Acta Biomaterialia 7 (2011) 27692781

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Review

Nanoscale hydroxyapatite particles for bone tissue engineering

Hongjian Zhou, Jaebeom Lee
Department of Nanomedical Engineering, College of Nanoscience and Nanotechnology, Pusan National University, Miryang 627-706, Republic of Korea

a r t i c l e

i n f o

a b s t r a c t
Hydroxyapatite (HAp) exhibits excellent biocompatibility with soft tissues such as skin, muscle and gums, making it an ideal candidate for orthopedic and dental implants or components of implants. Synthetic HAp has been widely used in repair of hard tissues, and common uses include bone repair, bone augmentation, as well as coating of implants or acting as llers in bone or teeth. However, the low mechanical strength of normal HAp ceramics generally restricts its use to low load-bearing applications. Recent advancements in nanoscience and nanotechnology have reignited investigation of nanoscale HAp formation in order to clearly dene the small-scale properties of HAp. It has been suggested that nanoHAp may be an ideal biomaterial due to its good biocompatibility and bone integration ability. HAp biomedical material development has beneted signicantly from advancements in nanotechnology. This feature article looks afresh at nano-HAp particles, highlighting the importance of size, crystal morphology control, and composites with other inorganic particles for biomedical material development. 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Article history: Received 7 December 2010 Received in revised form 11 March 2011 Accepted 16 March 2011 Available online 1 April 2011 Keywords: Biomaterials Bioceramics Nanoscale particles Hydroxyapatite Bone tissue engineering

1. Introduction Bone is a natural organicinorganic ceramic composite consisting of collagen brils containing embedded, well-arrayed, nanocrystalline, rod-like inorganic materials 2550 nm in length [1 3]. Structural order in bone occurs at several hierarchical levels and reects the materials and mechanical properties of its components (Fig. 1). Hydroxyapatite (HAp) is chemically similar to the inorganic component of bone matrix a very complex tissue with general formula Ca10(OH)2(PO4)6. The close chemical similarity of HAp to natural bone has led to extensive research efforts to use synthetic HAp as a bone substitute and/or replacement in biomedical applications [4,5]. Tissue engineering is intensively researching solutions that have the potential to reduce the complications related to current treatment methods. Tissue engineering can be dened as an interdisciplinary eld that applies the principles of engineering and life sciences to develop biological substitutes that restore, maintain or improve tissue function [6]. This concept involves three main strategies: the use of isolated cells or cell substitutes to replace limited functions of the tissue; utilization of tissue-inducing substances such as growth factors; and scaffolds to direct tissue development. An ideal scaffold for bone tissue engineering is a matrix that acts as a temporary substrate allowing cell growth and tissue development. This occurs initially in vitro and eventually in vivo. The scaffold should be able to mimic the structure and biological function of the native extracellular matrix (ECM) in terms of both chemical
Corresponding author.
E-mail address: jaebeom@pusan.ac.kr (J. Lee).

composition and physical structure. Scaffolds used for tissue engineering applications should also be biocompatible; able to provide appropriate mechanical support; exhibit favorable surface properties such as promoting adhesion, proliferation and differentiation of cells; and provide an environment in which cells can maintain their phenotypes. Recently, HAp has been used for a variety of biomedical applications, including matrices for drug release control and bone tissue engineering materials [8,9]. Since HAp has chemical similarity to the inorganic component of bone matrix, synthetic HAp exhibits strong afnity to host hard tissues. Chemical bonding with the host tissue offers HAp a greater advantage in clinical applications compared to most other bone substitutes such as allografts or metallic implants [10]. The main advantages of synthetic HAp are its biocompatibility, slow biodegradability in situ, and good osteoconductive and osteoinductive capabilities [1,11]. A study by Taniguchi et al. showed that sintered HAp exhibits excellent biocompatibility with soft tissues such as skin, muscle and gums. Such capabilities have made HAp an ideal candidate for orthopedic and dental implants or components of implants. Synthetic HAp has been widely used to repair hard tissues. Common uses include bone repair, bone augmentation, as well as coating of implants or acting as llers in bone or teeth [1218]. However, the low mechanical strength of normal HAp ceramics restricts its use mainly to low load-bearing applications. Recent advances in nanoscience and nanotechnology have reignited interest in the formation of nanosized HAp and the study of its properties on the nanoscale. Nanocrystalline HAp powders exhibit improved sinterability and enhanced densication due to greater surface area, which may improve fracture toughness, as well as other mechanical

1742-7061/$ - see front matter 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2011.03.019

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Fig. 1. The hierarchical structure of bone at its various length scales. The microstructure of cortical bone consists of osteons with Haversian canals and lamellae, and at the nanoscale, the structural units are collagen bers composed of bundles of mineralized collagen brils. Copyright Elsevier and reproduced with permission [7].

properties [11]. Moreover, nano-HAp, compared to coarser crystals, is expected to have better bioactivity [19]. Thus, nano-HAp particles can be utilized for engineered tissue implants with improved biocompatibility over other implants. Nanotechnology has the potential to signicantly benet development of HAp biomedical materials. To our knowledge, several reviews of nanocrystalline calcium orthophosphates have been published in recent years. For example, Dorozhkin et al. [20,21] reviewed the current state of technology and recent developments of various nanosized and nanocrystalline calcium orthophosphates, involved in synthesis and characterization as well as biomedical and clinical applications. Moseke et al. [22] reviewed the synthesis and properties of tetracalcium phosphate (TTCP) in biomaterial applications such as cements, sintered ceramics and coatings on implant metals; Johnson et al. [18] reviewed the compression, exural and tensile properties of calcium phosphate (CaP) and CaPpolymer composites for applications in bone replacement and repair; Tran et al. [23] summarized studies that have demonstrated enhanced in vitro and in vivo osteoblast functions (e.g. adhesion, proliferation, synthesis of bone-related proteins and deposition of calcium-containing mineral) on nanostructured metals, ceramics, polymers, and composites. After reviewing these feature articles to avoid any redundancy, we focus on calcium orthophosphate, and characterize its properties in the condition of nano-HAp with different morphologies and porous structures-materials that offer great promise as bone substitutes and/or replacements in biomedical applications. Moreover, we summarize how composites of HAp and other inorganic nanomaterials can enhance the bioactivity and biocompatibility of HAp an area that has become the focus of recent research. The remainder of this feature article is organized into ve sections. In the Section 2, the synthesis of morphologically different nano-HAps is introduced. Section 3 discusses the fabrication of the porous structure of nano-HAp. Section 4 reviews the bio-orthopedic properties of nanoscale HAp for application in bone tissue engineering. Section 5 introduces composites of HAp and other inorganic nanomaterials for enhancing the bioactivity and biocompatibility of HAp. Finally, in Section 6, we provide a summary and our own perspectives on this active area of research. 2. Synthesis of nanoscale HAp HAp (Ca10(OH)2(PO4)6) nano- and microcrystals with multiform morphologies (separated nanowires, nanorods, microspheres,

