Gradient collagen/nanohydroxyapatite composite scaffold: Development and characterization

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Gradient collagen/nanohydroxyapatite composite scaffold:Development and characterization

Chaozong Liu a,*, Zhiwu Han b, J.T. Czernuszka a

a Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH, UKb Key Laboratory of Bionics Engineering, Ministry of Education of China, Jilin University, People’s Republic of China

Received 17 April 2008; received in revised form 11 September 2008; accepted 23 September 2008Available online 17 October 2008

Abstract

This paper reports an in situ diffusion method for the fabrication of compositionally graded collagen/nanohydroxyapatite (HA) com-posite scaffold. The method is diffusion based and causes the precipitation of nano-HA crystallites in situ. A collagen matrix acts as atemplate through which calcium ions (Ca2+) and phosphate ions (PO4

3�) diffuse and precipitate a non-stoichiometric HA. It wasobserved that needle-like prismatic nano-HA crystallites (about 2 � 2 � 20 nm) precipitated in the interior of the collagen template ontothe collagen fibrils. Chemical and microstructural analysis revealed a gradient of the Ca to P ratio across the width of the scaffold tem-plate, resulting in the formation of a Ca-rich side and a Ca-depleted side of scaffold. The Ca-rich side featured low porosity and agglom-erates of the nano-HA crystallites, while the Ca-depleted side featured higher porosity and nano-HA crystallites integrated with collagenfibrils to form a porous network structure.� 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Collagen; Hydroxyapatite; Gradient scaffold; Microstructure

1. Introduction

Tissue engineering involves seeding of cells onto a three-dimensional (3D) scaffold, followed by culture within asuitable environment and finally implantation into thebody when a mature matrix is formed [1,2]. The scaffoldis one of the most important components in this process[3,4], serving initially as a physical support structure thataffects cell processes such as migration and proliferation[5,6]. Collagen and hydroxyapatite (HA) both have meritsas scaffold materials for bone regeneration [3,4]. Researchhas shown that a combination of collagen and HA wouldprovide an appropriate scaffold material for bone tissueculture. Many efforts have been made to fabricate a com-

posite collagen/HA scaffold for bone tissue engineering[7–12], and the microstructure, biodegradation andmechanical properties of such a composite could be tai-lored by carefully controlling the processing condition[13–17].

Tissues in nature exhibit gradients across a spatial vol-ume, in which each identifiable layer has specific functionsto perform so that the whole tissue/organ can behave nor-mally [18]. Many tissues are anatomically merged intoneighbouring tissues/organs, often via a non-specific transittissue. The transit tissue shows a progressive change inboth structure and composition, spatially arranged in orderto maintain the appropriate connections [19]. It acts as a‘‘connector” that attaches two neighbouring tissuetogether. At each end, the transit tissue is typically struc-turally or functionally identical to the tissues with whicheach end is connected [20]. The intermediate part of thetransit tissue typically has a distinct and unique structureor architecture that is related to its mechanical function,including the mechanical coupling of the two tissues to

1742-7061/$ - see front matter � 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.actbio.2008.09.022

* Corresponding author. Present address: Advanced Materials andBiomaterials Research Centre, School of Engineering, The RobertGordon University, Schoolhill, Aberdeen AB10 1FR, UK. Tel.: +44 775131 6611.

E-mail address: [email protected] (C. Liu).

Available online at www.sciencedirect.com

Acta Biomaterialia 5 (2009) 661–669

www.elsevier.com/locate/actabiomat


which it is attached [2]. For example, in diarthodial joints,the articular cartilage is supported by a subchondral boneplate which merges into the underlying cancellous bone.Here the subchondral bone, which connects the articularcartilage to the cancellous bone, has a significant mechan-ical function in transmitting loads from the cartilage intothe underlying cancellous bone [21]. The bone of diaphysisconsists of cancellous bone covered with a shell of corticalbone [22,23]. The flat bones of the skull have a middle layerof cancellous bone sandwiched between two relatively thicklayers of cortical bone. The cortical bone merges into thecancellous bone via a transit region, i.e. the junction region.Even the cortical and cancellous bone are a gradient systemwith respect to their structure and composition andmechanical property [24,25].

