Downloaded from rsta.royalsocietypublishing.

org on May 5, 2012

Biomimetic hydroxyapatite-containing composite nanofibrous substrates for bone tissue engineering

J. Venugopal, Molamma P. Prabhakaran, Yanzhong Zhang, Sharon Low, Aw Tar Choon and S. Ramakrishna Phil. Trans. R. Soc. A 2010 368, 2065-2081 doi: 10.1098/rsta.2010.0012

References

This article cites 65 articles, 1 of which can be accessed free

http://rsta.royalsocietypublishing.org/content/368/1917/2065.ful l.html#ref-list-1
Article cited in: http://rsta.royalsocietypublishing.org/content/368/1917/2065.full.html# related-urls

Subject collections

Articles on similar topics can be found in the following collections biomedical engineering (131 articles) materials science (108 articles)

Email alerting service

Receive free email alerts when new articles cite this article - sign up in the box at the top right-hand corner of the article or click here

To subscribe to Phil. Trans. R. Soc. A go to: http://rsta.royalsocietypublishing.org/subscriptions

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

Phil. Trans. R. Soc. A (2010) 368, 20652081 doi:10.1098/rsta.2010.0012

REVIEW

Biomimetic hydroxyapatite-containing composite nanobrous substrates for bone tissue engineering

B Y J. V ENUGOPAL1, *, M OLAMMA P. P RABHAKARAN1 , Y ANZHONG Z HANG1,2 , S HARON L OW3 , A W T AR C HOON3 AND S. R AMAKRISHNA1,2
and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, Block E3-05-12, 2 Engineering Drive 3, Singapore 117576, Republic of Singapore 2 Department of Bioengineering, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, Peoples Republic of China 3 StemLife Sdn BhD, Kuala Lumpur, Malaysia
The fracture of bones and large bone defects owing to various traumas or natural ageing is a typical type of tissue malfunction. Surgical treatment frequently requires implantation of a temporary or permanent prosthesis, which is still a challenge for orthopaedic surgeons, especially in the case of large bone defects. Mimicking nanotopography of natural extracellular matrix (ECM) is advantageous for the successful regeneration of damaged tissues or organs. Electrospun nanobre-based synthetic and natural polymer scaffolds are being explored as a scaffold similar to natural ECM for tissue engineering applications. Nanostructured materials are smaller in size falling, in the 1100 nm range, and have specic properties and functions related to the size of the natural materials (e.g. hydroxyapatite (HA)). The development of nanobres with nano-HA has enhanced the scope of fabricating scaffolds to mimic the architecture of natural bone tissue. Nanobrous substrates supporting adhesion, proliferation, differentiation of cells and HA induce the cells to secrete ECM for mineralization to form bone in bone tissue engineering. Our laboratory (NUSNNI, NUS) has been fabricating a variety of synthetic and natural polymer-based nanobrous substrates and synthesizing HA for blending and spraying on nanobres for generating articial ECM for bone tissue regeneration. The present review is intended to direct the readers attention to the important subjects of synthetic and natural polymers with HA for bone tissue engineering.
Keywords: electrospinning; synthetic and natural polymers; nanobres; hydroxyapatite; bone tissue engineering
1 Nanoscience

*Author for correspondence (nnijrv@nus.edu.sg). One contribution of 14 to a Theme Issue Advanced processing of biomaterials.

2065

This journal is 2010 The Royal Society

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

2066

J. Venugopal et al.

1. Introduction
Electrospinning processes have attracted a great deal of attention as a way to mimic the structure of natural extracellular matrix (ECM) by means of producing bres down to 5 nm. This technique is used to fabricate nanobrous structures from natural and synthetic polymers such as collagen (Col), gelatin, chitosan (CTS), silk broin, poly( DL-lactide-co-glycolide), poly(lactide), polyurethane, polycaprolactone, etc. Rapid progress in nanotechnology and its far-reaching developments have triggered the use of nanostructures as scaffolds for bone tissue engineering. Fabrication of nanobres is one of the most promising techniques for designing polymer nanobres in tissue engineering. There are several scaffold fabrication techniques, namely electrospinning (random, aligned, core shell and vertical nanobres), self-assembly, phase separation, melt-blown and template synthesis (Venugopal et al. 2008a). Of these techniques, electrospinning has been the most widely used technique recently, and it also seems to be demonstrating promising results for tissue engineering applications. Electrospinning has emerged as an elegant and leading technique to create bres with diameters in the range of micrometres down to a few nanometres cost-effectively for tissue engineering. Tissue engineering is the application of knowledge and expertise from a multidisciplinary eld, to develop and manufacture therapeutic products that use the combination of matrix scaffolds with viable human cell systems or cell-responsive biomolecules derived from such cells, for repair, restoration or regeneration of cells or tissues damaged by injury or disease (Venugopal et al. 2008a,b). The concept of tissue engineering using three-dimensional scaffolds has certain advantages over direct cell injection to the tissues: (i) three-dimensional scaffolds may replace the missing or damaged infrastructure (ECM) in the infarct area and provide temporary support for self or implanted cells, (ii) by tissue engineering, one can control the size, shape, strength and composition of the graft in vitro, and (iii) tissue engineering provides a solution to the problem of bone defects and can be used to replace the injured bone. The objective was to develop a scaffold for tissue engineering that is (i) highly porous with large interconnected pores (to facilitate mass transport), (ii) hydrophilic (to enhance cell attachment), (iii) structurally stable (to withstand the shearing forces during bioreactor cultivation), (iv) degradable (to provide ultimate biocompatibility of the tissue graft), and (v) elastic (to enable transmission of contractile forces). The scaffold structure determines the transport of nutrients, metabolites and regulatory molecules to and from the cells, whereas the scaffold chemistry has an important role in cell attachment and differentiation. Mechanical properties of the scaffold should ideally match those of the native tissue, to provide mechanical integrity of the forming tissue and to support an in vivo-like mechanotransduction between cells and their environment (Radisic et al. 2007). The role of biomaterials in tissue engineering is to act as a scaffold for cells to attach and organize into tissues. The ECM is a complex arrangement of proteins and polysaccharides such as Col, hyaluronic acid, proteoglycans, glycosaminoglycans and elastin. The structure and morphology of the non-woven nanobre matrix were found to closely match the structure of ECM of natural tissue (Li et al. 2002; Venugopal et al. 2008a). Every year, millions of people suffer from bone defects arising from trauma, tumour or bone diseases, and many people are dying because of insufcient bone substitute. One main driving force to explore nanomaterials in bone tissue
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