microowers and microsheets) have been successfully synthesized by many powder processing techniques, including solgel synthesis [2428], solid state reactions [29], co-precipitation [30], hydrothermal reactions [31], microemulsion syntheses [32] and mechanochemical synthesis [33]. 2.1. HAp nanoparticles From a practical application perspective, suitable nano- or micromaterials with specic morphologies not only need to be capable of being synthesized in large quantities with a desired composition, reproducible size and structure, but also of being prepared and assembled using environmentally responsible techniques. Recently, environmentally friendly synthetic methodologies, including molten-salt synthesis, hydrothermal processing, biomimic synthesis and template synthesis, have been implemented as viable techniques for the synthesis of a range of materials [34,35]. Nanosized HAp particles can be prepared by a variety of techniques such as mechanochemical synthesis [36], combustion preparation [37] and various wet chemistry techniques [38,39]. Among the most reported precipitation processes, chemical agents such as citric acid [40,41], amino acids [42] and ethylenediaminetetraacetic acid (EDTA) [43,44] have been used to mediate HAp nucleation and crystal growth. These modiers exert signicant control over crystal morphology due to afnity between the modifying agent and the HAp crystals. However, there has been less focus on the precipitation kinetics of nucleation and growth, which are related to the degree of supersaturation, SHAp. This value can be calculated as follows:

IAPHAp ; K sp

where IAPHAp is the ionic activity product expressed as:

3 5 3 IAPHAp Ca2 5 PO3 4 OH cCa2 cPO3 cOH :
4

The brackets represent ion concentrations of the respective species and c values are the activity coefcients of the ions. Ksp is the solubility product of HAp. With a higher degree of supersaturation, a greater driving force for precipitation, i.e. a faster precipitation rate, was expected with increasing IAPHAp [45]. Biological mineralization (or biomineralization) is the process of in vivo inorganic material formation. The new theory of aggre-

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gation-based crystal growth [46] and the new concept of the mesocrystal [47] highlight the roles of nanoparticles in biological crystal engineering. Mimicking the formation of natural CaP, hard tissues contribute signicantly to the biological function of engineered materials. Many advances have been made in biomaterial research with the rapid growth of nanotechnology. The study of CaPs is a specic area in nanotechnology, which may be applied readily in the repair of hard and soft skeletal tissues [4850]. Mollazadeh et al. [51] prepared HAp crystals using an in situ biomimetic process in the presence of polyvinyl alcohol (PVA). They systematically investigated the effects of polymer amount and molecular weight on the physical properties of HAp crystals. The results indicated that the development (size and shape) of HAp nanocrystals precipitated in an aqueous solution of PVA was inversely related to the polymer molecular weight (i.e. the smallest crystallite size was observed with the highest PVA molecular weight). It is thought that HAp formation is initiated through the interaction of Ca2+ ions with the negative side groups on the polymer surface. The larger number of reaction sites in the higher molecular weight PVA polymer led to a higher number of HAp nuclei, and therefore a smaller crystallite size. The hydrothermal method, a typical solution-based approach, has proven to be an effective and convenient process to prepare various inorganic materials with diverse, controllable morphologies and architectures [5255]. The environmentally acceptable advantages of this method include easily controllable reaction conditions, relatively large scale and high yield in terms of quantity of the desired products, and frequent use of water as the reaction medium. Zhang et al. [56] developed a general strategy for the synthesis of HAp nano- and microstructures using water as a reaction medium through a simple hydrothermal process. Several dominant morphologies were achieved (nanorods, nanowires, microsheets, burr-like microspheres and microowers). The pH value plays a crucial role in obtaining Ca10(OH)2(PO4)6 samples with various morphologies. The use of trisodium citrate also has an important

inuence on product shape. Possible formation mechanisms for Ca10(OH)2(PO4)6 nano- and microcrystals with diverse morphology are presented in Fig. 2. A strong blue emission peak at approximately 428 nm was observed at room temperature, and the photoluminescence (PL) intensity of this emission varied with Ca10(OH)2(PO4)6 morphology over a range of pH values. CO2 radicals in the HAp lattice interstitials may be responsible for self-activated luminescence. These types of phosphors do not contain metal ions as activators and contain no toxic elements; thus, they are considered environmentally friendly luminescent materials. The microsized Ca10(OH)2(PO4)6 sample prepared at pH 5.0 with strong blue emission, spherical morphology, non-aggregation and high crystallinity can potentially be used as a new, efcient and environmentally friendly blue luminescent material. Qiu et al. [57] prepared nanocrystalline HAp by precipitation with the aid of ultrasonic atomization using Ca(NO3)24H2O and (NH4)2HPO4 as raw materials. The results showed that the synthesis method used in this study can effectively shorten reaction time while improving powder homogeneity compared to other published methods. It was also found that addition of a small amount of the surfactant glycine during precipitation synthesis can reduce HAp nanoparticle agglomeration. However, Poinern et al. [1] and LeGeros [11] developed a chemical route to synthesize HAp using calcium nitrate and potassium hydrogen phosphate as the main raw materials. This hydrothermal method also used ultrasonic irradiation followed by heat treatment to manufacture nanosized HAp. This study has shown that nano-HAp particles with a spherical morphology in the nanometer range (approximately 30 nm 5%) can be synthesized using a hydrothermal chemical precipitation method incorporating low-power sonic irradiation. The crystallinity and morphology of HAp depends on the Ca/P ratio, ultrasonic irradiation and temperature. An ultrasonic power of 50 W and temperatures in the range of 400 C are sufcient to produce the nano-HAp. This offer an economic route for the synthesis of nano-HAp with a strong scale-up capability.