However, current approaches to developing scaffoldsgive little consideration to the gradient behaviours of thetissues. Most of the scaffolds reported in the literature areporous solids of uniform composition and pore structurewhich have been used to culture a single tissue. Few ofthe reported scaffolds can provide an appropriate substrateto facilitate formation of tissue for regions where tissuesare attached to each other, where each region differs interms of its resident cell type and composition.

This study seeks to develop a gradient scaffold to pro-duce a gradient in the mineral content for use in the tissueengineering of ‘‘connector” tissue to replicate the interfacebetween soft and hard tissues. In this study, a gradient col-lagen/nano-HA composite scaffold, in terms of its structureand composition, was fabricated by using an in situ precip-itation technique. The microstructure and chemical compo-sitions of the obtained scaffolds have been examined. Thereported graded scaffold has potential applications in theculture of tissues with gradient properties, such as osteo-chondral bone.

2. Materials and methods

2.1. Preparation of a collagen template matrix and

crosslinking treatment

Bovine Achilles tendon collagen type I (Sigma–Aldrich,UK) was used for the fabrication of the template matrix.Collagen dispersion of 4% w/v was prepared by addingthe respective mass of collagen I in a 0.05 M acetic acidsolution (pH 3.2) and homogenizing it on ice. The mixturewas then degassed in a bell jar and stored at 4 oC beforeuse. The collagen matrix was made by casting the collagendispersion into a PTFE mould (20 mm diameter and 2 mmheight) and freeze at �30 oC, followed by dehydration inethanol and critical point drying with liquid carbon dioxideresults in a dry collagen scaffold. The dried matrix was fur-ther subjected to dehydrothermal treatment (DHT) at105 oC under a vacuum pressure of less than 0.1 mbar for72 h prior to additional chemical crosslinking treatmentin the presence of lysine, as reported elsewhere [26,27]. Inbrief, the DHT-treated samples were subsequently

immersed and incubated in 50 mM 2-morpholinoethanesulphonic acid (MES) solution (pH 5.5) containing 2.5 Mlysine for 1 h at room temperature. Then 1 ml MES solu-tion containing 40 mM 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) and 20 mM N-Hydroxysuccinimide (NHS) was added. After incubationfor 24 h, the scaffolds were removed from the solution,washed with distilled water and freeze dried.

2.2. Preparation of gradient collagen/HA composite scaffold

A 250 ml quantity of 50 mM calcium chloride solutionwas added to a 1000 ml glass flask which was held at37 �C in a water bath, adjusted to the required pH eitherusing 50 mM hydrochloric acid solution or 50 mM sodiumhydroxide (NaOH) solution. Then 250 ml of 30 mM diso-dium hydrogen phosphate solution was added dropwiseover a time period of 2 h under stir vigorously using a mag-netic flyer. The precipitates were collected by centrifugingthe suspension at 4 g and washed in distilled water, thenfreeze dried to obtain the hydroxyapatite powder. Thepowder was further analysed by X-ray photoelectron spec-troscopy (XPS) to determine the Ca/P ratios.

A modified diffusion method was used for the fabrica-tion of the gradient nano-HA/collagen composite scaffold[28,29]. Fig. 1 shows a schematic representation of theexperimental set-up capable of precipitating calcium phos-phate nanocrystallites within the collagen scaffold. Theabove obtained collagen matrix was sandwiched betweentwo nylon meshes, draped over a polystyrene bottle filledwith 150 mM disodium hydrogen phosphate (Na2HPO4)solution and locked in place with a rubber band. The bottlewas inverted and placed in a water bath filled with 50 mMcalcium chloride (CaCl2�2H2O) solution kept at 37 oC.Both the calcium and the phosphate solution contained0.1 M sodium chloride to provide a constant backgroundionic strength and were adjusted to the required pH value(8.5 in this study). Once in contact, the PO4

3� ions fromthe phosphate solution and the Ca2+ ions from the calcium

Fig. 1. Schematic of biomimetic in situ nano-HA precipitation within acollagen scaffold to make a gradient HA/collagen composite scaffold.

