Phil. Trans. R. Soc. A (2010) 368, 2065–2081doi:10.1098/rsta.2010.0012

REVIEW

Biomimetic hydroxyapatite-containingcomposite nanofibrous substrates for bone

tissue engineeringBY J. VENUGOPAL1,*, MOLAMMA P. PRABHAKARAN1, YANZHONG ZHANG1,2,

SHARON LOW3, AW TAR CHOON3 AND S. RAMAKRISHNA1,2

1Nanoscience and Nanotechnology Initiative, Faculty of Engineering,National University of Singapore, Block E3-05-12, 2 Engineering Drive 3,

Singapore 117576, Republic of Singapore2Department of Bioengineering, College of Chemistry, Chemical Engineering

and Biotechnology, Donghua University, Shanghai, People’s Republic of China3StemLife Sdn BhD, Kuala Lumpur, Malaysia

The fracture of bones and large bone defects owing to various traumas or natural ageing isa typical type of tissue malfunction. Surgical treatment frequently requires implantationof a temporary or permanent prosthesis, which is still a challenge for orthopaedicsurgeons, especially in the case of large bone defects. Mimicking nanotopography ofnatural extracellular matrix (ECM) is advantageous for the successful regeneration ofdamaged tissues or organs. Electrospun nanofibre-based synthetic and natural polymerscaffolds are being explored as a scaffold similar to natural ECM for tissue engineeringapplications. Nanostructured materials are smaller in size falling, in the 1–100 nm range,and have specific properties and functions related to the size of the natural materials (e.g.hydroxyapatite (HA)). The development of nanofibres with nano-HA has enhanced thescope of fabricating scaffolds to mimic the architecture of natural bone tissue. Nanofibroussubstrates supporting adhesion, proliferation, differentiation of cells and HA induce thecells to secrete ECM for mineralization to form bone in bone tissue engineering. Ourlaboratory (NUSNNI, NUS) has been fabricating a variety of synthetic and naturalpolymer-based nanofibrous substrates and synthesizing HA for blending and sprayingon nanofibres for generating artificial ECM for bone tissue regeneration. The presentreview is intended to direct the reader’s attention to the important subjects of syntheticand natural polymers with HA for bone tissue engineering.

Keywords: electrospinning; synthetic and natural polymers; nanofibres; hydroxyapatite;bone tissue engineering

*Author for correspondence (nnijrv@nus.edu.sg).

One contribution of 14 to a Theme Issue ‘Advanced processing of biomaterials’.

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1. Introduction

Electrospinning processes have attracted a great deal of attention as a way tomimic the structure of natural extracellular matrix (ECM) by means of producingfibres down to 5 nm. This technique is used to fabricate nanofibrous structuresfrom natural and synthetic polymers such as collagen (Col), gelatin, chitosan(CTS), silk fibroin, poly(DL-lactide-co-glycolide), poly(lactide), polyurethane,polycaprolactone, etc. Rapid progress in nanotechnology and its far-reachingdevelopments have triggered the use of nanostructures as scaffolds for bone tissueengineering. Fabrication of nanofibres is one of the most promising techniques fordesigning polymer nanofibres in tissue engineering. There are several scaffoldfabrication techniques, namely electrospinning (random, aligned, core shell andvertical nanofibres), self-assembly, phase separation, melt-blown and templatesynthesis (Venugopal et al. 2008a). Of these techniques, electrospinning has beenthe most widely used technique recently, and it also seems to be demonstratingpromising results for tissue engineering applications. Electrospinning has emergedas an elegant and leading technique to create fibres with diameters in the rangeof micrometres down to a few nanometres cost-effectively for tissue engineering.