Fig. 2. Schematic for the formation and morphology evolution mechanism of Ca5(PO4)3OH samples with various morphologies based upon different pH values [56].

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Li et al. [58] studied the crystalline behaviors of HAp inuenced by different forms of citrates and explained this behavior using the classical theory of crystal nucleation and growth in solutions. They found that the supersaturation value decreased, i.e. the speed of crystal nucleation decreased, with increasing citric acid monohydrate (C6H8O7H2O, CiA) concentration or with decreasing trisodium citrate (Na3C6H5O72H2O, NaCit) concentration. The presence of CiA inhibited the fast-growing of (0 0 l) planes and increased the crystallinity of (h k 0) planes. This resulted in homogeneously grown particles rather than the prismatic particles obtained when synthesis is performed without CiA. By raising the synthesis temperature, ner and more homogenously grown particles could be prepared in the presence of CiA. With increased ripening time, a higher crystallinity of HAp with lower amounts of CaO phase was obtained. This could be explained by crystal growth in solutions, i.e. prolonged maturation time resulting in a higher amount of unreacted Ca(OH)2 that can be involved in the crystal growth of CaP powders. Utilizing nanotechnology, calcium and phosphate can be manipulated at the molecular level and assembled to produce materials with unique structural and functional properties. The preparation of CaP powders with a particular morphology, stoichiometry, crystallinity and crystal size distribution is important in biomedicine and materials science. Various processes have been employed to prepare CaP powders, including co-precipitation [59], solgel process [60,61], spray pyrolysis [62], hydrothermal synthesis [63,64], emulsion processing [65,66] and mechanochemical method [67]. Among these methods, the solgel method has received the most attention because of its well-known inherent advantages, namely homogeneous molecular mixing, low processing temperature, and ability to generate nanocrystalline powders, bulk amorphous monolithic solids and thin lms [68]. Natarajan et al. [69] prepared nanosized HAp particles using the solgel method from the water-based solution of calcium and phosphorus precursors. In that study, two calcium precursors, i.e. calcium nitrate tetrahydrate and calcium acetate, were chosen. The inuence of aging period, pH, viscosity and sintering temperature on the crystallinity and morphology of HAp particles were investigated for the two calcium precursors and a triethyl phosphate precursor. The morphology of nano-HAp when the phosphorous precursor was used was dependent on the type of calcium precursor used. Gopi et al. [70] reported the synthesis and characterization of nano-HAp powders by a novel ultrasonic coupled solgel synthesis. The resulting powders were sintered by conventional means at different temperatures. These results show that nano-HAp powders synthesized by ultrasonic coupled solgel synthesis showed a remarkable particle size reduction compared with the conventional solgel method, hence; these powders could be used as a coating material in biomedical applications. With the increasing need to develop clean, non-toxic and environmentally friendly techniques, HAp powders have been extracted using bioproducts such as corals, cuttlesh shells, natural gypsum, natural calcite and bovine bone [71,72]. Chemical analysis has shown that these products, typically considered as biowaste, are rich sources of calcium in the form of carbonates and oxide. One such biowaste is chicken eggshells. Every day, a million tonnes of eggshells are generated as biowaste around the globe. Eggshell represents $11% of the total egg weight and is primarily composed of calcium carbonate ($94%), calcium phosphate ($1%) and organic matter ($4%) [37]. Besides being economically cheap and plentiful in nature, eggshells have been shown to be biocompatible with the human body during implantation but not osteoconductive. Hence, converting these powders into HAp prior to implantation is advantageous. Sanosh et al. [73] reported a simple solgel precipitation technique to synthesize nano-HAp powders using CaO derived from chicken eggshells. Their study shows that biowaste egg shells

can effectively be utilized for synthesizing pure nano-HAp powders. Particle formation by solgel is a very complex process. It involves nucleation, growth, aggregation and agglomeration [74,75]. Lee et al. [76] also synthesized highly sinterable, nanosized HAp powders using a wet chemical route with recycled eggshell and phosphoric acid as calcium and phosphorous sources, respectively. Raw eggshell was easily converted to CaO by the calcining process, and phosphoric acid was mixed with the calcined eggshell by a wet ball-milling method. The observed phases of the powder synthesis process were dependent on the mixing ratio (wt.%) of the calcined eggshell to phosphoric acid and the heating temperature. The ball-milled, nanosized HAp powder, which has an average particle size of 70 nm, is fully densied at 1300 C for 1 h. The Ca/P ratio for the stoichiometric composition of HAp was controlled by adjusting the mixing ratio. In addition, HAp can be successfully produced from recycled eggshells, seashells and phosphoric acid. The phases obtained depend on the ratio of calcined eggshells/seashells to phosphoric acid, the calcination temperature and the mechanochemical activation method (ball milling or attrition milling) [77]. Mostaghaci et al. [78] synthesized nanosized HAp using an Iranian strain of Serratia. Results showed that the optimum powder production was achieved at approximately pH 8 and a temperature of 37 C. The powder particles were single crystals and ranged in size from 25 to 30 nm. Moreover, particle shapes and the sizes were relatively uniform and exhibited lower agglomeration relative to conventional methods. This powder could be used in the regeneration of bone defects, fabrication of medical implants, and as a vector for pharmaceutical and biological materials such as genes. 2.2. HAp nanorods and nanoakes Bone is a composite, consisting of HAp nanorods embedded in a collagen matrix. In the biomineralization process of vertebrate hard tissues, some specic molecules control the nucleation and growth of inorganic crystals (HAp), resulting in the formation of hierarchical structure of teeth and bones with superior mechanical properties [79,80]. Thus, controlled syntheses of apatitic crystals with various morphologies have been the focus of intensive research in order to understand more completely their biomineralization and utility in industrial and biomedical applications [81]. Inspired by biomineralization, molecular manipulation of inorganic crystals with organic growth modiers gradually developed into a powerful tool for the design of novel tissue engineering materials [82,83]. The moleculetemplate combination exerts signicant control over crystal morphology and has been discussed in many articles [8487]. A variety of organic molecules such as hexadecyltrimethyl-ammonium bromide (CTAB), sodium dodecyl sulfate (SDS), amino acids, proteins and monosaccharides have been used to synthesize HAp with brous- and ake-like morphologies [86,88 92]. Kalita et al. [93] synthesized bioactive HAp (Ca10(PO4)6(OH)2) ceramic powder in the lower end of the nanoregime using microwave radiation, which offers several advantages. The applied microwave power of 600 W, pH of the suspension, mole ratio of Ca/P in the starting chemicals, and the chelating effect of EDTA served as important factors in the synthesis of nanocrystalline HAp powder. Results conrmed a highly crystalline nanopowder (530 nm) with elemental composition of Ca and P in the HAp phase and possessing mixed (elliptical and rod-shaped) morphology. Tari et al. [94] reported that using poly(sodium 4-styrene sulfonate) (PSSS) as a nucleation- and growth-controlling agent resulted in the precipitation of well-crystallized HAp nanoparticles through microemulsion. During PSSS mixing with a calcium precursor, rod-like micelles were formed, which control the morphology and crystallization of nano-HAp. The investigations showed that

