In situ processing and properties of nanostructured hydroxyapatite/alginate composite
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- Wang, L., Li, Y. & Li, C. J Nanopart Res (2009) 11: 691. doi:10.1007/s11051-008-9431-y
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A series of hydroxyapatite/alginate (HA/Alg) nanocomposites with alginate amounts varying from 10 to 40 wt% were prepared through in situ hybridization technique. The inorganic phase in the composites was carbonate-substituted HA with low crystallinity. The crystallinity of HA decreased with the increase of alginate content. HA crystallites were needle-like in shape with a typical size of 20 to 50 nm in length and 5 nm in width. FT-IR spectroscopy indicated that the chemical interaction occurred between the mineral phase and the polymer matrix. As compared to pure HA without alginate, the composites showed more homogeneous microstructures, where HA nanocrystals were well embedded in alginate matrix. Among all the samples, the composite containing 30 wt% alginate exhibited a highly ordered three-dimensional network, similar to natural bone’s microstructure.
KeywordsHydroxyapatiteAlginateNanocompositeIn situ hybridizationMicrostructureNanocrystalsNanomaterials
Recently, dramatic increases in aging population and osseous defects originating from sports and traffic-related trauma, infections, and skeletal diseases have significantly stimulated the exploitation of artificial bone graft materials (Bigi et al. 1998; Murugan and Ramakrishna 2004). An ideal bone substitute should be able to provide bony bonding with surrounding living tissues, to serve as a scaffold with sufficient mechanical strength and to stimulate growth and formation of new bone tissues (Zhao et al. 2002; Tampieri et al. 2005). Besides, it should be biodegradable so that it can degrade gradually and ultimately be replaced by newly formed bone tissues. Directing efforts towards the development of desirable synthetic scaffold materials in view of the specific challenges posed by rapid advances in bone tissue engineering will undoubtedly be rewarding.
Natural bone has been well documented as a complex tissue comprising approximately 70 wt% of hydroxyapatite (HA) and 30 wt% of collagen matrix, in which nanosized HA particles are embedded within organic matrix in a well-organized manner (Bigi et al. 2002; Wang 2003). Natural bone’s ubiquitous hybrid composition and inimitable highly ordered hierarchical structure have presented us valuable clues to achieve intelligent scaffold materials. Therefore, increasing interest has been focused on the development of novel bio-inspired HA/polymer composites attempting to create devices capable of reproducing the complex hierarchical structure of native bone (Dujardin and Mann 2002; Mann and Ozin 1996; Stupp and Braun 1997). Several natural polymers with good biodegradability and bioactivity are commonly used, such as collagen (Du et al. 2000; Kikuchi et al. 2001; Zhang et al. 2003a, b), gelatin (Bigi et al. 1998; Chang et al. 2003), chitosan (Kong et al. 2005; Murugan and Ramakrishna 2004; Yamaguchi et al. 2001), silk fibroin (Furuzono et al. 2000; Wang et al. 2002, 2004), alginate (Lin and Yeh 2004; Maruyama et al. 1995; Ribeiro et al. 2004; Sivakumar and Rao 2003; Teng et al. 2006), and chondroitin sulfate (Rhee and Tanaka 2002).
Alginate (Alg) is a natural acidic polysaccharide derived from brown sea algae (Sivakumar and Rao 2003; Lin and Yeh 2004; Teng et al. 2006). It is a linear anionic copolymer containing (1-4)-linked β-d-mannuronic acid (M units) and (1-4)-linked α-l-guluronic acid (G units) residues. Alginate is highly biocompatible, biodegradable, hydrophilic, and relatively economical and has sufficient adhesiveness, plasticity, and hemostatic property. Therefore, alginate has been widely utilized in wound dressing, surgical or dental impression, and tissue engineering. Several reports in literature addressed the preparation of HA/Alg composite scaffolds as bone filler via different techniques such as phase separation, droplet extrusion, or dispersion polymerization (Lin and Yeh 2004; Maruyama et al. 1995; Ribeiro et al. 2004; Sivakumar and Rao 2003; Teng et al. 2006). Maruyama et al. reported that a mixture of HA powder and saline solution of sodium alginate showed good osteoconductivity to bridge the gap between implants and cortical bone without any adverse reaction (Maruyama et al. 1995). A HA/Alg composite with a highly interconnected porous structure and considerable mechanical properties was prepared by Lin and Yeh which displayed appreciable osteoblastic cell attachment (Lin and Yeh 2004). Sotome et al. and Zhang et al. demonstrated that the incorporation of alginate into HA/collagen composite improved the mechanical properties and promoted active tissue invasion and bone formation in vitro (Sotome et al. 2004; Zhang et al. 2003a).
A common feature of the above synthesis procedure lies in that the conventional mechanical mixing of as-prepared HA granules with Ca2+ crosslinked alginate was involved. However, lack of physicochemical homogeneity and adequate interfacial bonding between the mineral and polymer phases still remains an issue of concern over HA/Alg composites obtained by this method (Yamaguchi et al. 2001; Sailaja et al. 2003). As we know, natural bone apatite is formed in a physiological environment in the presence of collagen, Ca2+, PO43−, and other mineral ions (Gibson and Bonfield 2002; Liou et al. 2005). Inspired by this naturally occurring biomineralization, recently, material researchers have exploited in situ hybridization aimed at producing chemically and biologically improved HA-polymer composites as bone substitute materials. The advantage of this method over the mechanical mixing is that the binding strength at the inorganic–organic interface can be enhanced by the established chemical interaction between HA and the polymer matrix. However, little attention has been given to the in situ synthesis of the HA/Alg composite.