Review. Biomimetic HA substrates

2067

engineering applications is that tissues in the human body are nanostructures, clearly opening the gates for numerous opportunities to improve medicine. The functional treatment of fracture non-union and bone loss associated with trauma, cancer and revision joint arthroplasty has become increasingly common for orthopaedic surgeons and remains a signicant challenge in the eld of musculoskeletal injury annually, and there are over several million orthopaedic procedures performed each year (Market Dynamics 2008). The global orthopaedic market is estimated by Espicom to have been worth approximately US$37.1 billion in 2008, following a growth of 9.7 per cent over the previous year. Excluding arthroscopy and other segments (operating theatre equipment and supplies), the market totalled US$29 billion, having grown by 10.7 per cent over the previous year. To solve these problems, synthetic and natural polymeric nanobrous scaffolds are the alternative for bone tissue engineering applications; they should be biocompatible with the surrounding biological uids and tissues, biodegradable and highly porous with interconnected spaces, and favourable for the diffusion of nutrients as well as migration of a large number of cells. Bone tissue engineering is a rapidly expanding research area providing a new and promising approach for bone repair and regeneration. Good sterilizability, storability and processability, as well as relatively low cost, are also of great importance to permit clinical applications. Unfortunately, no articial biomaterial is available yet which embodies all these requirements, and it is unlikely that it will appear in the near future. Until now, most of the available biomaterials appear to be either predominantly osteogenic or osteoinductive or else purely osteoconductive (Meyer et al. 2004). The current challenge in bone tissue engineering is to fabricate bioarticial bone graft mimicking the ECM with effective bone mineralization, resulting in the regeneration of fractured or diseased bones. Today, nanotechnology and nanoscience approaches to scaffold design and functionalization are beginning to expand the market for tissue engineering, which forms the basis for a highly protable niche within the industry. This review describes recent advances for the fabrication of biomimetic nanobrous scaffolds with hydroxyapatite (HA) for bone tissue engineering.

2. Hydroxyapatite
Hydroxyapatite is a major mineral component of calcied tissues (bone and teeth). Synthetic HA (Ca10 (PO4 )6 (OH)2 ) has been used extensively as an implant material for bone substitute owing to its excellent osteoinductive properties (Rameshbabu et al. 2005). HA-enhanced surface properties (such as increased surface area and charge and the ability to alter adsorption of chemical species) could be used to promote cell response and proliferation to induce mineralization in bone tissue engineering. Hydroxyapatite has been used in a variety of biomedical applications such as matrices for controlled drug release, bone cements, tooth paste additive, dental implants, etc. Calcium phosphate biomaterials (HA/tricalcium phosphate) with appropriate three-dimensional geometry are able to bind and concentrate endogenous bone morphogenetic proteins (BMPs) in circulation and may become osteoinductive (Yuan et al. 1998) and can be an effective carrier of bone cells. Nanohydroxyapatite (nano-HA) can be prepared by co-precipitation and precipitation using emulsion, template and
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

2068

J. Venugopal et al.

solgel techniques, but these methods need highly controlled parameters such as reactant concentration, pH and temperature of the aqueous solution; microwave processing is a simple method for HA preparation (Rameshbabu et al. 2005). Synthetic-substituted HA is only starting to be developed in elaborate tailored biomaterials, and some of them have been shown to exhibit improved biological properties compared with stoichiometric HA (Porter et al. 2004). Currently, biocomposites with calcium orthophosphates incorporated as either a ller or a coating (or both) or into a biodegradable polymer matrix in the form of particles or bres are increasingly considered for using as bone tissue engineering scaffolds owing to their improved physical, biological and mechanical properties (Hutmacher et al. 2007; Dorozhkin 2009). In addition, such biocomposites could full general requirements for the next generation of biomaterials; these should combine the bioactive and bioresorbable properties to activate in vivo mechanisms of tissue regeneration, thus stimulating the body to heal itself and leading to the replacement of implants by regenerating tissues (Hench & Polak 2002).

3. Natural polymers/hydroxyapatite
One of the major challenges in developing porous scaffolds for bone tissue engineering is the conicting interest between porosity, mechanical strength and biodegradability. Stable osteoblast cell adhesion is largely mediated by integrins and heterodimeric receptors that interact with ECM proteins such as bronectin, vitronectin, brinogen and Col, allowing the cell to respond to its extracellular environment and modulating the cellular events that regulate remodelling of bone (Gronowicz & McCarthy 1996; Chang et al. 1998). Nanoscaled features such as surface roughness and topography of nanocrystalline bioceramics and nanobrous scaffolds promote cell behaviour such as adhesion, proliferation, migration and differentiated functions. Natural bone being an innate example of inorganicorganic biocomposites consists of approximately 70 wt% inorganic crystals, mainly HA, and 30 wt% organic matrix, mainly Col type I. Structurally, it is hierarchically organized from macro-, micro-, to nano-scale, where the basic building blocks were pinpointed to be a plate-like HA nanocrystal incorporated into Col nanobres (Glimcher 1959; Weiner & Traub 1986). Hydroxyapatite is considered to be a structural template for the bone mineral phase and also a major inorganic mineral component of bone, and is commonly used as a bioceramic ller in polymer-based bone substitute because of its higher level of bioactivity and biocompatibility (Hong et al. 2005).

(a) Collagen/hydroxyapatite
Col is a major ECM component that possesses brous structure with bre bundles of varying diameters (50500 nm). The nanometre size feature inuences cell behaviours by allowing cells to attach to diameters smaller than the cell size of the bres. Cells seeded on this structure tend to maintain normal phenotypic shape and guided growth according to nanobre orientation. The main idea in biomimetic approaches is to control and fabricate the morphology and composition of developed biomaterials, in which the nanocrystallites of inorganic compounds are being dispersed with preferential orientation in the organic matrices. Owing to its large potential in biomedical applications, many studies
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