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the obtained HAp nanorods have an average width and length of about 30 and 200 nm, respectively. Shanthi et al. [95] reported that CTAB is used as the capturing material for successfully prepared nano-HAp rods at ambient temperature and normal atmospheric pressure. A cytotoxicity test using a normal cell line indicated the biocompatible range of the nanocrystalline HAp and its fairly non-toxic nature. Calcium phosphates comprise a large family of compounds with important biological applications-including osteologic implant for coatings, grafts, scaffolds and bone cavity llings, and vehicles for drug, protein and gene delivery-due to their similarity with the mineral constituents of human bones and teeth. The properties of calcium phosphate, including bioactivity, biocompatibility, solubility, mechanical properties and absorption, can be tailored over wide ranges by controlling particle composition, size, morphology and assembly. For these reasons, it is of great importance to develop synthesis methods focused on the precise control of particle size, morphology and chemical composition. Jiang et al. [96] presented an approach using CaP that offered careful size and structural control via a template-guided process. In their study, HAp nanorods and well-aligned hybrid HAp composites were prepared via a template-guided synthesis procedure (a schematic illustration of the formation of HAp nanorods is shown in Fig. 3). First, phosphate ester was used as a structure-directing agent for preparing HAp nanorods. After hydrothermal treatment, CaP nanorods with well-controlled particle size and porosity can be obtained. Second, carboxymethyl cellulose (CMC) molecules were found to be effective in controlling particle size and the subsequent alignment of HAp. HAp composites with well-organized microstructures were prepared using CMC. CMC has carboxyl groups that can attract Ca2+ ions, thereby guiding the growth of HAp grains. These proposed processes can also be applied to the preparation of CaP materials with well-controlled microstructures using other similar templates for a wide range of applications. HAp is the basic component of natural bone. In the bone formation process, HAp mineralization is controlled by collagen, which is a special protein containing an ionic group that can interact with HAp, and a dispersive group, which can stabilize HAp in the physiological environment. However, dental structures are also composed of HAp, which is needle-like and forms under the control of proteins. Nearly the entire biological mineralization process is a crystallization process controlled by organic components.

Recently, considerable attention has been paid to the biomimetic mineralization process because this process may lead to the fabrication of novel materials that cannot be produced by conventional methods [97101]. Zhang et al. [102] reported the mineralization of nano-HAp on self-assembled collagen; the nanocomposite formed has a similar structure and biocompatibility to natural bone. Tang et al. [103] reported the fabrication of nano-HAp under the control of amino acids and found that both bone mimetic platelike and dental-mimetic rod-like nano-HAp can be produced under the control of different amino acids. This method makes it possible to control nano-HAp mineralization by purely synthetic polymers rather than natural biomacromolecules. Since the synthetic polymer has a relatively low cost and can be more easily designed, nano-HAp synthesized by this method has many potential applications. Yao et al. [104] employed double-hydrophilic block copolymer (DHBC) poly-(vinylpyrrolidone)-b-poly(vinylpyrrolidone-alt-maleicanhydride)-b-poly-(vinylpyrrolidone) (PVP-bP(NVP-alt-MAn)-b-PVP) to synthesize a biomimetic template for HAp nanocrystal synthesis. Needle-like HAp nanocrystals can be formed in the presence of PVP108P(NVP-MAn)28PVP108, as shown in Fig. 4. Compared to the HAp nanocrystals formed in the presence of poly-(vinylpyrrolidone) (PVP) homopolymer, those formed with DHBC are more stable and do not precipitate in water after preparation. The crystallization process and the morphology of the nal nano-HAp crystals can be controlled by adjusting the DHBC molecular structure. Cho et al. [105] prepared nanosized HAp powders with high crystallinity and appropriate stoichiometry by a hightemperature ame spray pyrolysis process from spray solutions containing PEG. The mean sizes of HAp powders obtained from PEG spray solutions were changed from several tens to several hundred of nanometers according to the PEG concentrations in the spray solutions at a post-treatment temperature of 800 C. HAp powders post-treated at a low temperature of 400 C had a ber-like morphology. On the other hand, post-treated HAp powders at temperatures of 600 and 1000 C had a rod-like morphology with a low aspect ratio and spherical-like morphology, respectively. The mean sizes of HAp powders post-treated at temperatures of 600 and 1000 C were 32 and 213 nm, respectively. The application of nanostructured HAp with different morphologies for bone tissue engineering has been introduced above. However, several other applications of nano-HAp are also in progress. Surface modications of HAp nanoparticles have been performed

Fig. 3. Schematic illustration of the formation of HAp nanorods. Copyright Elsevier and reproduced with permission [97].