Tissue engineering is the application of knowledge and expertise from amultidisciplinary field, to develop and manufacture therapeutic products thatuse the combination of matrix scaffolds with viable human cell systems orcell-responsive biomolecules derived from such cells, for repair, restoration orregeneration of cells or tissues damaged by injury or disease (Venugopal et al.2008a,b). The concept of tissue engineering using three-dimensional scaffolds hascertain advantages over direct cell injection to the tissues: (i) three-dimensionalscaffolds may replace the missing or damaged infrastructure (ECM) in the infarctarea and provide temporary support for self or implanted cells, (ii) by tissueengineering, one can control the size, shape, strength and composition of the graftin vitro, and (iii) tissue engineering provides a solution to the problem of bonedefects and can be used to replace the injured bone. The objective was to developa scaffold for tissue engineering that is (i) highly porous with large interconnectedpores (to facilitate mass transport), (ii) hydrophilic (to enhance cell attachment),(iii) structurally stable (to withstand the shearing forces during bioreactorcultivation), (iv) degradable (to provide ultimate biocompatibility of the tissuegraft), and (v) elastic (to enable transmission of contractile forces). The scaffoldstructure determines the transport of nutrients, metabolites and regulatorymolecules to and from the cells, whereas the scaffold chemistry has an importantrole in cell attachment and differentiation. Mechanical properties of the scaffoldshould ideally match those of the native tissue, to provide mechanical integrity ofthe forming tissue and to support an in vivo-like mechanotransduction betweencells and their environment (Radisic et al. 2007). The role of biomaterials in tissueengineering 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 structureand morphology of the non-woven nanofibre matrix were found to closely matchthe 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 insufficientbone substitute. One main driving force to explore nanomaterials in bone tissue

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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 withtrauma, cancer and revision joint arthroplasty has become increasingly commonfor orthopaedic surgeons and remains a significant challenge in the field ofmusculoskeletal injury annually, and there are over several million orthopaedicprocedures performed each year (Market Dynamics 2008). The global orthopaedicmarket is estimated by Espicom to have been worth approximately US$37.1billion in 2008, following a growth of 9.7 per cent over the previous year.Excluding arthroscopy and ‘other’ segments (operating theatre equipment andsupplies), the market totalled US$29 billion, having grown by 10.7 per centover the previous year. To solve these problems, synthetic and natural polymericnanofibrous scaffolds are the alternative for bone tissue engineering applications;they should be biocompatible with the surrounding biological fluids and tissues,biodegradable and highly porous with interconnected spaces, and favourablefor 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 newand promising approach for bone repair and regeneration. Good sterilizability,storability and processability, as well as relatively low cost, are also of greatimportance to permit clinical applications. Unfortunately, no artificial biomaterialis available yet which embodies all these requirements, and it is unlikely that itwill appear in the near future. Until now, most of the available biomaterialsappear to be either predominantly osteogenic or osteoinductive or else purelyosteoconductive (Meyer et al. 2004). The current challenge in bone tissueengineering is to fabricate bioartificial bone graft mimicking the ECM witheffective bone mineralization, resulting in the regeneration of fractured or diseasedbones. Today, nanotechnology and nanoscience approaches to scaffold design andfunctionalization are beginning to expand the market for tissue engineering, whichforms the basis for a highly profitable niche within the industry. This reviewdescribes recent advances for the fabrication of biomimetic nanofibrous scaffoldswith hydroxyapatite (HA) for bone tissue engineering.

2. Hydroxyapatite

Hydroxyapatite is a major mineral component of calcified tissues (bone andteeth). Synthetic HA (Ca10(PO4)6(OH)2) has been used extensively as animplant material for bone substitute owing to its excellent osteoinductiveproperties (Rameshbabu et al. 2005). HA-enhanced surface properties (such asincreased surface area and charge and the ability to alter adsorption of chemicalspecies) could be used to promote cell response and proliferation to inducemineralization in bone tissue engineering. Hydroxyapatite has been used in avariety of biomedical applications such as matrices for controlled drug release,bone cements, tooth paste additive, dental implants, etc. Calcium phosphatebiomaterials (HA/tricalcium phosphate) with appropriate three-dimensionalgeometry are able to bind and concentrate endogenous bone morphogeneticproteins (BMPs) in circulation and may become osteoinductive (Yuan et al. 1998)and can be an effective carrier of bone cells. Nanohydroxyapatite (nano-HA) canbe prepared by co-precipitation and precipitation using emulsion, template and