Review. Biomimetic HA substrates

2069

reported the preparation of bone-like biocomposites of HA and bioactive organic components such as Col, gelatin, chondroitin sulphate, CTS and amphiphilic peptide by direct precipitation methods (Kikuchi et al. 2001; Chen et al. 2002), poly(lactic acid) through a solvent-cast technique (Liao et al. 2004) and polyamide by a solution method (Wei et al. 2003). Col and HA have potential in mimicking natural ECM and in replacing diseased skeletal bones. More attention has been focused on HA because its crystallographic structure is similar to that of inorganic compounds found in natural bone; it has been investigated extensively owing to its excellent biocompatibility, bioactivity and osteoconductivity. Bone tissue contains high levels of type I Col and several non-collagenous proteins (such as osteopontin, bone sialoprotein and osteocalcin) that distinguish it from other types of tissues. The size of the bone mineral is around 50 nm in length, 25 nm in width and 25 nm in thickness (Sachlos et al. 2006). These crystals are oriented with their long crystallographic c-axis parallel to each other and aligned with Col tropocollagen molecules (Weiner & Traub 1989). Col is easily degraded and resorbed by the body and allows good attachment to cells. However, its mechanical properties are relatively low (E 100 MPa) in comparison with bone (E 25 GPa; Clarke et al. 1993), and it is, therefore, highly cross-linked or found in composites such as Colglycosaminoglycans for skin regeneration (OBrien et al. 2004) or Col/HA for bone remodelling (Venugopal et al. 2008ae). Col and HA devices signicantly inhibited the growth of bacterial pathogens, the frequent cause of prosthesis-related infection, compared with poly(lactide-co-glycolide) devices (Carlson et al. 2004). Electrostatic co-spinning of nanocomposite bres of polymers with a nano-HA composite system in native bone is the special orientation between HA and Col molecules. However, more efforts are required in the area of nanobrous Col/HA composite, for exactly mimicking the complex nanostructured architecture of Col matrix with c-axis orientation of nano-HA particles (Thomas et al. 2006). Col supports the cells for adhesion and proliferation, and HA acts as a chelating agent for mineralization of osteoblasts in bone tissue regeneration (Landis et al. 1993). Biologically inspired biocomposites of Col and nano-HA for bone substitute have a long history in the biomedical eld (Wahl et al. 2007). A combination of Col and nano-HA materials is bioactive, osteoconductive and osteoinductive, seems to be a natural choice for bone grafting and mimics the bone components. Nano-to-microscale nanoHA/Col alignment of composite was similar to that of natural bone and thus might have been identied as bone by the attached cells (Du et al. 1999). Itoh et al. (2001) prepared a novel HA/Col composite biomaterial using cold isostatic pressure by the co-precipitation method that has a chemical composition and crystallinity similar to that of bone and induces the development of osteogenic cells for remodelling of bone. Skeletal bones comprise mainly Col (predominantly type I) and carbonatesubstituted HA; both are osteoconductive components. Thus, an implant manufactured from such components is likely to behave similarly and to be of more use than a monolithic device. Indeed, both type I Col and HA were found to enhance osteoblast differentiation, but, combined together, they were shown to accelerate osteogenesis. A composite matrix when embedded with human osteoblastic cells showed better osteoconductive properties than monolithic HA and produced calcication of identical bone matrix (Wang et al. 1995).
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

2070

J. Venugopal et al.

(a)

(b)

HV mag WD spot 10.0 KV 10000x 16.3 mm 30

10 m collagen

mag HV WD spot det 10.0 KV 5000x 10.3 mm 30 ETD

20 m

Figure 1. Electrospun nanobrous substrate interaction with human foetal osteoblast (hFOB) cells. (a) Col nanobres (bre diameter: 272 0.63 nm) and (b) Col/HA nanobres with hFOB mineralization.

More interest has been focused on HA because its crystallographic properties are similar to those of inorganic components found in natural bone. It has been investigated extensively owing to its excellent biocompatibility, bioactivity and osteoconductivity (Li et al. 2007). In addition, Col/HA composites proved to be biocompatible both in humans and in animals (Wahl & Czernuszka 2006). Kikuchi et al. (2001) fabricated an articial bone material with bone-like nanostructure and chemical composition, a composite comprising HA and Col was synthesized under biomimetic conditions through a self-organization mechanism between HA and Col. The HA/Col composite fabricated and demonstrated bone-like orientation, so that c-axes of HA nanocrystals were regularly aligned along Col brils (Porter et al. 2005). The literature review suggests that the bone bonding ability of calcium phosphate ceramics occurs by partial dissolution of ceramic, resulting in the elevated concentration of calcium (Ca2+ ) and phosphate (PO3 ) 4 ions within the local environment. Investigations have suggested that there is a threshold concentration of calcium ions required to stimulate the subsequent activity of osteoblasts (Nishikawa et al. 2005). The HA/Col composite, designed to simulate bone tissue, is produced using atelocollagen to reduce antigenicity by condensing Ca(OH)2 /H3 PO4 suspension (Venugopal et al. 2008c). Venugopal et al. (2008c) found that the mineral deposition on Col/HA composite nanobrous scaffolds cultured with osteoblasts was signicantly higher than that on Col nanobrous scaffolds (gure 1a) during 10 days of culture period. Compared with Col/HA nanobrous scaffolds, the mineral deposition was signicantly lower (by up to 56 per cent) in Col nanobrous scaffolds. The Col/HA composite nanobrous scaffolds have huge potential for cell adhesion and growth, as well as stimulating the cells for mineralization, exhibiting functional activity of osteoblasts for bone tissue engineering (gure 1b). These studies showed that the Col/HA composite nanobrous scaffold has great potential for bone tissue regeneration.
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

Review. Biomimetic HA substrates

2071

The main practical problems with Col are its cost, and poor denition of commercial sources of this material makes it difcult to follow up on wellcontrolled processing. Col was replaced by gelatin (Gel), a protein produced by partial hydrolysis of Col extracted from skin, bone, cartilage, ligaments, etc. Mixing Gel with other synthetic polymers, sometimes called bioarticial polymeric materials, has frequently been adopted by other researchers. This approach is feasible to reduce the potential problem of cytotoxin, as a result of using a chemical cross-linking reagent, but also provides a compromise solution for overcoming the shortcomings of synthetic and natural polymers, that is, producing a new biomaterial with excellent biocompatibility and improved mechanical and physical/chemical properties (Venugopal et al. 2008e). Liu et al. (2009) reported the fabrication of three-dimensional nanobreGel/apatite composite scaffolds, which mimic both nanoscale architecture and chemical composition of natural bone ECM. Three-dimensional nanobrous Gel scaffolds with well-dened macropores were prepared by using a new thermally induced phase separation and porogen leaching technique (Liu et al. 2009). The deposition of a biomimetic apatite layer throughout the porous structure of threedimensional scaffolds is an effective method for controlling the surface topography and chemistry within large, complex structures. These scaffolds have excellent biocompatibility and mechanical properties and showed enhanced osteoblast adhesion, proliferation and differentiation suitable for bone tissue engineering. Although Col is used as a common matrix for scaffold design, HA seems to be a rational strategy for preparing a nanobrous biocomposite that compositionally and structurally resembles bone for engineering of bone tissue; however, Col alone is less ideal in terms of mechanical properties and biostability owing to its rapid degradation in the biological environment and problems related to its antigenicity. Recently, a few studies have explored the feasibility of replacing Col with other natural biopolymers (e.g. silk and CTS) to prepare the HA containing electrospun biomimetic composite bres for potential osteoregenerative applications, while the electrospinnability of the natural biopolymers of interest is no longer an issue under consideration in tissue engineering.

