive tapes. When the pressure in the analysis chamber was 3.4E−09 Torr, the chemical compositions of them were analyzed by XPS on a PHI Quantera SXM spectrometer (Japan) with an X-ray source (Al Ka, 1486.7 eV photons). All binding energies (BE) were referred to the C1s hydrocarbon peak at 284.8 eV. A survey spectrum (0–1000 eV) was recorded and high-resolution spectra for the Ca2p band were obtained. 2.9. FT-IR spectroscopy The Chi, Chi/HA/Col, and HA/Col samples were frozen in a refrigerator at − 20 °C for 12 h and then lyophilized in a freeze drier. The infrared spectra of HA/Col, Chi and Chi/HA/Col materials were measured with an ATR FT-IR (Nicolet 560, USA) spectrophotometer. Each spectrum was acquired via accumulation of 256 scans with a resolution of 4 cm−1. An IR spectral range of 400–4000 cm−1 was analyzed. 3. Results 3.1. Phase transition of the Chi/HA/Col system We first examined the sol–gel transition of the Chi/HA/Col system in a water bath of 37 ± 0.5 °C. The solutions were flowable viscous liquids and were injectable through a syringe at room temperature. When the solutions were heated to 37 °C, they transformed into gels that were nonflowing. The viscosity of the Chi/HA/Col system as a function of time is shown in Fig. 1. It is shown that the viscosity of the system begun to
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Fig. 1. The viscosity change of the Chi/HA/Col system as a function of time at 37 °C. The left photo shows the sol state of the Chi/HA/Col system, and the right photo shows the gel state of the Chi/HA/Col system.
increase within 10 min at a temperature of 37 °C, which indicated that the solution turned into a gel quickly. 3.2. Surface morphology of porous structures To examine the inner structure of the Chi/HA/Col hydrogels, SEM was conducted after the hydrogel was freeze-dried. The inner structure in the Chi/HA/Col hydrogel is roughly reflected by Fig. 2. Pores were formed after the hydrogel was freeze-dried [33]. As the mineralized collagen fibrils were introduced into the chitosan matrix, the surface of the chitosan matrix showed rough crystal topography [Fig. 2(c)]. The mineralized collagen fibrils were seen to be uniformly dispersed on the surface of the wall [Fig. 2(c)]. 3.3. TEM Fig. 3 shows the microstructure of the Chi/HA/Col composite and reveals the mineralized collagen fibrils entrapped in the chitosan matrix. The mineralized collagen fibrils were about 6 nm in diameter with varied length. The mineralized collagen fibrils were parallel
Fig. 2. SEM images of the freeze-dried Chi/HA/Col hydrogel. Magnification: (a) 500×; (b) 2000×; and (c) 5000×. Arrows represent a mineral coating with rough crystal topography on the pore wall surface. The Cross Star is the smooth surface of the chitosan matrix.
Fig. 3. The TEM image shows arrays of the mineralized collagen fibrils in the Chi/HA/Col composite. Insert is the SAED pattern of the mineralized collagen fibrils. The black flower is the center of the selected area and the diameter of the area is about 200 nm. Long arrow indicates the longitude direction of collagen fibrils.
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Fig. 6. XPS detail scan of Ca (2p) for the Chi/HA/Col composite.
Fig. 4. X-ray diffraction patterns: (a) Chi; (b) Chi/HA/Col; and (c) HA/Col.
assembled into fibril bundles, along a straight line. The SAED pattern of the mineralized collagen fibrils demonstrates that the mineral phase is hydroxyapatite, the (002) and (004) planes are oriented parallel to the long axis of Col fibrils (long arrow) [30,34].
3.4. XRD
3.5. XPS XPS wide scan (Fig. 5) identified carbon, nitrogen, oxygen, calcium (from HA), and phosphor (from HA and β-glycerophosphate) as the major constituents of the Chi/HA/Col system. The determination of the atomic concentrations obtained by XPS on the upper surface (10 nm) of the HA/Col composite gave a Ca/P ratio of 1.63. The measured Ca/P was lower than the theoretical one, and it was consistent with the literature [36]. For the Chi/HA/Col surface, a decreased Ca/P ratio (0.59) was observed corresponding to the addition of β-glycerophosphate with phosphor. Fig. 6 shows the XPS Ca2p spectrum, which has a doublet separated by ~3.5 eV in binding energy for Chi/HA/Col; the primary peak is located at 350.7 eV and the secondary peak is located at 347.2 eV. The XPS spectrum tell us the surface electron binding states of Ca, which is coordinated with both of PO3− 4 of HA nanocrystals and the RCOO− group on the surface of Col molecule [37].