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

3. Natural polymers/hydroxyapatite

One of the major challenges in developing porous scaffolds for bone tissueengineering is the conflicting interest between porosity, mechanical strength andbiodegradability. Stable osteoblast cell adhesion is largely mediated by integrinsand heterodimeric receptors that interact with ECM proteins such as fibronectin,vitronectin, fibrinogen and Col, allowing the cell to respond to its extracellularenvironment and modulating the cellular events that regulate remodelling ofbone (Gronowicz & McCarthy 1996; Chang et al. 1998). Nanoscaled featuressuch as surface roughness and topography of nanocrystalline bioceramics andnanofibrous scaffolds promote cell behaviour such as adhesion, proliferation,migration and differentiated functions. Natural bone being an innate exampleof inorganic–organic biocomposites consists of approximately 70 wt% inorganiccrystals, mainly HA, and 30 wt% organic matrix, mainly Col type I. Structurally,it is hierarchically organized from macro-, micro-, to nano-scale, where the basicbuilding blocks were pinpointed to be a plate-like HA nanocrystal incorporatedinto Col nanofibres (Glimcher 1959; Weiner & Traub 1986). Hydroxyapatite isconsidered to be a structural template for the bone mineral phase and also a majorinorganic mineral component of bone, and is commonly used as a bioceramic fillerin polymer-based bone substitute because of its higher level of bioactivity andbiocompatibility (Hong et al. 2005).

(a) Collagen/hydroxyapatite

Col is a major ECM component that possesses fibrous structure with fibrebundles of varying diameters (50–500 nm). The nanometre size feature influencescell behaviours by allowing cells to attach to diameters smaller than the cellsize of the fibres. Cells seeded on this structure tend to maintain normalphenotypic shape and guided growth according to nanofibre orientation. The mainidea in biomimetic approaches is to control and fabricate the morphology andcomposition of developed biomaterials, in which the nanocrystallites of inorganiccompounds are being dispersed with preferential orientation in the organicmatrices. Owing to its large potential in biomedical applications, many studies

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reported the preparation of bone-like biocomposites of HA and bioactive organiccomponents such as Col, gelatin, chondroitin sulphate, CTS and amphiphilicpeptide 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 havepotential in mimicking natural ECM and in replacing diseased skeletal bones.More attention has been focused on HA because its crystallographic structureis similar to that of inorganic compounds found in natural bone; it has beeninvestigated extensively owing to its excellent biocompatibility, bioactivity andosteoconductivity.

Bone tissue contains high levels of type I Col and several non-collagenousproteins (such as osteopontin, bone sialoprotein and osteocalcin) that distinguishit from other types of tissues. The size of the bone mineral is around 50 nmin length, 25 nm in width and 2–5 nm in thickness (Sachlos et al. 2006). Thesecrystals are oriented with their long crystallographic c-axis parallel to each otherand aligned with Col tropocollagen molecules (Weiner & Traub 1989). Col is easilydegraded and resorbed by the body and allows good attachment to cells. However,its mechanical properties are relatively low (E ∼ 100 MPa) in comparison withbone (E ∼ 2–5 GPa; Clarke et al. 1993), and it is, therefore, highly cross-linkedor found in composites such as Col–glycosaminoglycans for skin regeneration(O’Brien et al. 2004) or Col/HA for bone remodelling (Venugopal et al.2008a–e). Col and HA devices significantly inhibited the growth of bacterialpathogens, the frequent cause of prosthesis-related infection, compared withpoly(lactide-co-glycolide) devices (Carlson et al. 2004). Electrostatic co-spinningof nanocomposite fibres of polymers with a nano-HA composite system in nativebone is the special orientation between HA and Col molecules. However, moreefforts are required in the area of nanofibrous Col/HA composite, for exactlymimicking the complex nanostructured architecture of Col matrix with c-axisorientation of nano-HA particles (Thomas et al. 2006). Col supports the cells foradhesion and proliferation, and HA acts as a chelating agent for mineralization ofosteoblasts in bone tissue regeneration (Landis et al. 1993). Biologically inspiredbiocomposites of Col and nano-HA for bone substitute have a long history in thebiomedical field (Wahl et al. 2007). A combination of Col and nano-HA materialsis bioactive, osteoconductive and osteoinductive, seems to be a natural choicefor bone grafting and mimics the bone components. Nano-to-microscale nano-HA/Col alignment of composite was similar to that of natural bone and thusmight have been identified as ‘bone’ by the attached cells (Du et al. 1999). Itohet al. (2001) prepared a novel HA/Col composite biomaterial using cold isostaticpressure by the co-precipitation method that has a chemical composition andcrystallinity similar to that of bone and induces the development of osteogeniccells for remodelling of bone.