(b) Hydroxyapatite/silk protein

Silks are considered to be the most promising natural protein-type replacement for Cols in bone tissue engineering because of their biocompatibility, slow degradation and excellent mechanical performance. In the past few years, different natural silks (e.g. silkworm silk Bombyx mori and spider dragline silk Nephila clavipes) have been processed for making nanoscale bres via electrospinning (Jin et al. 2002; Kim et al. 2003; Ohgo et al. 2003; Zarkoob et al. 2004). For example, to improve the electrospinnability of silk solutions and to avoid potential inuences of using strong polarity organic solvents such as hexauoroisopropanol (Zarkoob et al. 2004), hexauoroacetone (Kim et al. 2003) and formic acid (Ohgo et al. 2003) on biocompatibility, Kaplans group (Jin et al. 2002) used an allaqueous process for silk electrospinning by blending regenerated silk broin with a bre forming agent, poly(ethylene oxide) (PEO), at a ratio from 1 to 2 . 4 3 A post-electrospinning treatment on the silk broin bres with methanol was then performed to induce a structurally conformational transition to the original b-sheet to render water insolubility in uses. Following this success, Li et al. (2006)
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

2072

J. Venugopal et al.

recently developed silk-based composite nanobres with the incorporation of HA nanoparticles (approx. 5 wt%) and BMP-2 (3 mg mg1 of silk broin) growth factor to realize enhanced bone formation outcomes from culturing human bone marrow-derived mesenchymal stem cells (MSCs). They found that the inclusion of BMP-2 and HA nanoparticles within the electrospun silk broin bres resulted in the highest calcium deposition and upregulation of BMP-2 transcript levels, compared with other electrospun silk-based scaffolds including silk/PEO, silk/PEO extracted, silk/PEO/BMP-2 and silk/PEO/HA. Li et al. (2005) employed different approaches to prepare silk-based composite bres, in which the inorganic apatite component was selectively grown on and associated with the electrospun brous silk broin matrix by a subsequent mineralization process using an alternative soaking method. Acidic protein poly( L-aspartate) (poly-Asp) as a molecular recognition motif was rst introduced into the electrospun silk broin bres to enable subsequent nucleation and crystal growth of apatite on the bre surface during the mineralization process. This approach is an alternative route for preparing composite nanobres to avoid the electrospinnability problem encountered in the electrospinning of those HAcontaining solutions, which eventually leads to the formation of apatite-coated broin composite bres, which is somewhat different from that obtained in the components hybridizing method (Li et al. 2006; Zhang & Lim 2008) and the nanostructure of hierarchical bone (Landis et al. 1993; Rho et al. 1998) in terms of distribution and morphology of the HA nanocrystals in the composite bres.

(c) Hydroxyapatite/chitosan
Amino polysaccharide CTS is derived from the structural biopolymer chitin that exists abundantly in crustacean shells (e.g. crabs) and plays a key role similar to that of collagen in higher animals. CTS possesses plenty of notable attributes such as structural similarity to glycosaminoglycan found in bone, osteoconductivity, excellent biocompatibility, tailorable biodegradability, low immunogenicity and better mechanical properties. Thus, it has become of great interest as one of the most attractive natural biopolymer matrices and alternatives to collagens for bone tissue engineering (Yamaguchi et al. 2001; Muzzarelli & Muzzarelli 2002; Hu et al. 2004). However, there are immense challenges in converting a bulk HA/CTS nanocomposite or hybrid into a brous form by electrospinning owing to the poor electrospinnability of the CTS itself as well as the adverse effect of the non-electrospinnable HA nanoparticles (and their aggregates) contained in the spinning dope. Formulating a robust CTS solution system consequently appears to be a prerequisite to generate nanobrous HA/CTS scaffolds. As a result of the noted obstacles in electrospinning, so far there are very limited attempts of using nanobrous HA/CTS for bone tissue engineering (Rusu et al. 2005; Yang et al. 2008). By using an ultra-high-molecular-weight PEO as the bre-forming aiding agent, Zhang et al. (2008a) demonstrated that CTS nanobres could be prepared easily with a minimum PEO loading ratio of up to 5 wt%. This enabled an attempt to develop HA/CTS composite nanobres for potential application in bone repair and regeneration (Rusu et al. 2005). A modied two-step approach (Zhang et al. 2008b) that combines the in situ co-precipitation synthesis route (Yamaguchi et al. 2001), which is likely to overcome the usual problem of nanoparticles
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

Review. Biomimetic HA substrates

2073
(b)

Ca + P + CTS

(a)

HA/CTS

co-precipitation

HA/CTS nanocomposites (c)

HA/CTS solution

electrospinning (d )

(e)

(f)

Figure 2. Electrospun nanobrous HA/CTS composite scaffolds for bone tissue engineering. (a) The co-precipitation method was employed for the synthesis of HA/CTS nanocomposite; (b) electrospinning of thus obtained HA/CTS nanocomposite by dissolving dilute acetic aciddominant solvent system; (c) SEM image of the co-precipitated HA/CTS nanocomposite with needle-shaped HA nanoparticles being incorporated evenly within the CTS matrix; (d) electrospun HA/CTS nanobres with an average diameter of ca 200 nm; (e) biomineralization of hFOBs on the nanobrous composite substrate of HA/CTS and (f ) apatite-like morphology of the deposited minerals in (e), visualized at a high magnication.

Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

2074

J. Venugopal et al.

agglomeration, with the electrospinning process was adopted for the preparation of HA/CTS nanobres containing a higher (30 wt%) loading of HA nanoparticles (gure 2). Despite the fact that a dilute acetic acid-dominated solvent system was used for dissolving CTS, results from the selected area of electron diffraction and X-ray diffraction analysis indicated that the crystalline nature of HA remains, thus implying a minor inuence of the used acid solvent system on the crystalline structure of HA incorporated within the co-precipitated HA/CTS nanocomposite. Bone formation ability assessed by conducting [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt] (MTS) and alizarin red staining (ARS) assays of human foetal osteoblasts (hFOBs) cultured for up to 15 days showed that the HA/CTS nanobrous scaffolds had signicantly encouraged bone formation-oriented outcomes compared with the electrospun CTS alone scaffolds. Biomineralization in the form of synthesizing apatite-like granular minerals, as reported elsewhere (Yoshimoto et al. 2003; Li et al. 2006), was similarly observed with the post-cultured cell-scaffold constructs (gure 2e,f ). The two-step method would pave the way for developing other sophisticated biomimetic CTS-based nanobrous composite scaffolds for functional bone tissue engineering.

4. Synthetic polymers/hydroxyapatite and synthetic/natural polymer/hydroxyapatite composites

Biocomposite nanobres fabricated by electrospinning mimic the nanoscale structure of ECM, and organize and provide signals for the cellular response (Gupta et al. 2009). The material for bone graft fabrication needs to provide structural integrity within the body and nally breakdown leaving new tissue formation. The material should modulate the cellular function to promote tissue regeneration in addition to mimicking the mechanical properties of the bone. Nonbiodegradable electrospun polymers such as polyurethane and polyesterurethane possess good mechanical properties, but they might interfere with tissue turnover owing to their slow degradation properties (Lee et al. 2005; Riboldi et al. 2005). Non-woven poly-e-caprolactone (PCL) nanobrous scaffolds were developed by Yoshimoto et al. (2003) and seeded with MSCs from the bone marrow of neonatal rats with osteogenic supplements. The penetration of cells along with abundant ECM deposits was observed in the cellpolymer constructs by these researchers, favouring potential scaffolds for bone tissue engineering. In contrast, the shrinkage behaviour of natural polymeric (Col and gelatin) scaffolds is a major phenomenon problematic in bone tissue regeneration (Liao et al. 2004). Preliminary in vitro and in vivo studies using nano-HA/Col composite also showed the material as bioactive and biodegradable, but its weak mechanical properties remain a major hindrance for practical bone tissue engineering. PCL/HA, PCL/Col/HA, PCL/Gel/HA, poly- L-lactic acid (PLLA)/Col/HA and poly(3-hydroxy-butyrate-co-3-hydroxyvalerate (PHBV)/HA were fabricated by various research groups as a substitute for bone tissue engineering (Ito et al. 2005; Venugopal et al. 2007, 2008d,e; Prabhakaran et al. 2009). The PCL/HA nanobrous scaffolds treated with plasma enhance the wettability and thus accelerate the biodegradation rate of nanobrous scaffolds. PCL/HA plasmatreated nanobre shows mineral formation on the surface of osteoblast cell
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