Fig. 4 shows the X-ray diffraction spectra of HA/Col, Chi and Chi/ HA/Col. The inorganic phase in HA/Col is HA (JCPDS 9-432) and there are no other phases of calcium phosphate in the sample. The (002), (211), (310), (222), (213) and (004) diffraction peaks of HA, centered at approximately 25.87, 31.77, 39.81, 46.71, 49.46 and 53.14, respectively, are detected from the patterns of the HA/Col [Fig. 4(c)] and Chi/HA/Col [Fig. 4(b)]. Compared to the HA crystallites, the broadening of the diffraction peaks of HA/Col implied a small grain size and low crystallinity [35]. The pattern of the Chi/HA/Col presents the same broadening peaks, which is also like the pattern of the natural bone [35]. Because the pattern of the Chi/HA/Col presents the overlap of peaks of Chi and HA/Col, there is no significant difference of HA peaks between HA/Col [Fig. 4(c)] and Chi/HA/Col [Fig. 4(b)], it is demonstrated that the nano-sized HA crystallites were not changed by the sol–gel procedure. The (200), (220), and (222) diffraction peaks of NaCl, centered at approximately 31.82, 45.54 and 56.40 (JCPDS 5-0628), respectively, were detected from the patterns of Chi [Fig. 4(a)] and Chi/HA/Col [Fig. 4(b)]. In our experiment, NaHCO3 was added to adjust pH value; it reacted with HCl to form NaCl.
The FT-IR results of Chi, Chi/HA/Col, and HA/Col are shown in Fig. 7. The FT-IR spectrum of Chi/HA/Col appears as a superposition of the spectra of HA/Col and the Chi. Chi/HA/Col shows typical peaks of phosphate at 563 (assignment: ν4), 602 (assignment: ν4), 962 (assignment: ν1), and 1034 (assignment: ν3) cm−1. The weak bands at 1419 and 874 cm−1 are derived from carbonate ions, which indicates that PO3− sites in the HA/Col are replaced partially by 4 carbonate ions [38]. It is reasonable that CO2− is probably incorpo3 rated into the solution from the air during mineral precipitation [30].
Fig. 5. XPS wide scan spectra for the Chi/HA/Col composite.
Fig. 7. FT-IR spectra: (a) HA/Col; (b) Chi/HA/Col; and (c) Chi.
3.6. FT-IR
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4. Discussion In the present work, we observed a substantial change in the viscosity of Chi/HA/Col solutions as a function of time at 37 °C. The viscosity change as a function of time may suggest the formation or destruction of a structured network, implying a sol or gel state [39]. At low temperature, the Chi/HA/Col system forms an aqueous solution with low viscosity to complete the injection, but forms a gel at body temperature [8]. In situ gel-forming scaffolds can be expected as candidates for workable systemic minimally invasive scaffolds [39]. Moreover, it has been demonstrated that the Chi system could act as a suitable biocompatible scaffold for entrapped rat bone marrow mesenchymal stem cells in vivo [14]. As with all organs in the body, bone tissue has a hierarchical organization over length scales that span several orders of magnitude from macro-scale (centimeter) to nanostructured (extracellular matrix or ECM) components [1]. Weiner and Wagner have identified seven discrete levels of hierarchical organization in bone [40,41]. The first level of hierarchy consists of the molecular components: water, HA, collagen, and other proteins. Not only are bone crystallites extremely small, they are often described as “poorly crystalline” because of the broad X-ray diffraction peaks (relative to synthetic HA), which is thought to arise from the incorporation of impurities, such as carbonate