Skeletal bones comprise mainly Col (predominantly type I) and carbonate-substituted HA; both are osteoconductive components. Thus, an implantmanufactured from such components is likely to behave similarly and to be ofmore use than a monolithic device. Indeed, both type I Col and HA were foundto enhance osteoblast differentiation, but, combined together, they were shownto accelerate osteogenesis. A composite matrix when embedded with humanosteoblastic cells showed better osteoconductive properties than monolithicHA and produced calcification of identical bone matrix (Wang et al. 1995).

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Figure 1. Electrospun nanofibrous substrate interaction with human foetal osteoblast (hFOB)cells. (a) Col nanofibres (fibre diameter: 272 ± 0.63 nm) and (b) Col/HA nanofibres with hFOBmineralization.

More interest has been focused on HA because its crystallographic propertiesare similar to those of inorganic components found in natural bone. It has beeninvestigated extensively owing to its excellent biocompatibility, bioactivity andosteoconductivity (Li et al. 2007). In addition, Col/HA composites proved to bebiocompatible both in humans and in animals (Wahl & Czernuszka 2006). Kikuchiet al. (2001) fabricated an artificial bone material with bone-like nanostructureand chemical composition, a composite comprising HA and Col was synthesizedunder biomimetic conditions through a self-organization mechanism betweenHA and Col. The HA/Col composite fabricated and demonstrated bone-likeorientation, so that c-axes of HA nanocrystals were regularly aligned along Colfibrils (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 isa threshold concentration of calcium ions required to stimulate the subsequentactivity of osteoblasts (Nishikawa et al. 2005). The HA/Col composite, designedto simulate bone tissue, is produced using atelocollagen to reduce antigenicity bycondensing Ca(OH)2/H3PO4 suspension (Venugopal et al. 2008c).

Venugopal et al. (2008c) found that the mineral deposition on Col/HAcomposite nanofibrous scaffolds cultured with osteoblasts was significantly higherthan that on Col nanofibrous scaffolds (figure 1a) during 10 days of cultureperiod. Compared with Col/HA nanofibrous scaffolds, the mineral deposition wassignificantly lower (by up to 56 per cent) in Col nanofibrous scaffolds. The Col/HAcomposite nanofibrous scaffolds have huge potential for cell adhesion and growth,as well as stimulating the cells for mineralization, exhibiting functional activityof osteoblasts for bone tissue engineering (figure 1b). These studies showed thatthe Col/HA composite nanofibrous scaffold has great potential for bone tissueregeneration.

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The main practical problems with Col are its cost, and poor definition ofcommercial sources of this material makes it difficult to follow up on well-controlled processing. Col was replaced by gelatin (Gel), a protein producedby partial hydrolysis of Col extracted from skin, bone, cartilage, ligaments,etc. Mixing Gel with other synthetic polymers, sometimes called bioartificialpolymeric materials, has frequently been adopted by other researchers. Thisapproach is feasible to reduce the potential problem of cytotoxin, as a resultof using a chemical cross-linking reagent, but also provides a compromisesolution for overcoming the shortcomings of synthetic and natural polymers,that is, producing a new biomaterial with excellent biocompatibility andimproved mechanical and physical/chemical properties (Venugopal et al. 2008e).Liu et al. (2009) reported the fabrication of three-dimensional nanofibre-Gel/apatite composite scaffolds, which mimic both nanoscale architecture andchemical composition of natural bone ECM. Three-dimensional nanofibrous Gelscaffolds with well-defined macropores were prepared by using a new thermallyinduced phase separation and porogen leaching technique (Liu et al. 2009). Thedeposition of a biomimetic apatite layer throughout the porous structure of three-dimensional scaffolds is an effective method for controlling the surface topographyand chemistry within large, complex structures. These scaffolds have excellentbiocompatibility and mechanical properties and showed enhanced osteoblastadhesion, proliferation and differentiation suitable for bone tissue engineering.

Although Col is used as a common matrix for scaffold design, HA seems to be arational strategy for preparing a nanofibrous biocomposite that compositionallyand structurally resembles bone for engineering of bone tissue; however, Col aloneis less ideal in terms of mechanical properties and biostability owing to its rapiddegradation in the biological environment and problems related to its antigenicity.Recently, a few studies have explored the feasibility of replacing Col with othernatural biopolymers (e.g. silk and CTS) to prepare the HA containing electrospunbiomimetic composite fibres for potential osteoregenerative applications, whilethe electrospinnability of the natural biopolymers of interest is no longer an issueunder consideration in tissue engineering.