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Fig. 4. TEM images of HAp nanocrystals templated by PVP108P(NVP-MAn)28PVP108 after the 1 day (a), 8 day (b) and 13 day (c) preparations. Copyright ACS and reproduced with permission [104].

in order to modulate their colloid stability, prevent dissolution in the case of low pH, prevent inammation, serve as an intermediate layer to allow strong bond formation between HAppolymer matrices, and potentially enhance its bioactivity or improve its conjugation ability with special functional groups [106108]. HAp nanoparticles have also served as non-viral carriers for drug delivery and gene therapy because of their established biocompatibility, ease of handling and well-known adsorption afnity [109112]. Furthermore, HAp nanoparticles can be stably loaded with radioisotopes [111]. After loading with genes or drugs by adsorption, HAp nanoparticles provide a protective environment that shields them from degradation while providing a convenient pathway for cell membrane penetration and the controlled release of the genes/drugs [112]. The research results indicate the potential of nano-HAp in gene delivery and as drug carriers [112,113]. Readers who are interested in learning more may refer to several elegant and more comprehensive reviews, e.g. those by Dorozhkin [20,21,114]. 3. Porous structure of nanoscale HAp HAp ceramics have been widely used as articial bone substitutes because of their high biocompatibility, bioafnity and osteoconductibility. However, induction of bone growth into HAp blocks is unsatisfactory, because it is very slowly replaced by host bone after implantation. For this reason, porous bodies and granules of HAp ceramics have been developed and have been widely used in clinical settings. However, due to the closed structure of conventional porous HAp, which has non-uniform pore geometry and low interpore connections, it is very difcult for implant pores to become completely lled with newly formed host bone [115]. However, porous HAp ceramics with highly interconnecting structures have been developed, and osteoconduction can occur deep inside such ceramics. Wang et al. [116] systematically studied the effects of electrical polarization of porous HAp ceramics using two types of cylindrical porous HAp ceramics with high and low interpore connections (HAp-H and HAp-L, respectively) on bone ingrowth. Electrical polarization was effective in enhancing bone ingrowth through all the pores of HAp-H implants; however, this advantage was not apparent in the HAp-L implants. This suggested that enhanced bone ingrowth into HAp porous bodies due to electrical polarization may be a co-operative interaction between the osteoconductivity of HAp porous bodies and the enhanced osteogenic cell activity induced by large charges stored on pore surfaces. The size and density of interpore connections, as well as those of pores, are important factors for osteoconduction into the central area of porous HAp. Diverse characteristics, such as pore size, pore shape, pore interconnectivity and total porosity of the scaffold, are considered important factors for successful tissue regeneration [117,118]. Microwave heating has also been applied to fabricate interconnective porous structured bodies by foaming as-synthe-

sized, calcium-decient HAp (Ca-decient HAp) precipitate containing H2O2. The porous bodies were sintered by a microwave process with activated carbon as the embedding material to prepare nano- and submicron-structured ceramics. The study results suggest that porous carbonated biphasic CaP ceramics with a nanostructure promote osteoblast adhesion, proliferation and differentiation [119]. In conclusion, porous carbonated biphasic calcium phosphate (BCP) ceramics with a nanostructure are simple and quick to prepare using microwaves. Compared to those produced by conventional sintering, carbonated BCP ceramics may be better bone graft materials. Since synthesized HAp is very brittle, it cannot be used for loadbearing bone replacements. Hence, implant materials composed of hard and soft phases (composite materials) are used for total bone replacement [120]. Composites of HAp with polymers such as polymethyl methacrylate, poly(3-hydroxybutyrate-co-3-hydroxyvaleate) and polyacrylic acid show improved mechanical properties, as well as good biocompatibility and bioactivity [121,122]. Degradable and non-degradable polymers are used in controlled drug delivery, scaffolds for tissue engineering, wound dressing, cosmetic skin masks and protective clothing. Imai, Furuichi and co-workers [122,123] reported hierarchical laminated architecture and porous structures after calcinations of the polymer composite (poly(acrylic acid)) at 700 C. Polyacrylamide hydrogel is a biomaterial and non-degradable water-based polymer used as tissue ller. Joshy et al. [124] investigated mineralization of HAp in a UV-polymerized acrylamide gel matrix by varying precursor concentration and pH (pH 810). During polymerization, diammonium hydrogen phosphate ions were implanted in the gel matrix and subsequently immersed in calcium nitrate solution. Thin, laminated, macroporous structures, embedded with nanospheres and ribbons of HAp were mineralized. The HAp was found to be oriented along the c-axis, which could lead to preferential binding of acidic proteins on its surface. The laminated structures displayed a resorbable nature, whereas ake-like structures obtained at higher concentrations were found to be bioactive. This composite could be an alternative to the use of silicone gel to avoid the long-term risk of brosis and migration when implanted. Muthutantri et al. [125] developed a novel fabrication technique, a combination of slurry dipping and electrospraying, to produce HAp foams as potential matrices for bone tissue engineering applications. Slurrydipped and electrosprayed scaffolds for different time intervals were compared with foams prepared by the individual methods of dipping and electrospraying. Signicant differences in strut crack distribution and strut thickness and porosity were observed on sintered foams prepared under various conditions. All sintered structures had average porosities in the range of 8494% and desirable pore interconnections, whereas the combined method produced foams of uniform pore distribution, thicker struts and improved mechanical properties.