and nonstoichiometry of the biogenic mineral [22]. The FT-IR spectrum (Fig. 7) showed that both phosphate and carbonate groups were present in the Chi/HA/Col specimens. XRD results delineated the presence of n-HA in the Chi/HA/Col specimens. All the peaks in Fig. 4b–c were in cognate with the peaks associated with natural bone [35]. The second level is formed by the mineralization of collagen fibrils. This platelet-reinforced fibril composite is described by Weiner and Wagner [40] as containing parallel plate-like HA crystals with their c-axis aligned with the long axis of the fibril [40,41]. To determine the intra-fibrillar crystal arrangement, we examined individual mineralized fibrils with SAED. As shown in Fig. 3, small HA crystals associated with Col fibrils show a strong preferred orientation of their c-crystallographic axis. The third level of hierarchy is composed of arrays of these mineralized collagen fibrils. These fibrils are rarely found isolated but rather associated as bundles, often aligned along their long axis [40,41]. As shown in the TEM image (Fig. 3), the mineralized collagen fibrils are found associated as bundles, aligned along their long axis. The arrangement of the mineralized collagen fibrils is consistent with the third hierarchical level of organization of natural bone as proposed by Weiner and Wagner [40,41]. The design of biomaterials with surface properties similar to physiological bone would undoubtedly aid the formation of new bone at the tissue/biomaterial interface and, therefore, improve orthopedic implant efficacy [18]. XPS analysis (Fig. 5) gives the composition information of the very top surface layer; in addition to C, P and O, Ca was also found on the surface of the Chi/HA/Col specimens in significant amounts. The Chi/HA/Col system can be expected as candidates for workable systemic minimally invasive scaffolds with surface properties similar to physiological bone based on SEM, TEM and XPS results. As the conceptual approach of employing the Chi/HA/Col composites is not fully lucid, the preliminary results render further investigations on the extensive plexus of biological pathways, gene and protein expressions associated with mineralization and bone cells. 5. Conclusion A new type of an in situ-forming bone repair material, Chi/HA/Col composite is fabricated with a biomimetic strategy. The Chi/HA/Col
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solution rapidly forms a stable gel at body temperature and can be injected directly into cavities, even for irregular shape and size, in a minimally invasive manner. The nano-composite shows some features of natural bone both in main composition and hierarchical microstructure. This composite, combined with bone-like feature and injectability, provided a promising scaffold in both traditional bonedefect repair and in bone tissue engineering. Acknowledgements The authors are grateful for the financial support from the National Natural Science Foundation of China (50772052 and 51072090) and the Doctor Subject Foundation of the Ministry of Education of China under Grant (20070003004). References [1] M.M. Stevens, Mater. Today 11 (2008) 18. [2] J.D. Kretlow, S. Young, L. Klouda, M. Wong, A.G. Mikos, Adv. Mater. 21 (2009) 3368. [3] E. Ruel-Gariepy, J.C. Leroux, Eur. J. Pharm. Biopharm. 