(b) Hydroxyapatite/silk protein

Silks are considered to be the most promising natural protein-type replacementfor Cols in bone tissue engineering because of their biocompatibility, slowdegradation and excellent mechanical performance. In the past few years, differentnatural silks (e.g. silkworm silk Bombyx mori and spider dragline silk Nephilaclavipes) have been processed for making nanoscale fibres via electrospinning(Jin et al. 2002; Kim et al. 2003; Ohgo et al. 2003; Zarkoob et al. 2004). Forexample, to improve the electrospinnability of silk solutions and to avoid potentialinfluences of using strong polarity organic solvents such as hexafluoroisopropanol(Zarkoob et al. 2004), hexafluoroacetone (Kim et al. 2003) and formic acid (Ohgoet al. 2003) on biocompatibility, Kaplan’s group (Jin et al. 2002) used an all-aqueous process for silk electrospinning by blending regenerated silk fibroin witha fibre forming agent, poly(ethylene oxide) (PEO), at a ratio from 1

4 to 23 .

A post-electrospinning treatment on the silk fibroin fibres with methanol wasthen performed to induce a structurally conformational transition to the originalb-sheet to render water insolubility in uses. Following this success, Li et al. (2006)

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recently developed silk-based composite nanofibres with the incorporation of HAnanoparticles (approx. 5 wt%) and BMP-2 (3 mg mg−1 of silk fibroin) growthfactor to realize enhanced bone formation outcomes from culturing humanbone marrow-derived mesenchymal stem cells (MSCs). They found that theinclusion of BMP-2 and HA nanoparticles within the electrospun silk fibroin fibresresulted in the highest calcium deposition and upregulation of BMP-2 transcriptlevels, 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 compositefibres, in which the inorganic apatite component was selectively grown on andassociated with the electrospun fibrous silk fibroin matrix by a subsequentmineralization process using an alternative soaking method. Acidic proteinpoly(L-aspartate) (poly-Asp) as a molecular recognition motif was first introducedinto the electrospun silk fibroin fibres to enable subsequent nucleation and crystalgrowth of apatite on the fibre surface during the mineralization process. Thisapproach is an alternative route for preparing composite nanofibres to avoidthe electrospinnability problem encountered in the electrospinning of those HA-containing solutions, which eventually leads to the formation of apatite-coatedfibroin composite fibres, which is somewhat different from that obtained in thecomponents hybridizing method (Li et al. 2006; Zhang & Lim 2008) and thenanostructure of hierarchical bone (Landis et al. 1993; Rho et al. 1998) in termsof distribution and morphology of the HA nanocrystals in the composite fibres.

(c) Hydroxyapatite/chitosan

Amino polysaccharide CTS is derived from the structural biopolymer chitinthat exists abundantly in crustacean shells (e.g. crabs) and plays a key rolesimilar to that of collagen in higher animals. CTS possesses plenty of notableattributes such as structural similarity to glycosaminoglycan found in bone,osteoconductivity, excellent biocompatibility, tailorable biodegradability, lowimmunogenicity and better mechanical properties. Thus, it has become ofgreat interest as one of the most attractive natural biopolymer matrices andalternatives to collagens for bone tissue engineering (Yamaguchi et al. 2001;Muzzarelli & Muzzarelli 2002; Hu et al. 2004). However, there are immensechallenges in converting a bulk HA/CTS nanocomposite or hybrid into a fibrousform by electrospinning owing to the poor electrospinnability of the CTS itselfas well as the adverse effect of the non-electrospinnable HA nanoparticles (andtheir aggregates) contained in the spinning dope. Formulating a robust CTSsolution system consequently appears to be a prerequisite to generate nanofibrousHA/CTS scaffolds. As a result of the noted obstacles in electrospinning, so farthere are very limited attempts of using nanofibrous HA/CTS for bone tissueengineering (Rusu et al. 2005; Yang et al. 2008).