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A new direct rapid prototyping process called low-temperature deposition manufacturing (LDM) was proposed to fabricate scaffolds [126]. This process integrated extrusion/jetting and phase separation and can therefore fabricate scaffolds with hierarchical porous structures, creating an ideal environment for new tissue growth. Scanning electron microscopy (SEM) images of fabricated scaffold structures with different polymer matrices are shown in Fig. 5. The interconnected computer-designed macropores allow cells in new tissues to grow throughout the scaffold. Moreover, the parameter-controlled micropores allow nutritional components in, and metabolic wastes out. The macrocellular morphology, microcellular morphology, porosity and mechanical properties of poly(a-hydroxy acid)-tricalcium phosphate (TCP) composite scaffolds prepared by the proposed method were investigated. These highly controllable scaffolds may play an important role in tissue engineering. LDM could also be combined with multinozzle deposition or cell deposition to accurately control materials or cells point-by-point. Sinha et al. [127] report a novel method of producing HApPVA microspheres suitable for biomedical application. Spray drying is a well-established industrial process that produces ne ceramic powders. Integrating this with a method akin to biomineralization provides a direct route to produce HAp microspheres with highly controlled morphological features. 4. Bio-orthopedic properties of nanoscale HAp Bone substitutes are required to repair segmental defects caused by the removal of infected tissue or bone tumors. The most desirable form of bone substitutes, in such cases, is autologous bone. However, autografts are not always available and may result in morbidity at the donor site. An allograft is preferred in some cases, but the possible immune response and disease (i.e. human immunodeciency virus (HIV) or hepatitis B) transmission are detrimental to the recipient [128]. Bone graft substitutes have attracted much attention because of their advantages over both autografts and allografts [129]. In the case of a bone, an optimized biomaterial should be as biomimetic as possible, i.e. it should consist of poorly crystalline, carbonate-substituted apatite with sufcient mechanical properties [130]. HAp articial bones are now widely used in clinical practice and show satisfying repair function in a series of studies [131133]. Nonetheless, there are some weaknesses, such as weak intensity and slow degradation. To strengthen HAp, researchers have carried out omnidirectional investigations and the challenge in HAp development is whether biological properties can match mechanical properties [134]. Recently, our group reported HAp coating on scratched areas of a human tooth and HAp disks by the immersion method in a HAp colloidal solution (620 lm average diameter dispersed in deionized water) [135]. The surface morphologies of the scratched area

after immersion for 13 months were investigated and showed that the damaged surfaces were remarkably recovered. The mechanical property and chemical stability of the HAp coating layers on both specimens were then determined via the Vickers hardness test and concentration measurement of extracted Ca2+ ions, respectively, after strong acidic treatment. The cellular behavior of mouse calvaria-derived pre-osteoblastic cells (MC3T3-E1) was also examined on the HAp layers regenerated on micro-scratched HAp disks for the purpose of their potential applications on maxillofacial bone conservation and reconstruction for prosthetic dentistry, and articial disk preparation of a vertebral column. These results of HAp coating on the scratched areas of the tooth suggest that this technique could be suitable for the development of longterm prevention of micro-cleavage and tooth health supporters to reduce discoloration, and for other maxillofacial and orthopedic applications. Another interesting result published by our group concerned the regeneration of a micro-scratched tooth enamel layer by nanoscale HAp solution [136]. Nanoscale HAp powders with a mean particle size of 200 nm were used to regenerate the enamel layers of damaged teeth. An articially scratched tooth was immersed in a nanoscale HAp powder suspension in deionized water (70 wt.% HAp) at 37 C for a period of 13 months. After 3 months, the scratched surface was nally inlaid with HAp and the roughness increased from 2.80 to 5.51. Moreover, the hardness of the newly generated HAp layer on the crown was similar to that of the innate layer. Ca2+ and PO3 4 ions from the HAp powders dissolved in deionized water were precipitated on the tooth to produce cement pastes on the enamel surface due to its high degree of recrystallization, resulting in a hard, newly regenerated HAp layer on the enamel layer. This nanoscale HAp powder solution might be used to heal decayed teeth as well as to develop toothwhitening materials. In addition, our group reported the clinical evaluation of SB-1 as a synthetic bone in sinus bone grafting. The SB-1 used consists of synthetic HAp with 5001400 lm particle size and has the same chemical composition as the inorganic part of human bones. Rabbits with damaged bones were treated surgically with SB-1 and the progress of bone recovery was monitored using X-ray techniques. The result showed that the SB-1 was well integrated with the surrounding host bone and promoted induction of new bone, leading to bonding with newly formed bone and recovery of damaged bone tissue. In particular, it was found through an in vivo study with rabbit that SB-1 was superior in terms of bonding with host bones as well as induction and integration with new bone compared to the allografts and bone grafts with low HAp contents. Zhu et al. [128] evaluated the osteoconductive properties of nano-HAp material and its potential application as articial bone in repairing bone defects, and attempted to analyze the scientic

Fig. 5. SEM micrographs of scaffolds fabricated with different polymer matrices. Copyright ACS and reproduced with permission [126].

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basis of these properties. Their animal model of bone defects was based on the bilateral radius of 39 New Zealand white rabbits, which were randomly divided into an experimental group (bone defect repaired with nano-HAp articial bone), a control group (bone defect repaired with HAp articial bone) and a blank group (defect left empty). The experimental group was found to stimulate a callus bonier than the control and blank groups. These differences in bone conduction are statistically signicant (P < 0.05). Therefore, nano-HAp articial bone can potentially be used for bone defect treatment. Osteosarcoma is a primary malignant bone tumor, most prevalent in children and adolescents, and is usually highly aggressive and eventually lethal. Despite multimodal therapies, there is no effective approach to treat this malignant disease. Shi et al. [137] observed the biological response of osteosarcoma cells to two kinds of nano-Hap: nano-HAp-S and nano-HAp-L. These nanospheres have the same crystallinity (phase) and morphology but differ in size. Cells treated with both forms of nano-HAp were inhibited and mainly led to apoptotic cell death. The caspase-9dependent intrinsic apoptotic pathway plays a role in this. Interestingly, the suppression and apoptosis of osteosarcoma cells were directly related to the size of nanoparticles, and the larger-sized nano-HAp was more effective than the smaller particles. In the collagen matrix, tens to hundreds of these nano-blocks combine into self-assembled biomaterials that have remarkable physical and chemical features such as unique mechanical strength, insensitivity to growth/dissolution and exible structures [138,139]. Thus, features of smaller HAp nanoparticles may more closely resemble features of HAp during biomineralization than features of the larger HAp particles that are conventionally used. Therefore, nanoHAp may promote osteoblast adhesion, proliferation and synthesis of alkaline phosphatase and lead to rapid repair of hard tissue injury [140,141]. Nano-HAp has been reported to be a better candidate in biomedical applications. Cai et al. [142] prepared nano-HAp, typically