Review. Biomimetic HA substrates

2075

(a)

(b)

mag WD spot det HV 10.0 KV 5000x 16.3 mm 30 ETD

20 m

HV mag WD spot det 10.0 KV 5000x 10.5 mm 30 ETD

20 m PCL

Figure 3. Mineralization of hFOBs on PCL/HA plasma-treated nanobrous substrates. (a) PCL nanobres (bre diameter: 276 56 nm) and (b) hFOB-secreted mineral deposition on the surface of the PCL/HA.

layers within 6 days of culture and morphology similar to that of HA of the natural bone (gure 3). On fabricating the composites of HA with biodegradable polyesters such as PCL, PLLA produced a scaffold with better mechanical properties, whereas HA provided excellent bioactivity and osteoconductive properties (Hamadouche & Sedel 2000; LeGeros 2002). Moreover, the optimal performance of a composite material is achieved when the small particles (HA) are uniformly dispersed throughout the scaffolds and interact strongly within the organic matrix. Electrospun Gel/PCL nanobres were fabricated by mixing a 1 : 1 ratio of Gel and PCL with Gel concentrations ranging from 2.5 to 12.5%w/v and the membrane showed improved mechanical properties and favourable wettability compared with Gel or PCL membrane (Zhang et al. 2005). Bone marrow stem cells (BMSCs) grown on the surface of these scaffolds were found to migrate inside the scaffold up to 114 mm within a week of culture, showing their biocompatibility better than PCL nanobres. Figure 4 shows the cellular ingrowth after 4 days of osteoblasts cultured on biocomposite nanobrous scaffolds. While loosely interlaced brous structure and the weak nanoscale bres can provide the least obstruction and matched mechanical properties for cell movements, it seems that the presence of appropriate molecular signals on the nanobre surface can also guide or attract the living cells to enter into the matrix through their amoeboid movement (Venugopal et al. 2008e). HA/PLLA composites have been described to full certain requirements for its application as a suitable substrate for bone tissue engineering (Liao et al. 2004). The compressive strength of the HA/PLLA composite has also been described as reaching the lower limit of natural cancellous bone (approx. 1 MPa). Implant experiments showed quick healing of large segmental defects after 12 weeks of scaffold implantation and also showed appropriate bone substitute material for ingrowth of the new bone. However, Prabhakaran et al. (2009) fabricated PLLA/HA and PLLA/Col/HA nanobres by electrospinning and showed that the biocomposite PLLA/Col/HA nanobres are superior to PLLA/HA nanobres for effective bone regeneration and mineralization. Moreover, the tensile strength
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

2076

J. Venugopal et al.

(a)

(b)

mag HV WD spot det 10.0 KV 5000x 16.7 mm 30 ETD

20 m PCL/HA/Gel

mag HV WD spot det 10.0 KV 5000x 16.2 mm 30 ETD

20 m

Figure 4. Field emission SEM micrographs of hFOB interaction with a nanobrous scaffold after 6 days of culture. (a) PCL/HA/Gel nanobres (bre diameter 358 58 nm) and (b) PCL/HA/Gel/osteoblasts with mineral deposition on the surface.

of these electrospun PLLA/Col/HA scaffolds was higher than that of the collagen brous matrix (1.68 MPa) prepared by Thomas et al. (2007) and even PCL/HA scaffolds fabricated by Venugopal et al. (2008d). These authors seeded hFOBs on the nanobres and studied the cell proliferations (MTS assay), alkaline phosphatase (ALP) activity and mineralizations (ARS staining and SEM evaluation) on these nanobrous scaffolds. The ALP activity was 25 per cent higher on PLLA/Col/HA scaffolds than on PLLA/HA scaffolds after 20 days of cell culture. The bone nodule formation of hFOBs cultured on different electrospun nanobres was characterized by ARS staining, and the mineralization was found to be 57 per cent higher on PLLA/Col/HA scaffolds than on PLLA/HA scaffolds. Elemental analysis by EDX measurement primarily consisted of calcium and phosphorus deposition, showing that osteoblasts seeded on PLLA/Col/HA nanobres formed mineralized tissue that primarily consists of Ca and P deposits. Scaffolds with HA-containing polymeric composites enhanced the formation of new bone tissue with increased osteoblast adhesion, osteointegration and calcium mineral deposition on its surface (Prabhakaran et al. 2009). PCL/HA/Col nanobres with bre diameters of 373 191 nm were electrospun by Venugopal et al. (2007), with pore sizes of 235 mm, providing a large surface area-to-volume ratio for cell attachment and sufcient space for bone ingrowth and nutrient transportation. The interconnected porous structure of PCL/HA/Col nanobres provided the mechanical support and facilitated ECM production for bone tissue formation. Nanobrous scaffolds with controlled morphology suitable for bone tissue engineering were also proved by Gupta et al. (2009) by electrospraying of HA nanoparticles on poly- L-lactic acid-co-ecaprolactone (PLACL)/Gel nanobres. These authors carried out electrospraying and electrospinning simultaneously to produce PLACL/Gel/HA nanobres and, at the same time, compared the mechanical and cellular properties with those of electrospun PLACL/Gel/HA-blended nanobres. Electrospun PLACL/Gel/HA (blend) nanobres showed HA nanoparticles embedded inside the polymer
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