By using an ultra-high-molecular-weight PEO as the fibre-forming aidingagent, Zhang et al. (2008a) demonstrated that CTS nanofibres could be preparedeasily with a minimum PEO loading ratio of up to 5 wt%. This enabled an attemptto develop HA/CTS composite nanofibres for potential application in bone repairand regeneration (Rusu et al. 2005). A modified two-step approach (Zhang et al.2008b) that combines the in situ co-precipitation synthesis route (Yamaguchiet al. 2001), which is likely to overcome the usual problem of nanoparticles

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(c)

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(d )

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HA/CTS

HA/CTSnanocomposites

HA/CTSsolution

Ca + P + CTS

Figure 2. Electrospun nanofibrous 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 acid-dominant solvent system; (c) SEM image of the co-precipitated HA/CTS nanocomposite withneedle-shaped HA nanoparticles being incorporated evenly within the CTS matrix; (d) electrospunHA/CTS nanofibres with an average diameter of ca 200 nm; (e) biomineralization of hFOBs onthe nanofibrous composite substrate of HA/CTS and (f ) apatite-like morphology of the depositedminerals in (e), visualized at a high magnification.

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agglomeration, with the electrospinning process was adopted for the preparationof HA/CTS nanofibres containing a higher (30 wt%) loading of HA nanoparticles(figure 2). Despite the fact that a dilute acetic acid-dominated solvent system wasused for dissolving CTS, results from the selected area of electron diffraction andX-ray diffraction analysis indicated that the crystalline nature of HA remains,thus implying a minor influence of the used acid solvent system on the crystallinestructure of HA incorporated within the co-precipitated HA/CTS nanocomposite.Bone formation ability assessed by conducting [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-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 nanofibrous scaffolds hadsignificantly encouraged bone formation-oriented outcomes compared with theelectrospun CTS alone scaffolds. Biomineralization in the form of synthesizingapatite-like granular minerals, as reported elsewhere (Yoshimoto et al. 2003;Li et al. 2006), was similarly observed with the post-cultured cell-scaffoldconstructs (figure 2e,f ). The two-step method would pave the way for developingother sophisticated biomimetic CTS-based nanofibrous composite scaffolds forfunctional bone tissue engineering.

4. Synthetic polymers/hydroxyapatite and synthetic/naturalpolymer/hydroxyapatite composites

Biocomposite nanofibres fabricated by electrospinning mimic the nanoscalestructure of ECM, and organize and provide signals for the cellular response(Gupta et al. 2009). The material for bone graft fabrication needs to providestructural integrity within the body and finally breakdown leaving new tissueformation. The material should modulate the cellular function to promote tissueregeneration in addition to mimicking the mechanical properties of the bone. Non-biodegradable electrospun polymers such as polyurethane and polyesterurethanepossess good mechanical properties, but they might interfere with tissue turnoverowing to their slow degradation properties (Lee et al. 2005; Riboldi et al. 2005).Non-woven poly-e-caprolactone (PCL) nanofibrous scaffolds were developed byYoshimoto et al. (2003) and seeded with MSCs from the bone marrow of neonatalrats with osteogenic supplements. The penetration of cells along with abundantECM deposits was observed in the cell–polymer constructs by these researchers,favouring potential scaffolds for bone tissue engineering. In contrast, the shrinkagebehaviour of natural polymeric (Col and gelatin) scaffolds is a major phenomenonproblematic in bone tissue regeneration (Liao et al. 2004). Preliminary in vitroand in vivo studies using nano-HA/Col composite also showed the material asbioactive and biodegradable, but its weak mechanical properties remain a majorhindrance for practical bone tissue engineering.

PCL/HA, PCL/Col/HA, PCL/Gel/HA, poly-L-lactic acid (PLLA)/Col/HAand poly(3-hydroxy-butyrate-co-3-hydroxyvalerate (PHBV)/HA were fabricatedby 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/HAnanofibrous scaffolds treated with plasma enhance the wettability and thusaccelerate the biodegradation rate of nanofibrous scaffolds. PCL/HA plasma-treated nanofibre shows mineral formation on the surface of osteoblast cell

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Figure 3. Mineralization of hFOBs on PCL/HA plasma-treated nanofibrous substrates. (a) PCLnanofibres (fibre diameter: 276 ± 56 nm) and (b) hFOB-secreted mineral deposition on the surfaceof the PCL/HA.