20 5, 40 10 and 80 12 nm in diameter, and studied their effects on proliferation of two bone-related cells: bone marrow mesenchymal stem cells (MSCs) and osteosarcoma cells (U2OS). Cell culture experiments showed improved cytophilicity of the nanophase mineral relative to conventional HAp. Greater cell viability and proliferation of MSCs were measured for nano-HAp, remarkably so for the 20 nm particles. Interestingly, the growth of osteosarcoma cells was inhibited by nano-Hap, and 20 nm particles were the best retardant. Nano-HAp has been suggested to exhibit favorable cell proliferation to optimize biological functionality, for which particle size is believed to play a key role. These in vitro ndings are of great signicance for understanding the cytophilicity and biological activity of nanoparticles during biomineralization. Li et al. [143] investigated the effects on highly malignant melanoma cells of nano-HAp particles with different morphologies. Three types of HAp particles with different morphologies were synthesized and co-cultured with highly malignant melanoma cells using phosphate-buffered saline (PBS) as a control. A precipitation method with or without citric acid addition as a surfactant was used to produce rod-like nano- and micron-sized HAp particles, respectively, and a novel oil-in-water emulsion method was employed to prepare ellipse-like nano-HAp particles. Experiment results indicated that the particle nanoscale effect, rather than particle morphology of HAp, was more effective in inhibiting highly malignant melanoma cell proliferation. Appleford et al. [144] found that the bone formation and angioconductive potential of HAp scaffolds closely matched to trabecular bone in a canine segmental defect after 3 and 12 weeks of implantation. Histomorphometric comparisons were made between naturally forming trabecular bone (control) and defects implanted with scaffolds fabricated with micro-size HAp (M-HAp) and nanosized HAp (N-HAp) ceramic surfaces. As shown in Fig. 6, no signicant differences were identied between the two HAp scaffolds; however, signicant bone in-growth was observed after 12 weeks with

Fig. 6. Bone tissue cross-section of N-HA scaffold under phase contrast (a) 3 and (c) 12 weeks post-surgery with corresponding cross-polarized light micrographs shown in (b,d) representing birefringence of collagen strands. 200 original magnication. S, scaffold; M, mineralized bone; C, collagen birefringence; V, vessel. Copyright ACS and reproduced with permission [144].

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43.9 4.1% and 50.4 8.8% of the cross-sectional area lled with mineralized bone in M-HAp and N-HAp scaffolds, respectively. This study showed the potential of trabecular bone modeled, highly porous and interconnected HAp scaffolds for regenerative orthopedics. 5. Composites of HAp and inorganic nanomaterials The low fracture toughness and poor wear resistance of HAp can be improved by adding second-phase reinforcement. Carbon nanotubes (CNTs) have already shown their potential as effective reinforcements for HAp and other ceramics [145148] to improve fracture toughness. Reports are available on the processing of HApCNT composite coatings for orthopedic implants through plasma spraying [149152], laser surface alloying [153,154], electrophoretic deposition [155,156] and aerosol deposition [157]. In addition to conventional sintering [158,159] and hot isostatic pressing [160,161], spark plasma sintering (SPS) [161163] has also been employed to fabricate free-standing HApCNT composites. Omori et al. [161] reported consolidation of multiwalled (MW) CNTs by SPS followed by dip-coating with HAp and a second round of SPS consolidation of the coated preform. They observed a consolidated coating of HAp on CNTs without crack formation. Another study on SPS of HApCNT composites used nano-HAp powders and CNTs, mixed by ball milling, as the starting material. Sarkar et al. [162] found the fracture toughness of HApCNT composite to be as high as 1.27 MPa m0.5, with 2.5 vol.% CNT addition, which was a 30% increase over HAp. Xu et al. [163] performed extensive mechanical stirring to homogeneously mix CNTs with spray-dried HAp powder to prepare feedstock for SPS processing. The Youngs modulus has been reported to be 131 GPa with 2 vol.% CNT addition, although no direct comparison with HAp has been reported. The biocompatibility of CNT-reinforced HAp composite has also been studied by Xu et al. This group determined the benecial effect of CNT on osteoblast cell proliferation. Lahiri et al. [164] investigated CNT-reinforced HAp composite synthesized using SPS. Quantitative microstructural analysis suggests that CNTs play a role in grain boundary pinning and are responsible for improved densication and retention of nanostructure throughout the thickness of the sintered pellet. HAp crystal forms a coherent interface with CNTs, resulting in a strong interfacial bond. The uniform distribution of 4 wt.% CNTs in the HAp matrix, good interfacial bonding and ne HAp grain size help to improve fracture toughness by 92% and elastic modulus by 25% compared to a HAp matrix without CNT. Xiao et al. [165] introduced a simple and effective approach to functionalize MWCNTs by in situ deposition of HAp to improve hydrophilicity and biocompatibility. The scheme of biomineraliza-

tion mechanism of HApPEGMWCNT preparation is shown in Fig. 7. First, two types of pre-functionalized MWCNTs were prepared: acid-oxidized MWCNTs, and MWCNTs covalently modied by PEG. The inuence of acid-oxidation time, pre-phosphorylation and PEGylation of MWCNTs on in situ growth of HAp was further investigated in simulated body uid (SBF) with ionic concentrations of 2, 5 and 10 times, respectively, at 37 C for 24 h. The results showed that these factors have positive effects on HAp crystal growth, with PEGylation of MWCNTs playing a key role during deposition. Finally, the methyl thiazolyl tetrazolium (MU) assay was performed to evaluate cytotoxicity, which showed that PEGylated MWCNTs wrapped by HAp crystals have the best biocompatibility. Inspired by self-assembly of nano-HAp association with the 67 nm periodic microstructure of collagen, Liao et al. [166] used MWCNTs with an approximately 40 nm bamboo periodic microstructure as a template for nano-HAp deposition to form a nanoHApMWCNT composite. Spindle-shaped units consisting of an assembly of near-parallel, bril-like nano-HAp polycrystals were formed and oriented at a specic angle to the long axis of CNTs. Spindle-shaped units detached from the MWCNT template are able to maintain the ordered parallel structure of the nano-HAp polycrystal bril. Tang et al. [167] found that biomimetic synthesis of HAp on SDS functionalized MWCNTs by using an alternate soaking process (ASP) in Ca/P solutions. The results showed that nano-HAp crystals were formed on SDS functionalized MWCNTs, and mineralized MWCNTs remained in a dispersed state. HApMWCNT nanohybrids, combining the osteconductive properties of HAp and the excellent mechanical properties of MWCNTs, will provide a promising material for bone tissue engineering. Balani et al. [151] synthesized HAp-reinforced with submicrometer Al2O3 and CNTs as a coating on a Ti6Al4 V substrate via plasma spraying. The addition of Al2O3 and CNTs to HAp shows improvements in the hardness and elastic modulus by 65% and 50%, respectively, compared to HAp. Consequently, HApAl2O3 CNT coatings have been nano-scratched to determine their wear performance. Reinforcement of HAp by Al2O3 decreases wear volume by more than 13 times, whereas HApAl2O3CNT coating shows an even further decreased wear volume of 5 times compared to that of HApAl2O3 coating. Ajeesh et al. [168] prepared sintered iron oxideHAp nanocomposite ceramics from powders produced through a co-precipitation process. The phase purity and bioactivity of the composites were analyzed as a function of percentage of iron oxide in the composite. In all the prepared composites, HAp retains the same phase identity and high X-ray opacity as a composition containing 40 wt.% iron oxide. Increased cell viability and cell adhesion showed that the prepared composite offers considerable potential for bone tissue engineering

