ABSTRACT Hydroxyapatite(HAP)/Chitosan(CS) composite is a biocompatible and bioactive material for tissue engineering. A novel homogeneous HAP/CS composite scaffold was developed via lyophilization and in situ hydration. A model CS solution with a Ca/P atom ratio of 1.67 was prepared through titration and stirring so as to attain a homogeneous dispersion of HAP particles. After lyophilization and in situ hydration, rod-shaped HAP particles (5 μm in diameter) within the CS matrix homogeneously scattered at the pore wall of the CS scaffold. X-ray diffraction (XRD) and Fouri-er-Transformed Infrared spectroscopy (FTIR) confirmed the formation of HAP crystals. The compressive strength in the composite scaffold indicated a significant increment over a CS-only scaffold. Bioactivity in vitro was completed by immersing the scaffold in simulated body fluid (SBF), and the result suggested that there was an increase in apatite formation on the HAP/CS scaffolds. Biological in vivo cell culture with MC 3T3-E1 cells for up to 7 days demonstrated that a homogeneous incorporation of HAP particles into CS scaffold led to higher cell viability compared to that of the pure CS scaffold or the HAP/CS scaffold blended. The results suggest that the homogeneous composite scaffold with better strength, bioactivity and biocompatibility can be prepared via in vitro hydration, which may serve as a good scaffold for bone tissue engineering.
Cite this paper
nullH. Li, C. Zhou, M. Zhu, J. Tian and J. Rong, "Preparation and Characterization of Homogeneous Hydroxyapatite/Chitosan Composite Scaffolds via In-Situ Hydration," Journal of Biomaterials and Nanobiotechnology, Vol. 1 No. 1, 2010, pp. 42-49. doi: 10.4236/jbnb.2010.11006.
 Y. J. Seol, J. Y. Lee, Y. J. Park, Y. M. Lee, Y. Ku, I. C. Rhyu, S. J. Lee, S. B. Han and C. P. Chung, “Chitosan Sponges as Tissue Engineering Scaffolds for Bone Formation,” Biotechno Lett, Vol. 26, 2004, pp. 1037-1041.
 F. Zhao, W. L. Grayson, T. Ma, B. Bunnell and W. W. Lu “Effects of Hydroxyapatite in 3-D Chitosan–Gelatin Polymer Network On Human Mesenchymal Stem Cell Construct Development,” Biomaterials, Vol. 27, 2006, pp. 1859-1867.
 T. Kawakami, M. Antoh, H. Hasegawa, T. Yamagish, M. Ito and S. Eda, “Experimental Study On Osteoconductive Properties of a Chitosan-Bonded Hydroxyapatite Self- Hardening Paste,” Biomaterials, Vol. 13, 1992, pp. 759- 763.
 Z. Ge, S. Baguenard, L. Y. Lim, A. Wee and E. Khor “Hydroxyapatite-Chitin Materials as Potential Tissue Engineered Bone Substitutes,” Biomaterials, Vol. 25, 2004, pp. 1049-1058.
 Kong LJ, Gao Y, Lu GY, Gong YD, Zhao NM, Zhang X F. A study on the bioactivity of chitosan/nano- hydroxyapatite composite scaffolds for bone tissue engineering. Eur Poly J 2006; 42: 3171-3179.
 Hu Q, Li B, Wang M, Shen J. Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a potential material as internal fixation of bone fracture. Biomaterials 2004; 25: 779-785.
 Zhang Y, Zhang M. Synthesis and characterization ofmacroporous chitosan/calcium phosphate composite scaffolds for tissue engineering. J Biomed Mater Res 2001; 55: 304-312.
 J. M. Oliveira, M. T. Rodrigues, S. S. Silva, P. B. Malafaya, M. E. Gomes, C. A. Viegas, I. R. Dias, J. T. Azevedo, J. F. Mano and R. L. Reis, “Novel Hydro- xyapatite/Chitosan Bilayered Scaffold for Osteochondral Tissue-Engineering Applications: Scaffold Design and its Performance when Seeded with Goat Bone Marrow Stromal Cells,” Biomaterials 2006; 27: 6123-6137.
 Viala S, Freche M, Lacout JL. Preparation of a new organic mineral-composite: chitosan-hydroxyapatite. Ann Chin Sci Mater, 1998; 23: 69-72.
 Zhao F, Yin Y, Lu WW, Leong JC, Zhang W, Zhang J, Zhang M, Yao K. Preparation and histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan- gelatin network composite scaffolds. Biomaterials 2002; 23: 3227-3234.