The present study was designed to establish the feasibility of in situ synthesis of HA/Alg composite with chemical and structural homogeneity, and to gain more insight into the microstructure and physicochemical properties of the composite. In order to reach these scopes, a series of HA/Alg composites with different alginate contents were prepared via in situ hybridization. Moreover, the investigation into the influences of alginate content on the crystallographic properties and morphology of HA crystallites, the chemical interaction between HA and alginate, as well as the microstructure of the composites was carried out. This work is expected to provide a framework for the design and generation of HA-based composite materials for bone repair and remodeling.
Preparation of HA/Alg composites
Ca(OH)2, CaCl2, and 85 wt% H3PO4 of analytical grade were used without further purification. Sodium alginate (with a low viscosity of 0.25 Pas for its 2 wt% solution at 25 °C) was purchased from Sigma-Aldrich Inc., USA.
Notations for the samples and the amount of each starting material used
HA/Alginate weight ratio
Weight of Ca(OH)2 (g)
Weight of 85 wt% H3PO4 (g)
Weight of sodium alginate (g)
Crystalline phase composition of the as-prepared powders was confirmed by X-ray diffractometer (XRD, RINT PC1 Rigaku Co.) with CuKα radiation. Fourier transform infrared (FT-IR) spectra were recorded using a spectrometer (Nicolet AVATR360). An enhanced laser diffraction granulometer (Beckman Coulter LS230) was used to analyze particle size distribution. Morphology and size dimensions of HA crystallites were examined by a transmission electron microscope (TEM, JEOL JEM-2100). Microstructural observations were performed on a scanning electron microscope (SEM, JEOL JSM-6700F).
Results and discussion
Crystalline phase composition
Particle size distribution
Results of particle size distribution
Mean size (μm)
Chemical state analysis
Morphology of HA crystallite
Alginate is crosslinked with Ca2+ to form Ca–alginate complex with an “egg-box” structure through the ionic interaction between Ca2+ and the carboxyl group of G units located on the polymer chain (Lin and Yeh 2004; Teng et al. 2006). The Ca–alginate complexes are thought to affect the nucleation and growth of HA crystals. In the composites with low alginate content of 10 to 20 wt%, the influence of the alginate matrix is not very significant and thus the morphology of HA crystallites is not greatly changed. With alginate content increased to 30–40 wt%, larger number of Ca–alginate complexes with long polymer chains are formed, which in turn intensifies the steric hindrance of the adjacent complex macromolecules. The extended direction of the polymer chains of alginate macromolecules may be parallel to the elongated direction, ca. c-axis of HA crystallites. Consequently, HA crystallites show an apparent tendency to elongate and aggregate preferentially along c-axis.
Microstructure of HA/Alg composite
It is observed in HA/Alg composites that HA aggregates are embedded in the alginate matrix. The interfacial boundary between the mineral and organic phases is hardly distinguished, indicating the appreciable miscibility between them. The microstructure tends to be more homogeneous and compact with the increase of alginate content. In pure HA, HA90/Alg, and HA80/Alg, hollows with irregular shapes and variable sizes ranging from 50 nm to several hundred nanometers are observed among those large block aggregates, while fewer hollows are observed in HA70/Alg and HA60/Alg. Alginate acts as a binder for HA nanoparticles, and hence its content is of importance to determine the ultimate microstructure of the HA/Alg composite. Alginate content higher than 30 wt% is sufficient to permit complete fusion of HA crystallites into the alginate matrix. As reported, there is a problem always encountered with HA-based implant is that HA particles easily migrate from the implanted sites into surrounding tissues when mixed with patient’s body fluid and blood (Murugan and Ramakrishna 2004; Yamaguchi et al. 2001). The migration causes damage to healthy tissues and thus the therapeutic efficacy of the implant is not satisfactory. Bearing this in mind, the complete fusion of HA nanoparticles into the alginate matrix is beneficial for particulate immobilization.
In HA70/Alg, higher level of interpenetration between the HA nanoparticles and the alginate matrix is achieved so that a hierarchical three-dimensional network is built up. HA60/Alg shows a compact and homogeneous microstructure with smoother surface and uniform particle distribution. Among all the samples, HA70/Alg bears a very close microstructural resemblance to natural bone, and thereby, can be considered as an ideal candidate for healing bone defects.
HA/Alg nanocomposites with different HA/Alg weight ratios were synthesized by in situ hybridization. The inorganic component in the composite is identified as poorly crystalline HA containing carbonate ions, similar to biological apatite. The primary HA crystallites are needle-like and 20–50 nm long and around 5 nm wide. The crystallographic features of HA are affected by the involvement of alginate, i.e., the crystallinity of HA decreases and the crystallites tend to elongate along the c-axis upon increasing the alginate content. As confirmed by FT-IR spectroscopy, the notable blue shifts in the carboxyl group from alginate to the composites indicate that the chemical interaction takes place between the inorganic and polymeric constituents in the composites. The chemical interaction is assumed to be mediated by the chemical bonding between Ca2+ and the carboxyl group in alginate. The composites present a more compact microstructure than pure HA. The complete fusion of HA crystallites into the polymer matrix is achieved in the composites with the alginate content of 30 to 40 wt%, facilitating the immobilization of HA nanoparticles upon implantation. A three-dimensional network with a higher degree of structural organization is built up in the composite with HA/Alg weight ratio of 70/30. The innovative in situ processing might offer a promising future for HA/Alg composite materials in bone regeneration and implant fixation.
This work was supported by the national high technology research and development program of China (2006AA03Z358), and the Special Projects for Nanotechnology of Shanghai (0652 nm034).