58 (2004) 409. [4] A. Chenite, C. Chaput, D. Wang, C. Combes, M.D. Buschmann, C.D. Hoemann, J.C. Leroux, B.L. Atkinson, F. Binette, A. Selmani, Biomaterials 21 (2000) 2155. [5] L.M. Wang, J.P. Stegemann, Biomaterials 31 (2010) 3976. [6] S. Kim, S.K. Nishimoto, J.D. Bumgardner, W.O. Haggard, M.W. Gaber, Y.Z. Yang, Biomaterials 31 (2010) 4157. [7] Y.H. Cheng, S.H. Yang, W.Y. Su, Y.C. Chen, K.C. Yang, W.T.K. Cheng, S.C. Wu, F.H. Lin, Tissue Eng. A 16 (2010) 695. [8] Z. Huang, J. Tian, B. Yu, Y. Xu, Q. Feng, Biomed. Mater. 4 (2009). [9] C.D. Hoemann, H. El-Gabalawy, M.D. Mckee, Pathol. Biol. 57 (2009) 318. [10] S.M. Richardson, N. Hughes, J.A. Hunt, A.J. Freemont, J.A. Hoyland, Biomaterials 29 (2008) 85. [11] K.E. Crompton, J.D. Goud, R.V. Bellamkonda, T.R. Gengenbach, D.I. Finkelstein, M.K. Horne, J.S. Forsythe, Biomaterials 28 (2007) 441. [12] K.E. Crompton, D. Tomas, D.I. Finkelstein, M. Marr, J.S. Forsythe, M.K. Horne, J. Mater. Sci. Mater. Med. 17 (2006) 633. [13] S.M. Richardson, N.L. Hughes, A.J. Freemont, J.A. Hunt, J.A. Hoyland, Int. J. Exp. Pathol. 87 (2006) A20. [14] M.H. Cho, K.S. Kim, H.H. Ahn, M.S. Kim, S.H. Kim, G. Khang, B. Lee, H.B. Lee, Tissue Eng. A 14 (2008) 1099. [15] C.D. Hoemann, A. Chenite, J. Sun, M. Hurtig, A. Serreqi, Z. Lu, E. Rossomacha, M.D. Buschmann, J. Biomed. Mater. Res. A 83A (2007) 521. [16] C.D. Hoemann, J. Sun, A. Legare, M.D. McKee, M.D. Buschmann, Osteoarthritis Cartilage 13 (2005) 318. [17] S. Liao, C.K. Chan, S. Ramakrishna, Mat. Sci. Eng. C Bio. S 28 (2008) 1189. [18] T.J. Webster, R.W. Siegel, R. Bizios, Biomaterials 20 (1999) 1221. [19] X.M. Li, Q.L. Feng, F.Z. Cui, Mat. Sci. Eng. C Bio. S 26 (2006) 716. [20] X.M. Li, Q.L. Feng, X.H. Liu, W. Dong, F.H. Cui, Biomaterials 27 (2006) 1917. [21] S. Itoh, M. Kikuchi, Y. Koyama, K. Takakuda, K. Shinomiya, J. Tanaka, Biomaterials 23 (2002) 3919. [22] M.J. Olszta, X.G. Cheng, S.S. Jee, R. Kumar, Y.Y. Kim, M.J. Kaufman, E.P. Douglas, L.B. Gower, Mater. Sci. Eng. R 58 (2007) 77. [23] X.M. Li, C.A. van Blitterswijk, Q.L. Feng, F.Z. Cui, F.M. Watari, Biomaterials 29 (2008) 3306. [24] F.Z. Cui, Y. Li, J. Ge, Mater. Sci. Eng. R 57 (2007) 1. [25] X.M. Cheng, Y.B. Li, Y. Zuo, L. Zhang, J.D. Li, H.A. Wang, Mat. Sci. Eng. C Bio. S 29 (2009) 29. [26] J.J. Li, Y. Dou, J. Yang, Y.J. Yin, H. Zhang, F.L. Yao, H.B. Wang, K.D. Yao, Mat. Sci. Eng. C Bio. S 29 (2009) 1207. [27] F. Sun, B.K. Lim, S.C. Ryu, D. Lee, J. Lee, Mater. Sci. Eng., C 30 (2010) 789. [28] J.J. Li, D.W. Zhu, J.W. Yin, Y.X. Liu, F.L. Yao, K.D. Yao, Mater. Sci. Eng., C 30 (2010) 795. [29] O.C. Wilson, J.R. Hull, Mat. Sci. Eng. C Bio. S 28 (2008) 434. [30] W. Zhang, S.S. Liao, F.Z. Cui, Chem. Mater. 15 (2003) 3221. [31] F. Ganji, M.J. Abdekhodaie, A. Ramazani, J. Sol–Gel. Sci. Technol. 42 (2007) 47. [32] H.Y. Zhou, X.G. Chen, M. Kong, C.S. Liu, J. Appl. Polym. Sci. 112 (2009) 1509. [33] J. Zan, H.H. Chen, G.Q. Jiang, Y. Lin, F.X. Ding, J. Appl. Polym. Sci. 101 (2006) 1892. [34] W. Traub, T. Arad, S. Weiner, Proc. Natl Acad. Sci. USA 86 (1989) 9822. [35] S.S. Liao, F.Z. Cui, W. Zhang, Q.L. Feng, J. Biomed. Mater. Res. B 69B (2004) 158. [36] C.C. Chusuei, D.W. Goodman, M.J. Van Stipdonk, D.R. Justes, E.A. Schweikert, Anal. Chem. 71 (1999) 149. [37] M.C. Chang, J. Tanaka, Biomaterials 23 (2002) 3879. [38] B.O. Fowler, Inorg. Chem. 13 (1974) 194. [39] K.S. Kim, J.Y. Lee, Y.M. Kang, E.S. Kim, B. Lee, H.J. Chun, J.H. Kim, B.H. Min, H.B. Lee, M.S. Kim, Tissue Eng. A 15 (2009) 3201. [40] S. Weiner, H.D. Wagner, Annu. Rev. Mater. Sci. 28 (1998) 271. [41] L.C. Palmer, C.J. Newcomb, S.R. Kaltz, E.D. Spoerke, S.I. Stupp, Chem. Rev. 108 (2008) 4754.