Fig. 7. Scheme of biomineralization mechanism of the preparation of HApPEGMWCNTs. Copyright Elsevier and reproduced with permission [165].

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applications. Jiang et al. [169] designed and synthesized nano-HAp crystals carrying incorporated Fe ions by a co-precipitation method. The results showed that lattice substitution or incorporation occurs between Fe ions and nano-HAp, which is quite different from the mechanical mixture of Fe3O4 nanoparticles and nanoHAp crystals. The nano-FeHAp with a synthetic molar ratio of 1:2 (calculated by Fe3O4:HAp) exhibited a needle-like crystal shape and relatively good crystallinity. However, when the molar ratio was increased to 1:1, some Fe3O4 nanoparticles were separated from the nano-FeHAp crystals. Magnetization measurements showed that nano-FeHAp crystals were in a superparamagnetic state, proving that the existence of Fe ions in nano-HAp crystals contributed to the magnetic properties of nano-FeHAp crystals. As one component of biomedical composites, nano-FeHAp has the potential to promote bone regeneration. Bioactive nano-titania ceramics with excellent biomechanical compatibility and bioactivity were prepared by using HAp additive as a grain growth inhibitor. The mechanical properties of nanotitania ceramics were analogous to those of human bone. The amount of HAp additive played an important role in determining the grain/particle size of nano-titania ceramics, which had a great effect on osteoblast proliferation in cell culture experiments. Cell culture experiments also showed that the bioactive HAp additive itself also signicantly affected the cytocompatibility of nano-titania ceramics. These results indicated that the content of HAp additive not only had an effect on the bioactivity of nano-titania ceramics due to the bioactivity of the additive itself, but also had an effect both on the biomechanical compatibility and bioactivity of nano-titania ceramics by adjusting the grain/particle size of the ceramics. The HAp and ZrO2 nanopowders were prepared by chemical reactions and alcoholaqueous solution heating, respectively. The nanosized HAp-ZrO2 powders with a homogeneous distribution could then be synthesized by ball-milling. HAp-ZrO2 bioceramics with small grain sizes could be obtained by using hot-press sintering technology [170]. The in vitro biocompatibility of these bioceramics was studied, and the results showed that there was no reaction between HAp and ZrO2 powders-which may be attributed to the very short sintering time of the hot-press sintering- and that HAp-ZrO2 bioceramics possess non-toxic and non-allergenic properties. Biomaterials science has found many ways to enhance different properties of synthetic HAp by introducing various ionic substitutions into the apatite structure [171]. One of the most interesting substitutions is the incorporation of Si, which considerably improves the bioactivity of synthetic apatite [172]. The role of Si in bioactive processes involved in new bone tissue formation is well known [173]. Carlisle rst reported the presence Si traces during bone mineralization at early stages of calcication, and this has proven the inuence of an Si-decient diet in diseases such as osteoporosis [174]. Therefore, Si-substituted HAp (SiHA) was chosen as an appropriate candidate for the preparation of an improved CaP material and was subsequently produced by chemical synthesis [175,176]. Solla et al. [177] chemically synthesized SiHA and presented it as a new material with enhanced bioactivity. SiHAp lms were deposited by pulsed laser deposition (PLD), using targets composed of HAp mixtures with different Si-containing sources such as SiO2 and diatomaceous earth. Analysis revealed that Si is successfully incorporated into the HAp structure, as well as traces of other elements such as Na, Fe or K.

from advances in nanotechnology. Several methods for synthesizing HAp on the nanoscale have evolved in the past few decades. Due to the chemical similarity between HAp and mineralized bone of human tissue, synthetic HAp exhibits a strong afnity to host hard tissues. A signicant amount of research in this area is expected to be focused on the nanoscale for enhanced applications as resorbable scaffolds that can be replaced by endogenous hard tissues over time. In the future, the ability to functionalize surfaces with different molecules of varying natures and dimensions based on their means of attachment to cells, as well as the potential to nanostructure the surface physically, chemically and topographically, will enable selective targeting within biological systems to show specicity towards individual proteins and peptides. Understanding the inuence of nano-HAp particle size, crystal morphology controls and interfaces with cells from a higher level is essential for the future development of nanotechnology and biotechnology. Such an interdisciplinary approach is very complicated, and the effective collaboration of scientists from different disciplines is necessary. Acknowledgments This research was supported by the International Research and Development Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) of Korea, and National Fisheries Research and Development Institute (Grant Nos. K20091003000, FY2009, 20100434961-00). Appendix A. Figures with essential colour discrimination Certain gures in this article, particularly Figures 4, 6, 7, are difcult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio.2011.03.019. References
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6. Summary and perspective Nanophase HAp bioceramics have gained importance in the biomedical eld due to their superior biological and biomechanical properties. Development of HAp biomedical materials will benet

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