Review. Biomimetic HA substrates

2077

bres (diameter 198 107 nm), while the HA nanoparticles were found to be uniformly sprayed forming a layer of HA on the surface of electrospinelectrospray PLACL/Gel/HA nanobres (diameter 406 155 nm). The tensile stress for electrospunelectrospray PLACL/Gel/HA scaffolds was higher than that of the PLACL/Gel/HA (blend) scaffolds, and this was attributed to the fact that the electrospraying resulted in supercial dispersion of HA nanoparticles compared with a mechanically mixed blend. A signicant increase in hFOB proliferation was observed on PLACL/Gel/HA (spray) nanobres compared with PLACL/Gel/HA (blend) nanobres after 15 days of cell seeding. These scaffolds also showed 50 per cent higher mineralization than the PLACL/Gel/HA (blend) scaffolds, proving once again that the electrospraying method is superior to the blending technique for the fabrication of biomimetic nanobrous scaffolds for bone tissue engineering. The biocomposite nanobres contain amino, carboxyl and apatite molecules mostly to mimic the natural ECM for hFOBs to attach, proliferate and even migrate inside the scaffolds. The biocomposites are generally composed of a mixture of biodegradable polymers such as PLLA, PCL, PLACL, PEO, etc. with natural polymers such as Col, Gel, CTS along with nano-HA molecules. For example, Col (or Gel) supports the cells for proliferation, and HA acts as a chelating agent for the mineralization of osteoblasts for bone regeneration. PHBV nanobrous lm was fabricated by electrospinning and composited with HA by soaking in simulated body uid by Ito et al. (2005). Studying the degradation behaviour of HA/PHBV nanobrous lms using polyhydroxybutyrate depolymerase enzyme was faster, and cell adhesion on a nanobrous lm was also higher than that on a at lm. Nanobrous composites mimicking the bone components and characteristics were fabricated by Ngiam et al. (2009) by combining the method of electrospinning and mineralization processes. Preferential HA deposition on PLLA/Col nanobres with better early osteoblast attachment on mineralized nanobres was observed by these researchers. The collagen present in PLLA/Col nanobres had carboxyl groups, which favoured Ca2+ ion chelation. The alternative soaking method for mineralization was effective enough, and rapid HA formation on nanobres was achieved by increasing the concentrations of Ca and P ions up to a ratio of 1.66 (Ca/P) similar to that found in natural bone. The presence of nano-HA on the scaffolds had a greater inuence on the functionality of cells at early time points of culture, as observed by a higher level of intracellular protein production by cells. These authors concluded that the mineralization on nanobrous scaffolds could be a biomimetic method for exploitation of early osteoblast attachment and proliferation for bone tissue engineering.

5. Conclusions
This article described a number of materials/engineering approaches that are currently being explored in order to create systems that can impart clinical benets. They reect many of the signicant challenges for producing medical implants and therapies that are capable of achieving the regeneration of viable tissues and organs for implantation. Electrospinning offers a rapid, costeffective and convenient way for mass production of nanobres for fabricating
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

2078

J. Venugopal et al.

scaffolds with biomolecules and has been used across a broad range of biocomposite polymer systems and bone tissue engineering endeavours. Tissue engineers need to improve the biomechanical properties, and the cell binding sites of electrospun nanobres are paramount but they are also the major obstacle currently facing tissue engineers. The emerging and promising next generation of engineered tissues is relying on producing scaffolds with an informational function, e.g. material containing growth factor sequences that facilitate cell adhesion, proliferation and differentiation that are far better than in non-informational polymers. More studies remain to be done on the long journey between the laboratory and the clinics, and success in this eld depends on the effective cooperation of clinicians, chemists, biologists, bioengineers and materials scientists.
This study was supported by NUSNNI, National University of Singapore and StemLife Sdn Bhd, Kuala Lumpur, Malaysia.

References
Carlson, G. A., Dragoo, J. L., Samimi, B., Bruckner, D. A. & Benhaim, P. 2004 Bacteriostatic properties of biomatrices against common orthopaedic pathogens. Biochem. Biophys. Res. Commun. 321, 472478. (doi:10.1016/j.bbrc.2004.06.165) Chang, R., El-Amin, S. F., Hickok, N. J. & Tuan, R. S. 1998 Osteoblasts attachment is enhanced on collagen-coated orthopedic implant materials via alpha-2 and beta-1 integrins. Trans. Orthop. Res. Soc. 44, 517523. Chen, F., Wang, Z. C. & Lin, C. J. 2002 Preparation and characterization of nano-sized hydroxyapatite particles and hydroxyapatite/chitosan nanocomposite for use in biomedical materials. Mater. Lett. 57, 658662. (doi:10.1016/S0167-577X(02)00885-6) Clarke, K. I., Graves, S. E., Wong, A. T. C., Trift, J. T., Francis, M. J. O. & Czernuszka, J. T. 1993 Investigation into the formation and mechanical properties of a bioactive material based on collagen and calcium phosphate. J. Mater. Sci. Mater. Med. 4, 107110. (doi:10.1007/ BF00120378) Dorozhkin, S. V. 2009 Calcium orthophosphate-based biocomposites and hybrid biomaterials. J. Mater. Sci. 44, 23432387. (doi:10.1007/s10853-008-3124-x) Du, C., Cui, F. Z., Zhu, X. D. & de Groot, K. 1999 Three-dimensional nano-HAp/collagen matrix loading with osteogenic cells in organ culture. J. Biomed. Mater. Res. 44, 407415. (doi:10.1002/(SICI)1097-4636(19990315)44:4%3C407::AID-JBM6%3E3.0.CO;2-T) Glimcher, M. J. 1959 Molecular biology of mineralized tissues with particular reference to bone. Rev. Mod. Phys. 31, 359393. (doi:10.1103/RevModPhys.31.359) Gronowicz, G. & McCarthy, M. B. 1996 Response of human osteoblasts to implant materials: integrins mediated adhesion. J. Orthop. Res. 14, 878887. (doi:10.1002/jor.1100140606) Gupta, D., Venugopal, J., Mitra, S., Giri Dev, V. R. & Ramakrishna, S. 2009 Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts. Biomaterials 30, 20852094. (doi:10.1016/j.biomaterials.2008.12.079) Hamadouche, M. & Sedel, L. 2000 Ceramics in orthopaedics. J. Bone Joint Surg. Br. 82, 10951099. (doi:10.1302/0301-620X.82B8.11744) Hench, L. L. & Polak, J. M. 2002 Third-generation biomedical materials. Science 295, 10141017. (doi:10.1126/science.1067404) Hong, Z., Zhang, P. & Jing, X. 2005 Nano-composite of poly( L-lactide) and surface grafted hydroxyapatite: mechanical properties and biocompatibility. Biomaterials 26, 62966304. (doi:10.1016/j.biomaterials.2005.04.018) Hu, Q., Li, B., Wang, M. & Shen, J. 2004 Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