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
Biomimetic properties of an injectable chitosan/nano-hydroxyapatite/collagen composite Zhi Huang a, Qingling Feng a,⁎, Bo Yu b, Songjian Li b a b
Laboratory of Advanced Materials, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084, China Department of Orthopedics, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China
a r t i c l e
i n f o
Article history: Received 25 May 2010 Received in revised form 2 November 2010 Accepted 29 December 2010 Available online 8 January 2011 Keywords: Biomimetic Injectable Hydrogel In situ Bone
a b s t r a c t To meet the challenges of designing an injectable scaffold and regenerating bone with complex threedimensional (3D) structures, a biomimetic and injectable hydrogel scaffold based on nano-hydroxyapatite (HA), collagen (Col) and chitosan (Chi) is synthesized. The chitosan/nano-hydroxyapatite/collagen (Chi/HA/ Col) solution rapidly forms a stable gel at body temperature. It shows some features of natural bone both in main composition and microstructure. The Chi/HA/Col system can be expected as a candidate for workable systemic minimally invasive scaffolds with surface properties similar to physiological bone based on scanning electron microscopic (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) results. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Materials that enhance bone regeneration have a wealth of potential clinical applications from the treatment of nonunion fractures to spinal fusion [1]. Injectable materials, particularly those delivered in an aqueous solution, are considered ideal delivery vehicles for cells and bioactive factors and can also be delivered through minimally invasive methods and fill complex 3D shapes [2]. The thermosensitive approach can be advantageous for particular applications as it does not require organic solvents, co-polymerization agents, or an externally applied trigger for gelation [3]. Chitosan/β-glycerophosphate (C/GP) formulations undergo thermosensitive sol–gel transition at body temperature [4]. These formulations possess a physiological pH and can be held liquid below room temperature for encapsulating living cells [5–13]. Recent studies have demonstrated the application of the C/GP hydrogel as an injectable scaffold for tissue engineering [10,14–16]. Extracellular matrix (ECM) in natural tissues supports cell attachment, proliferation, and differentiation. Ideally the scaffold should mimic natural ECM as much as possible [17]. The design of scaffolds with surface properties similar to physiological bone would undoubtedly aid in the formation of new bone at the tissue/ biomaterial interface and, therefore, improve orthopedic/dental implant efficacy [18–20]. Bone is mainly composed of nanohydroxyapatite and collagen fibers, in which the c-axes of the HA ⁎ Corresponding author. Department of Materials Science and Engineering, Tsinghua University, Beijing 100084, China. Tel.: +86 10 62782770; fax: +86 10 62771160. E-mail address: [email protected] (Q. Feng). 0928-4931/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.12.014
are regularly aligned along the collagen fibers [21]. While considerable effort has gone into determining the relationship between collagen structure and mineral orientation, synthetic re-creation of this most fundamental level of bone structure has eluded the materials engineer seeking to fabricate bone-like composites. It would be desirable to mimic both the composition and structure of bone for synthetic bone graft substitutes [22,23]. Biomimetic bone materials can be used in conjunction with natural bone, to induce new bone tissue formation and promote bone remodeling. At present this is the most promising route for the repair of defects in natural bone [24–27]. The use of in situ gel-forming scaffolds from bioceramic and polymer components to support bone cell and tissue growth is a longstanding area of interest [1]. In our previous study, the feasibility of developing a thermosensitive and injectable chitosan solution in the presence of HA/Col (nHAC) was demonstrated [8]. It is likely that biofunctionalization strategies will approach to better integrate micron- and nanoscale features into designed scaffolds [1,28,29]. Little is known about the bone-like feature of the thermosensitive and injectable chitosan solution in the presence of HA/Col. In this paper, the main composition and microstructure were investigated. 2. Materials and methods 2.1. Materials Medical grade type I collagen was purchased from YierKang Company (China). Medical grade chitosan was provided by Shandong AK Biotech Ltd. (China). The degree of deacetylation of chitosan was
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estimated to be 95.6% by colloidal titration with polyvinyl sulfate potassium. The molecular weight was 2.5 × 105 Da determined by size exclusion chromatography using dextran as standards. Hydrated βglycerophosphate disodium salt (GP), (C 3 H 7 O 3 PO 3 Na 2 5H 2 O; Mw = 306), was from Sigma (USA). The chemicals in analytical pure grade were from Chemical Agents Co. Ltd., Beijing, China. The water used in the experiments was triply distilled. 2.2. Mineralization of collagen fibrils (HA/Col) Synthesis of HA/Col has been reported previously [30]. Collagen was diluted at a concentration of 0.6 mg/ml by 10 mM hydrochloric acid at 4 °C. CaCl2 solution (1.4 ml, 0.1 M) was added into 10 ml of collagen solution and maintained for 10 min after mixture. NaH2PO4 solution (0.84 ml, 0.1 M) was added and the pH was adjusted to 7.0 by 0.1 M NaOH solution. When the pH exceeded about 6.0, the solution became supersaturated and calcium phosphate started to precipitate with collagen. The solution was maintained at pH 7.0 for 1 h, after which the composite was harvested by centrifugation at 5000 rpm and suspended in deionized water to remove the salts. The centrifugation and suspension cycle was repeated 3 times. After the last suspension the sample was freeze-dried. The precipitate was ground into fine powder. 