layers within 6 days of culture and morphology similar to that of HAof the natural bone (figure 3). On fabricating the composites of HA withbiodegradable polyesters such as PCL, PLLA produced a scaffold withbetter mechanical properties, whereas HA provided excellent bioactivity andosteoconductive properties (Hamadouche & Sedel 2000; LeGeros 2002). Moreover,the optimal performance of a composite material is achieved when the smallparticles (HA) are uniformly dispersed throughout the scaffolds and interactstrongly within the organic matrix. Electrospun Gel/PCL nanofibres werefabricated by mixing a 1 : 1 ratio of Gel and PCL with Gel concentrations rangingfrom 2.5 to 12.5%w/v and the membrane showed improved mechanical propertiesand favourable wettability compared with Gel or PCL membrane (Zhang et al.2005). Bone marrow stem cells (BMSCs) grown on the surface of these scaffoldswere found to migrate inside the scaffold up to 114 mm within a week of culture,showing their biocompatibility better than PCL nanofibres. Figure 4 shows thecellular ingrowth after 4 days of osteoblasts cultured on biocomposite nanofibrousscaffolds. While loosely interlaced fibrous structure and the weak nanoscale fibrescan provide the least obstruction and matched mechanical properties for cellmovements, it seems that the presence of appropriate molecular signals on thenanofibre surface can also guide or attract the living cells to enter into the matrixthrough their amoeboid movement (Venugopal et al. 2008e).

HA/PLLA composites have been described to fulfil certain requirements for itsapplication as a suitable substrate for bone tissue engineering (Liao et al. 2004).The compressive strength of the HA/PLLA composite has also been described asreaching the lower limit of natural cancellous bone (approx. 1 MPa). Implantexperiments showed quick healing of large segmental defects after 12 weeksof scaffold implantation and also showed appropriate bone substitute materialfor ingrowth of the new bone. However, Prabhakaran et al. (2009) fabricatedPLLA/HA and PLLA/Col/HA nanofibres by electrospinning and showed thatthe biocomposite PLLA/Col/HA nanofibres are superior to PLLA/HA nanofibresfor effective bone regeneration and mineralization. Moreover, the tensile strength

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20 µm 20 µm

Figure 4. Field emission SEM micrographs of hFOB interaction with a nanofibrous scaffoldafter 6 days of culture. (a) PCL/HA/Gel nanofibres (fibre 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 thecollagen fibrous matrix (1.68 MPa) prepared by Thomas et al. (2007) and evenPCL/HA scaffolds fabricated by Venugopal et al. (2008d). These authors seededhFOBs on the nanofibres and studied the cell proliferations (MTS assay),alkaline phosphatase (ALP) activity and mineralizations (ARS staining andSEM evaluation) on these nanofibrous scaffolds. The ALP activity was 25 percent higher on PLLA/Col/HA scaffolds than on PLLA/HA scaffolds after 20days of cell culture. The bone nodule formation of hFOBs cultured on differentelectrospun nanofibres was characterized by ARS staining, and the mineralizationwas found to be 57 per cent higher on PLLA/Col/HA scaffolds than on PLLA/HAscaffolds. Elemental analysis by EDX measurement primarily consisted of calciumand phosphorus deposition, showing that osteoblasts seeded on PLLA/Col/HAnanofibres formed mineralized tissue that primarily consists of Ca and P deposits.Scaffolds with HA-containing polymeric composites enhanced the formation ofnew bone tissue with increased osteoblast adhesion, osteointegration and calciummineral deposition on its surface (Prabhakaran et al. 2009).

PCL/HA/Col nanofibres with fibre diameters of 373 ± 191 nm were electrospunby Venugopal et al. (2007), with pore sizes of 2–35 mm, providing a largesurface area-to-volume ratio for cell attachment and sufficient space for boneingrowth and nutrient transportation. The interconnected porous structure ofPCL/HA/Col nanofibres provided the mechanical support and facilitated ECMproduction for bone tissue formation. Nanofibrous scaffolds with controlledmorphology suitable for bone tissue engineering were also proved by Guptaet al. (2009) by electrospraying of HA nanoparticles on poly-L-lactic acid-co-e-caprolactone (PLACL)/Gel nanofibres. These authors carried out electrosprayingand electrospinning simultaneously to produce PLACL/Gel/HA nanofibres and,at the same time, compared the mechanical and cellular properties with those ofelectrospun PLACL/Gel/HA-blended nanofibres. Electrospun PLACL/Gel/HA(blend) nanofibres showed HA nanoparticles embedded inside the polymer