Review. Biomimetic HA substrates

2079

potential material as internal xation of bone fracture. Biomaterials 25, 779785. (doi:10.1016/ S0142-9612(03)00582-9) Hutmacher, D. W., Schantz, J. T., Lam, C. X. F. & Lim, T. C. 2007 State of the art and future directions of scaffold-based bone engineered from a biomaterials perspective. J. Tissue Eng. Regen. Med. 1, 245260. (doi:10.1002/term.24) Ito, Y., Hasuda, H., Kamitakahara, M., Ohtsuki, C., Tanihara, M., Kang, I. K. & Kwon, O. H. 2005 A composite of hydroxyapatite with electrospun biodegradable nanobers as a tissue engineering material. J. Biosci. Bioeng. 100, 4349. (doi:10.1263/jbb.100.43) Itoh, S., Kikuchi, M., Matsumoto, H. N., Tanaka, J. & Shinomiya, K. 2001 The biocompatibility of osteoconductive activity of a novel hydroxyapatite/collagen composite biomaterial, and its function as a carrier of rhBMP-2. J. Biomed. Mater. Res. 54, 445453. (doi:10.1002/1097-4636 (20010305)54:3%3C445::AID-JBM190%3E3.0.CO;2-9) Jin, H. J., Fridrikh, S. V., Rutledge, G. C. & Kaplan, D. L. 2002 Electrospinning Bombyx mori silk with poly(ethylene oxide). Biomacromolecules 3, 12331239. (doi:10.1021/bm025581u) Kikuchi, M., Itoh, S., Ichinose, S., Shinomiya, K. & Tanaka, J. 2001 Self-organization mechanism in a bone-like hydroxyapatite/collagen composite synthesized in vitro and its biological reaction in vivo. Biomaterials 22, 17051711. (doi:10.1016/S0142-9612(00)00305-7) Kim, S. H., Nam, Y. S., Lee, T. S. & Park, W. H. 2003 Silk broin nanober. Electrospinning, properties and structure. Polym. J. 35, 185190. (doi:10.1295/polymj.35.185) Landis, W. J., Song, M. J., Leith, A., McEwen, L. & McEwen, B. F. 1993 Mineral and organic matrix in normally calcifying tendon visualized in three dimensions by high voltage electron microscopic tomography and graphic image reconstruction. J. Struct. Biol. 110, 3954. (doi:10.1006/jsbi.1993.1003) Lee, C. H., Shin, H. J. & Cho, I. H. 2005 Nanober alignment and direction of mechanical strain affect the ECM production of human ACL broblast. Biomaterials 26, 12611270. (doi:10.1016/j.biomaterials.2004.04.037) LeGeros, R. Z. 2002 Properties of osteoconductive biomaterials: calcium phosphates. Clin. Orthop. 395, 8198. (doi:10.1097/00003086-200202000-00009) Li, W. J., Laurencin, C. T. & Ko, F. K. 2002 Electrospun nanobrous structure: a novel scaffold for tissue engineering. J. Biomed. Mater. Res. 60, 613621. (doi:10.1002/jbm.10167) Li, C., Jin, H. J., Botsaris, G. D. & Kaplan, D. L. 2005 Silk apatite composites from electrospun bers. J. Mater. Res. 20, 33743384. (doi:10.1557/jmr.2005.0425) Li, C., Vepari, C., Jin, H. J., Kim, H. & Kaplan, D. 2006 Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials 27, 31153124. (doi:10.1016/j.biomaterials.2006.01.022) Li, J., Chen, Y., Yin, Y., Yao, F. & Yao, K. 2007 Modulation of nano-hydroxyapatite size via formation on chitosangelatin network lm in situ. Biomaterials 28, 781790. (doi:10.1016/ j.biomaterials.2006.09.042) Liao, S. S., Cui, F. Z. & Feng, Q. L. 2004 Hierarchically biomimetic bone scaffold materials: nanoHA/collagen/PLA composite. J. Biomed. Mater. Res. B 69, 158165. (doi:10.1002/jbm.b.20035) Liu, X., Smith, L. A., Hu, J. & Ma, P. X. 2009 Biomimetic nanobrous gelatin/apatite composite scaffolds for bone tissue engineering. Biomaterials 30, 22522258. (doi:10.1016/ j.biomaterials.2008.12.068) Market Dynamics. 2008 Bone substitutes and growth factors. Rockville, MD: Datamonitor. Meyer, U., Joos, U. & Weismann, H. P. 2004 Biological and biophysical principles in extracorporeal bone tissue engineering. Part III. Int. J. Oral Maxillofac. Surg. 33, 635641. (doi:10.1016/ j.ijom.2004.04.006) Muzzarelli, C. & Muzzarelli, R. A. A. 2002 Natural and articial chitosan-inorganic composites. J. Inorg. Biochem. 92, 8994. (doi:10.1016/S0162-0134(02)00486-5) Ngiam, M., Liao, S., Patil, A. J., Cheng, Z., Yang, F., Gubler, M. J., Ramakrishna, S. & Chan, C. K. 2009 Fabrication of mineralized polymeric nanobrous composites for bone graft materials. Tissue Eng. Part A 15, 535546. Nishikawa, T., Masuno, K., Tominaga, K. & Tanaka, A. 2005 Bone repair analysis in a novel biodegradable hydroxyapatite/collagen implanted in bone. Implant. Dent. 14, 252260. (doi:10.1097/01.id.0000173628.00705.d0)
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