2.3. Synthesis of thermosensitive Chi/HA/Col composite The Chi/HA/Col composite solution was prepared in a clean room (grade 100) in sterile conditions. Firstly, chitosan solution was obtained by dissolving 0.2 g of chitosan in 9 ml of HCl solution (0.1 M). The pH of the chitosan solution was adjusted to 4.0 by adding droplets of NaHCO3 solution (1 M). The mineralized collagen was added at a weight ratio of HA/Col: Chi = 1:1. The solution was then stirred gently at room temperature for 4–6 h and was cooled down to 4 °C. To the resulting solution, the pH of the solution was adjusted to 7.0 by adding droplets of GP solution [30% (w/v), sterilized by filtration] [8]. Thermosensitive chitosan solution was prepared as a control group [8,31]. Briefly, the chitosan solution was obtained by dissolving 0.2 g of chitosan in 9 ml of HCl solution (0.1 M). The pH of the chitosan solution was adjusted to 4.0 by adding droplets of NaHCO3 solution (1 M). The solution was then stirred gently at room temperature for 4–6 h and was cooled down to 4 °C. To the resulting solution, the pH of the solution was adjusted to 7.0 by adding droplets of GP solution [30% (w/v), sterilized by filtration]. 2.4. Characterization of thermosensitivity A simple test tube inverting method [32] was employed to determine the occurrence of sol–gel transition. The sol phase was defined as flowing liquid and the gel phase as nonflowing gel when the hydrogel solution in the test tube was inverted. The sample (10 ml) was added into 20 ml tube to study sol–gel transition characteristics in a water bath of 37 ± 0.5 °C. At a predetermined interval, the tube was taken out and inverted to observe the state of the sample. The gelation point was determined by flow or no-flow criterion over 30 s with the test tube inverted. The sol–gel transition behavior of the Chi/HA/Col composite system was further illustrated by shear viscosity measurement on physica MCR300 Modular Compact Rheometer (Germany) using a cone-plane CC 27 geometry at 37 ± 0.1 °C. Solution aliquots of 18 ml were poured into the concentric cylinders and then covered with mineral oil in order to prevent evaporation during the measurements. Shear viscosity measurements were made at a fixed shear rate of 6.28 rad/s, and the acquisition rate was set up at two points per 1 min.
2.5. Morphology observation The Chi/HA/Col hydrogel was frozen in a refrigerator at −20 °C for 12 h and then lyophilized in a freeze drier. Lyophilized samples were placed on a double-sided tape, sputter-coated with gold and examined with a scanning electron microscope (LEO Gemini 1530 Field Emission Gun SEM, Germany). The accelerating voltage used in this study was 5 kV. 2.6. Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) The Chi/HA/Col hydrogel was frozen in refrigerator at − 20 °C for 12 h and then lyophilized in a freeze drier. Lyophilized samples were characterized using bright-field transmission electron microscopy to assess for mineral crystal morphology. Samples were embedded in epoxy resin and sectioned using the ultracut (Leika ultracut UCT) technique and then transferred onto Formvar-coated [4% (w/v)] copper grids. TEM observation was carried out with a JEOL 2010F instrument operated in a transmission mode at 200 kV. Electron diffraction of the crystals was conducted to identify calcium phosphate phase [30]. 2.7. X-ray diffraction The Chi, Chi/HA/Col, and HA/Col samples were frozen in a refrigerator at − 20 °C for 12 h and then lyophilized in a freeze drier. The samples were placed in a sample holder and the surface of the sample was flattened. The samples were placed in the XRD equipment (Rigaku D/max-RB diffractometer, Rigaku, Tokyo) with Cu anticathode. A diffraction range of 10°–60° (2θ) was selected and the XRD analysis was carried out at 8°/min. 2.8. X-ray photoelectron spectroscopy (XPS) analysis The dried scaffolds of Chi/HA/Col were cut into small slices and fixed on stubs with adhesive tapes. When the pressure in the analysis chamber was 3.4E−09 Torr, the chemical compositions of them were analyzed by XPS on a PHI Quantera SXM spectrometer (Japan) with an X-ray source (Al Ka, 1486.7 eV photons). All binding energies (BE) were referred to the C1s hydrocarbon peak at 284.8 eV. A survey spectrum (0–1000 eV) was recorded and high-resolution spectra for the Ca2p band were obtained. 2.9. FT-IR spectroscopy The Chi, Chi/HA/Col, and HA/Col samples were frozen in a refrigerator at − 20 °C for 12 h and then lyophilized in a freeze drier. The infrared spectra of HA/Col, Chi and Chi/HA/Col materials were measured with an ATR FT-IR (Nicolet 560, USA) spectrophotometer. Each spectrum was acquired via accumulation of 256 scans with a resolution of 4 cm−1. An IR spectral range of 400–4000 cm−1 was analyzed. 3. Results 3.1. Phase transition of the Chi/HA/Col system We first examined the sol–gel transition of the Chi/HA/Col system in a water bath of 37 ± 0.5 °C. The solutions were flowable viscous liquids and were injectable through a syringe at room temperature. When the solutions were heated to 37 °C, they transformed into gels that were nonflowing. The viscosity of the Chi/HA/Col system as a function of time is shown in Fig. 1. It is shown that the viscosity of the system begun to
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Fig. 1. The viscosity change of the Chi/HA/Col system as a function of time at 37 °C. The left photo shows the sol state of the Chi/HA/Col system, and the right photo shows the gel state of the Chi/HA/Col system.