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fibres (diameter 198 ± 107 nm), while the HA nanoparticles were found to beuniformly sprayed forming a layer of HA on the surface of electrospin–electrosprayPLACL/Gel/HA nanofibres (diameter 406 ± 155 nm). The tensile stress forelectrospun–electrospray PLACL/Gel/HA scaffolds was higher than that of thePLACL/Gel/HA (blend) scaffolds, and this was attributed to the fact that theelectrospraying resulted in superficial dispersion of HA nanoparticles comparedwith a mechanically mixed blend. A significant increase in hFOB proliferation wasobserved on PLACL/Gel/HA (spray) nanofibres compared with PLACL/Gel/HA(blend) nanofibres after 15 days of cell seeding. These scaffolds also showed50 per cent higher mineralization than the PLACL/Gel/HA (blend) scaffolds,proving once again that the electrospraying method is superior to the blendingtechnique for the fabrication of biomimetic nanofibrous scaffolds for bone tissueengineering.

The biocomposite nanofibres contain amino, carboxyl and apatite moleculesmostly to mimic the natural ECM for hFOBs to attach, proliferate and evenmigrate inside the scaffolds. The biocomposites are generally composed ofa mixture of biodegradable polymers such as PLLA, PCL, PLACL, PEO,etc. with natural polymers such as Col, Gel, CTS along with nano-HAmolecules. For example, Col (or Gel) supports the cells for proliferation, andHA acts as a chelating agent for the mineralization of osteoblasts for boneregeneration. PHBV nanofibrous film was fabricated by electrospinning andcomposited with HA by soaking in simulated body fluid by Ito et al. (2005).Studying the degradation behaviour of HA/PHBV nanofibrous films usingpolyhydroxybutyrate depolymerase enzyme was faster, and cell adhesion on ananofibrous film was also higher than that on a flat film.

Nanofibrous composites mimicking the bone components and characteristicswere fabricated by Ngiam et al. (2009) by combining the method ofelectrospinning and mineralization processes. Preferential HA deposition onPLLA/Col nanofibres with better early osteoblast attachment on mineralizednanofibres was observed by these researchers. The collagen present in PLLA/Colnanofibres had carboxyl groups, which favoured Ca2+ ion chelation. Thealternative soaking method for mineralization was effective enough, and rapidHA formation on nanofibres was achieved by increasing the concentrations of Caand P ions up to a ratio of 1.66 (Ca/P) similar to that found in natural bone. Thepresence of nano-HA on the scaffolds had a greater influence on the functionalityof cells at early time points of culture, as observed by a higher level of intracellularprotein production by cells. These authors concluded that the mineralizationon nanofibrous scaffolds could be a biomimetic method for exploitation of earlyosteoblast attachment and proliferation for bone tissue engineering.

5. Conclusions

This article described a number of materials/engineering approaches that arecurrently being explored in order to create systems that can impart clinicalbenefits. They reflect many of the significant challenges for producing medicalimplants and therapies that are capable of achieving the regeneration ofviable tissues and organs for implantation. Electrospinning offers a rapid, cost-effective and convenient way for mass production of nanofibres for fabricating

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scaffolds with biomolecules and has been used across a broad range ofbiocomposite polymer systems and bone tissue engineering endeavours. Tissueengineers need to improve the biomechanical properties, and the cell bindingsites of electrospun nanofibres are paramount but they are also the majorobstacle currently facing tissue engineers. The emerging and promising nextgeneration of engineered tissues is relying on producing scaffolds with aninformational function, e.g. material containing growth factor sequences thatfacilitate cell adhesion, proliferation and differentiation that are far better thanin non-informational polymers. More studies remain to be done on the longjourney between the laboratory and the clinics, and success in this field dependson the effective cooperation of clinicians, chemists, biologists, bioengineers andmaterials scientists.

This study was supported by NUSNNI, National University of Singapore and StemLife Sdn Bhd,Kuala Lumpur, Malaysia.

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