2080

J. Venugopal et al.

OBrien, F. J., Harley, B. A., Yannas, I. V. & Gibson, L. 2004 Inuence of freezing rate on pore structure in freeze dried collagen GAG scaffolds. Biomaterials 25, 10771086. (doi:10.1016/ S0142-9612(03)00630-6) Ohgo, K., Zhao, C., Kobayashi, M. & Asakura, T. 2003 Preparation of non-woven nanobers of Bombyx mori silk, Samia cynthia ricini silk and recombinant hybrid silk with electrospinning method. Polymer 44, 841846. (doi:10.1016/S0032-3861(02)00819-4) Porter, A., Patel, R. & Boneld, W. 2004 Effect of sintered silicate substituted hydroxyapatite on remodeling process at the bone implant interface. Biomaterials 225, 33033314. (doi:10.1016/ j.biomaterials.2003.10.006) Porter, A., Patel, N., Brooks, R. & Boneld, W. 2005 Effect of carbonate substitution on the ultrastructural characteristics of hydroxyapatite implants. J. Mater. Sci. Mater. Med. 16, 899907. (doi:10.1007/s10856-005-4424-1) Prabhakaran, M. P., Venugopal, J. & Ramakrishna, S. 2009 Electrospun nanostructured scaffolds for bone tissue engineering. Acta Biomater. 5, 28842893. (doi:10.1016/j.actbio.2009.05.007) Radisic, M., Park, H., Gerecht, S., Langer, R. & Vunjak-Novakovic, G. 2007 Biomimetic approach to cardiac tissue engineering. Phil. Trans. R. Soc. B 362, 13571368. (doi:10.1098/rstb.2007.2121) Rameshbabu, N., Rao, K. P. & Kumar, T. S. S. 2005 Accelerated microwave processing of nanocrystalline hydroxyapatite. J. Mater. Sci. 40, 63196323. (doi:10.1007/s10853-005-2957-9) Rho, J. Y., KuhnSpearing, L. & Zioupos, P. 1998 Mechanical properties and the hierarchical structure of bone. Med. Eng. Phys. 20, 92102. (doi:10.1016/S1350-4533(98)00007-1) Riboldi, S. A., Sampaolesi, M. & Neuenschwander, P. 2005 Electrospun degradable polyesterurethane membranes: potential scaffolds for skeletal muscle tissue engineering. Biomaterials 26, 46064615. (doi:10.1016/j.biomaterials.2004.11.035) Rusu, V. M., Ng, C. H., Wilke, M., Tiersch, B., Fratzl, P. & Peter, M. G. 2005 Size-controlled hydroxyapatite nanoparticles as self-organized organicinorganic composite materials. Biomaterials 26, 54145426. (doi:10.1016/j.biomaterials.2005.01.051) Sachlos, E., Gotora, D. & Czernuszka, J. T. 2006 Collagen scaffolds reinforced with biomimetic composite nano-sized carbonate-substituted hydroxyapatite crystals and shaped by rapid prototyping to contain internal microchannels. Tissue Eng. 12, 24792487. (doi:10.1089/ ten.2006.12.2479) Thomas, V., Dean, D. R. & Vohra, Y. K. 2006 Nanostructured biomaterials for regenerative medicine. Curr. Nanosci. 2, 155177. Thomas, V., Dean, D. R., Jose, M. V., Mathew, B., Chowdhury, S. & Vohra, Y. K. 2007 Nanostructured biocomposite scaffolds based on collagen coelectrospun with nanohydroxyapatite. Biomacromolecules 8, 631637. (doi:10.1021/bm060879w) Venugopal, J., Vadagama, P., Sampath Kumar, T. S. & Ramakrishna, S. 2007 Biocomposite nanobers and osteoblasts for bone tissue engineering. Nanotechnology 18, 055 101. (doi:10.1088/ 0957-4484/18/5/055101) Venugopal, J., Low, S., Choon, A. T. & Ramakrishna, S. 2008a Interaction of cells and nanobrous scaffolds in tissue engineering. J. Biomed. Mater. Res. B Appl. Biomater. 84, 3448. (doi:10.1002/jbm.b.30841) Venugopal, J., Prabhakaran, M. P., Low, S., Choon, A. T. & Ramakrishna, S. 2008b Nanotechnology for nanomedicine and delivery of drugs. Curr. Pharm. Des. 14, 21842200. (doi:10.2174/138161208785740180) Venugopal, J., Low, S., Choon, A. T., Sampath Kumar, T. S. & Ramakrishna, S. 2008c Mineralization of osteoblasts with electrospun collagen/hydroxyapatite nanobers. J. Mater. Sci. Mater. Med. 19, 20392046. (doi:10.1007/s10856-007-3289-x) Venugopal, J., Low, S., Choon, A. T., Kumar, A. B. & Ramakrishna, S. 2008d Electrospun-modied nanobrous scaffolds for the mineralization of osteoblast cells. J. Biomed. Mater. Res. 85A, 408417. (doi:10.1002/jbm.a.31538) Venugopal, J., Low, S., Choon, A. T., Bharath Kumar, A. & Ramakrishna, S. 2008e Nanobioengineered electrospun composite nanobers and osteoblasts for bone regeneration. Artif. Organs 32, 388397. (doi:10.1111/j.1525-1594.2008.00557.x) Wahl, D. & Czernuszka, J. T. 2006 Collagenhydroxyapatite composites for hard tissue repair. Euro. Cells Mater. 11, 4356.
Phil. Trans. R. Soc. A (2010)

Downloaded from rsta.royalsocietypublishing.org on May 5, 2012

Review. Biomimetic HA substrates

2081

Wahl, D. A., Sachlos, E., Liu, C. & Czernuszka, J. T. 2007 Controlling the processing of collagen hydroxyapatite scaffolds for bone tissue engineering. J. Mater. Sci. Mater. Med. 18, 201209. (doi:10.1007/s10856-006-0682-9) Wang, R. Z., Cui, F. Z., Lu, H. B., Wen, H. B., Ma, C. L. & Li, H. D. 1995 Synthesis of nanophase hydroxyapatite/collagen composite. J. Mater. Sci. Lett. 14, 490492. (doi:10.1007/BF00665911) Wei, J., Li, Y. B., Chen, W. Q. & Zuo, Y. 2003 A study on nanocomposite of hydroxyapatite and polyamide. J. Mater. Sci. 38, 33033306. (doi:10.1023/A:1026127018920) Weiner, S. & Traub, W. 1986 Organization of hydroxyapatite crystals within collagen brils. FEBS Lett. 206, 262266. (doi:10.1016/0014-5793(86)80993-0) Weiner, S. & Traub, W. 1989 Crystal size and organization in bone. Connect. Tissue Res. 21, 589595. (doi:10.3109/03008208909050015) Yamaguchi, I., Tokuchi, K., Fukuzaki, H., Koyama, Y., Takakuda, K. & Monma, H. 2001 Preparation and microstructure analysis of chitosan/hydroxyapatite nanocomposites. J. Biomed. Mater. Res. 55, 2027. (doi:10.1002/1097-4636(200104)55:1%3C20::AIDJBM30%3E3.0.CO;2-F) Yang, D. Z., Jin, Y., Ma, G. P., Chen, X. M., Lu, F. M. & Nie, J. 2008 Fabrication and characterization of chitosan/PVA with hydroxyapatite biocomposite nanoscaffolds. J. Appl. Polym. Sci. 110, 33283335. (doi:10.1002/app.28829) Yoshimoto, H., Shin, Y. M., Terai, H. & Vacanti, J. P. 2003 A biodegradable nanober scaffold by electrospinning and its potential for bone tissue engineering. Biomaterials 24, 20772082. (doi:10.1016/S0142-9612(02)00635-X) Yuan, H. Z., Yang, Z., Li, Y. & Zhang, X. 1998 Osteoinduction by calcium phosphate biomaterials. J. Mater. Sci. Mater. Med. 9, 723726. (doi:10.1023/A:1008950902047) Zarkoob, S., Eby, R. K., Reneker, D. H., Hudson, S. D., Ertley, D. & Adams, W. W. 2004 Structure and morphology of electrospun silk nanobers. Polymer 45, 39733977. (doi:10.1016/ j.polymer.2003.10.102) Zhang, Y. Z. & Lim, C. T. 2008 The development of biocomposite nanobers for tissue scaffolding applications. J. Miner. Metal Mater. Soc. 60, 4548. (doi:10.1007/s11837-008-0070-7) Zhang, Y. Z., Ouyang, H. W., Lim, C. T., Ramakrishna, S. & Huang, Z. M. 2005 Electrospinning of gelatin bers and gelatin/PCL composite brous scaffolds. J. Biomed. Mater. Res. B Appl. Biomater. 72, 156165. (doi:10.1002/jbm.b.30128) Zhang, Y. Z., Su, B., Ramakrishna, S. & Lim, C. T. 2008a Chitosan nanobers from an easily electrospinnable UHMWPEO-doped chitosan solution system. Biomacromolecules 9, 136141. (doi:10.1021/bm701130e) Zhang, Y. Z., Venugopal, J. R., El-Turki, A., Ramakrishna, S., Su, B. & Lim, C. T. 2008b Electrospun biomimetic nanocomposite nanobers of hydroxyapatite/chitosan for bone tissue engineering. Biomaterials 29, 43144322. (doi:10.1016/j.biomaterials.2008.07.038)

Phil. Trans. R. Soc. A (2010)