increase within 10 min at a temperature of 37 °C, which indicated that the solution turned into a gel quickly. 3.2. Surface morphology of porous structures To examine the inner structure of the Chi/HA/Col hydrogels, SEM was conducted after the hydrogel was freeze-dried. The inner structure in the Chi/HA/Col hydrogel is roughly reflected by Fig. 2. Pores were formed after the hydrogel was freeze-dried [33]. As the mineralized collagen fibrils were introduced into the chitosan matrix, the surface of the chitosan matrix showed rough crystal topography [Fig. 2(c)]. The mineralized collagen fibrils were seen to be uniformly dispersed on the surface of the wall [Fig. 2(c)]. 3.3. TEM Fig. 3 shows the microstructure of the Chi/HA/Col composite and reveals the mineralized collagen fibrils entrapped in the chitosan matrix. The mineralized collagen fibrils were about 6 nm in diameter with varied length. The mineralized collagen fibrils were parallel
Fig. 2. SEM images of the freeze-dried Chi/HA/Col hydrogel. Magnification: (a) 500×; (b) 2000×; and (c) 5000×. Arrows represent a mineral coating with rough crystal topography on the pore wall surface. The Cross Star is the smooth surface of the chitosan matrix.
Fig. 3. The TEM image shows arrays of the mineralized collagen fibrils in the Chi/HA/Col composite. Insert is the SAED pattern of the mineralized collagen fibrils. The black flower is the center of the selected area and the diameter of the area is about 200 nm. Long arrow indicates the longitude direction of collagen fibrils.
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Fig. 6. XPS detail scan of Ca (2p) for the Chi/HA/Col composite.
Fig. 4. X-ray diffraction patterns: (a) Chi; (b) Chi/HA/Col; and (c) HA/Col.
assembled into fibril bundles, along a straight line. The SAED pattern of the mineralized collagen fibrils demonstrates that the mineral phase is hydroxyapatite, the (002) and (004) planes are oriented parallel to the long axis of Col fibrils (long arrow) [30,34].
3.4. XRD
3.5. XPS XPS wide scan (Fig. 5) identified carbon, nitrogen, oxygen, calcium (from HA), and phosphor (from HA and β-glycerophosphate) as the major constituents of the Chi/HA/Col system. The determination of the atomic concentrations obtained by XPS on the upper surface (10 nm) of the HA/Col composite gave a Ca/P ratio of 1.63. The measured Ca/P was lower than the theoretical one, and it was consistent with the literature [36]. For the Chi/HA/Col surface, a decreased Ca/P ratio (0.59) was observed corresponding to the addition of β-glycerophosphate with phosphor. Fig. 6 shows the XPS Ca2p spectrum, which has a doublet separated by ~3.5 eV in binding energy for Chi/HA/Col; the primary peak is located at 350.7 eV and the secondary peak is located at 347.2 eV. The XPS spectrum tell us the surface electron binding states of Ca, which is coordinated with both of PO3− 4 of HA nanocrystals and the RCOO− group on the surface of Col molecule [37].
Fig. 4 shows the X-ray diffraction spectra of HA/Col, Chi and Chi/ HA/Col. The inorganic phase in HA/Col is HA (JCPDS 9-432) and there are no other phases of calcium phosphate in the sample. The (002), (211), (310), (222), (213) and (004) diffraction peaks of HA, centered at approximately 25.87, 31.77, 39.81, 46.71, 49.46 and 53.14, respectively, are detected from the patterns of the HA/Col [Fig. 4(c)] and Chi/HA/Col [Fig. 4(b)]. Compared to the HA crystallites, the broadening of the diffraction peaks of HA/Col implied a small grain size and low crystallinity [35]. The pattern of the Chi/HA/Col presents the same broadening peaks, which is also like the pattern of the natural bone [35]. Because the pattern of the Chi/HA/Col presents the overlap of peaks of Chi and HA/Col, there is no significant difference of HA peaks between HA/Col [Fig. 4(c)] and Chi/HA/Col [Fig. 4(b)], it is demonstrated that the nano-sized HA crystallites were not changed by the sol–gel procedure. The (200), (220), and (222) diffraction peaks of NaCl, centered at approximately 31.82, 45.54 and 56.40 (JCPDS 5-0628), respectively, were detected from the patterns of Chi [Fig. 4(a)] and Chi/HA/Col [Fig. 4(b)]. In our experiment, NaHCO3 was added to adjust pH value; it reacted with HCl to form NaCl.