 Correlo MV, Luciano FB, Mrinal B, Joao F M, Nuno MN, Ruis LR. Hydroxyapatite Reinforced Chitosan and Polyester Blends for Biomedical Applications. Macroml Mater Eng 2005; 290: 1157-1165.
 Shen X, Tong H, Jiang T, Zhu Z, Wan P, Hu J. Homogeneous chitosan/carbonate apatite/citric acid nano- composites prepared through a novel in situ precipitation method. Comp Sci Tech 2007; 67(11-12): 2238-2245.
 Kokubo T, Hanakawab M, Kawashita M, Minoda, Beppu T, MiyamotoT, NakamurT. Apatite formation on non-woven fabric of carboxymethylated chitin in SBF. Biomaterials, 2004; 25: 4485-4488.
 Tuzlakoglu K, Reis R L. Formation of bone-like apatite layer on chitosan fiber mesh scaffolds by a biomimetic spraying process. J Mater Sci Mater Med 2007; 18: 1279-1286.
 Guo ZH, Wei SY, Shedd B, Scaffaro R, Pereira T, Hahn HT. Particle surface engineering effect on the mechanical, optical and photoluminescent properties of ZnO/vinyl- ester resin nanocomposites. J Mater Chem 2007; 17: 806-813.
 Kuo MC, Tsai C, Huang JC, Chen M. PEEK composites reinforced by nano-sized SiO2 and Al2O3 particulates. Mater Chem Phy 2005; 90: 185-195.
 Kokubo T, Kushitani H, Ohtsuki C, Sakka S, Yamamuro T. Chemical reaction of bioactive glass and glass– ceramics with a simulated body fluid. J Mater Sci Mater Med, 1992; 1: 79-83.
 Rusua VM, Ng CH, Wilke M, Tierscha B, Fratzld P, Peter MG. Size-controlled hydroxyapatite nanoparticles as self-organized organic–inorganic composite materials. Biomaterials 2005; 26: 5414-5426.
 Pang YX, Bao X. Influence of temperature, ripening time and calcination on the morphology and crystallinity of hydroxyapatite nanoparticles. J Eur Ceram Soc 2003; 23: 1697-704.
 Anee TK, Palanichamy M, Ashok M. Influence of iron and temperature on the crystallization of calcium phosphates at the physiological pH. Mater Lett 2004; 58: 478-482.
 Chen CW, Riman RE, Tenhuisen KS, Brown K. Mechanochemical–hydrothermal synthesis of hydroxyapatite from nonionic surfactant emulsion precursors. JCryst Growth 2004; 270: 615-623.
 Koumoulidis GC, Katsoulidis AP, Ladavos AK, Pomonis PJ, Trapalis CC, Sdoukos AT, Vaimakis TC. Preparation of hydroxyapatite via microemulsion route. J Colloid Interf Sci, 2003; 259: 254-260.
 Li BQ, Hu QL, Qian XZ, Fang ZP, Shen JC. Bioabsorbable chitosan/hydroxyapatite composite rod for internal fixation of bone fracture prepared by in situ precipitation. Acta. Polym. Sin 2002; 6: 828-833.
 Kokubo T, Ito I, Huang T. P–rich layer formed on high–strength bioactive glass–ceramics A–W. J Biomed Mater Res 1990: 24: 331-343.
 Li P, Ohtsukl C, Kokubo T. Effects of ions in aqueous media on hydroxyapatite induction by silica gel and its relevance to bioactivity of bioactive glasses and glass/ ceramics. J Appl Biomater 1993: 4: 221-229.
 Posner AS. The mineral of bone. Clin Orthop Rel Res 1985;200: 87-93.
 Kokubo T. Apatite formation on surfaces of ceramics, metals and polymers in body environment. Acta Mater 1998; 7: 2519-2527.
 Lu X, Leng Y. Thyeoretical analysis of calcium phosphate precipitation in simulated body fluid. Biomaterials 2005; 26: 1097-1108.
 Matsumoto T, Okazaki M, Inoue M, Yamaguchi S, Kusunose T, Toyonaga T, Hamada Y, Takahashi J. Hydroxyapatite particles as a controlled release carrier of protein. Biomaterials 2004; 25: 3807-3812.
 Kokubo T. Apatite formation on surfaces of ceramics, metals and polymers in body environment. Acta Mater 1998; 46: 2519-2527.