The FT-IR results of Chi, Chi/HA/Col, and HA/Col are shown in Fig. 7. The FT-IR spectrum of Chi/HA/Col appears as a superposition of the spectra of HA/Col and the Chi. Chi/HA/Col shows typical peaks of phosphate at 563 (assignment: ν4), 602 (assignment: ν4), 962 (assignment: ν1), and 1034 (assignment: ν3) cm−1. The weak bands at 1419 and 874 cm−1 are derived from carbonate ions, which indicates that PO3− sites in the HA/Col are replaced partially by 4 carbonate ions [38]. It is reasonable that CO2− is probably incorpo3 rated into the solution from the air during mineral precipitation [30].
Fig. 5. XPS wide scan spectra for the Chi/HA/Col composite.
Fig. 7. FT-IR spectra: (a) HA/Col; (b) Chi/HA/Col; and (c) Chi.
3.6. FT-IR
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4. Discussion In the present work, we observed a substantial change in the viscosity of Chi/HA/Col solutions as a function of time at 37 °C. The viscosity change as a function of time may suggest the formation or destruction of a structured network, implying a sol or gel state [39]. At low temperature, the Chi/HA/Col system forms an aqueous solution with low viscosity to complete the injection, but forms a gel at body temperature [8]. In situ gel-forming scaffolds can be expected as candidates for workable systemic minimally invasive scaffolds [39]. Moreover, it has been demonstrated that the Chi system could act as a suitable biocompatible scaffold for entrapped rat bone marrow mesenchymal stem cells in vivo [14]. As with all organs in the body, bone tissue has a hierarchical organization over length scales that span several orders of magnitude from macro-scale (centimeter) to nanostructured (extracellular matrix or ECM) components [1]. Weiner and Wagner have identified seven discrete levels of hierarchical organization in bone [40,41]. The first level of hierarchy consists of the molecular components: water, HA, collagen, and other proteins. Not only are bone crystallites extremely small, they are often described as “poorly crystalline” because of the broad X-ray diffraction peaks (relative to synthetic HA), which is thought to arise from the incorporation of impurities, such as carbonate and nonstoichiometry of the biogenic mineral [22]. The FT-IR spectrum (Fig. 7) showed that both phosphate and carbonate groups were present in the Chi/HA/Col specimens. XRD results delineated the presence of n-HA in the Chi/HA/Col specimens. All the peaks in Fig. 4b–c were in cognate with the peaks associated with natural bone [35]. The second level is formed by the mineralization of collagen fibrils. This platelet-reinforced fibril composite is described by Weiner and Wagner [40] as containing parallel plate-like HA crystals with their c-axis aligned with the long axis of the fibril [40,41]. To determine the intra-fibrillar crystal arrangement, we examined individual mineralized fibrils with SAED. As shown in Fig. 3, small HA crystals associated with Col fibrils show a strong preferred orientation of their c-crystallographic axis. The third level of hierarchy is composed of arrays of these mineralized collagen fibrils. These fibrils are rarely found isolated but rather associated as bundles, often aligned along their long axis [40,41]. As shown in the TEM image (Fig. 3), the mineralized collagen fibrils are found associated as bundles, aligned along their long axis. The arrangement of the mineralized collagen fibrils is consistent with the third hierarchical level of organization of natural bone as proposed by Weiner and Wagner [40,41]. The design of biomaterials with surface properties similar to physiological bone would undoubtedly aid the formation of new bone at the tissue/biomaterial interface and, therefore, improve orthopedic implant efficacy [18]. XPS analysis (Fig. 5) gives the composition information of the very top surface layer; in addition to C, P and O, Ca was also found on the surface of the Chi/HA/Col specimens in significant amounts. The Chi/HA/Col system can be expected as candidates for workable systemic minimally invasive scaffolds with surface properties similar to physiological bone based on SEM, TEM and XPS results. As the conceptual approach of employing the Chi/HA/Col composites is not fully lucid, the preliminary results render further investigations on the extensive plexus of biological pathways, gene and protein expressions associated with mineralization and bone cells. 5. Conclusion A new type of an in situ-forming bone repair material, Chi/HA/Col composite is fabricated with a biomimetic strategy. The Chi/HA/Col
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