662 C. Liu et al. / Acta Biomaterialia 5 (2009) 661–669


solution diffuse through the collagen matrix in oppositedirections, owing to the effect of the concentration gradi-ent. Nano-HA crystallites precipitated within the collagenscaffold from the reaction of the Ca2+ and PO4

3� ionsbased on the following formula:

10CaCl2 þ 6Na2HPO4 þ 2H2O

! Ca10ðPO4Þ6ðOHÞ2 þ 12NaClþ 8HCl ð1Þ

The pH value of the calcium solution was measured duringthe precipitate process and the readings fed back into acomputer-controlled autoburette. The consequent drop inpH due to the reaction triggered the addition of titrantfrom the autoburettes to maintain the pH value of the solu-tion at 8.5 by adding of appropriate acidic or basic titrants.The precipitation was continued for 24 h, then the nano-HA/collagen composite scaffold was removed from thebottle and freeze dried.

2.3. Structural and chemical characterization

The microstructure of the samples was examined byscanning electron microscopy (SEM) using a JEOL JSM-840F scanning electron microscope operated at 5 kV, aftersputter deposition of a conductive platinum film (2 nm).The pore size and size distribution of the samples were ana-lysed by a high-resolution micro-X-ray computed tomogra-phy (l-CT) system (lCT 40, Scanco Medical, Switzerland)operated at a voltage of 55 kV and a current of 145 mA.

A DualBeamTM SEM/focused ion beam (FIB) system(FEI Nova 600 Nanolab) fitted for energy-dispersive X-ray analysis (EDAX) (EDAX Genesis), which combinesultra-high-resolution field emission SEM and precise FIBetch and deposition, was used for interior microstructureexamination and local elemental analysis, and for transmis-sion electron microscopy (TEM) sample preparation. Theobtained TEM samples were examined by a TEM system(JEOL 2000FX) in bright field mode to assess the morphol-ogy of the nanocrystallites. The electron diffraction pat-terns of the crystallites were used to identify the calciumphosphate phase. Local elemental analysis was carriedout by a fitted EDAX Genesis system to assist with thephase identification. The crystalline phase was investigatedby X-ray diffraction (XRD) using a Bruker D8 diffractom-eter operated at 40 kV and 40 mA with Cu Ka radiation(k = 0.15418 nm) over a 2h range of 5–70� at incrementsof 0.02�.

A 1 mg specimen was mixed with potassium bromidepowder and ground using an agate mortar and pestle.The resulting mixture was pressed into a transparent discwith a diameter of 13 mm. Fourier transform infrared(FTIR) spectrum of the sample was examined in transmis-sion mode using an FTIR spectrometer (Spectrum 2000,Perkin Elmer) in the range from 4000 to 400 cm�1 at a res-olution of 4 cm�1.

The surface chemistry of the samples was determinedusing an XPS system (VG ESCALAB5, VG Scientific

Ltd., UK). The X-ray source used was an Mg Ka line(HV = 1253.6 eV), operating at an emission voltage andcurrent of 14 kV and 20 mA, respectively. A survey scanin the range of 0–1000 eV was performed and high-resolu-tion spectra were also obtained.

3. Results

3.1. Effect of pH value on Ca/P ratio

The variation in the Ca/P molar ratio of the precipitateswith the pH value of the solution is shown in Fig. 2. Thisfigure shows that the Ca/P ratio increases with increasingstarting pH value within the investigated range of 5–11.Ca/P values of 1.45 and 1.81 were measured when the reac-tions were undertaken at pH 5 and 11, respectively. Theprecipitate exhibited a Ca/P ratio of 1.67, which is the stoi-chiometric composition (Ca/P = 1.67) of HA, when precip-itated at pH 8.5. This pH value was used for thepreparation of the gradient collagen/HA scaffold.

3.2. Microstructure of the scaffolds

As a template for the collagen/nano-HA composite scaf-fold, the collagen matrix obtained from the 4% collagendispersion had a porosity of 73% and featured an intercon-nected pore network, as revealed by micro-CT examination(Fig. 3(a)). The pores within the scaffold arise from the icecrystals that form during freezing of the collagen disper-sion. This forces the collagen to form aggregates in theinterstitial spaces and create an interconnected networkof collagen fibrils. A previous study has reported that thepore size of the scaffold can be adjusted by altering the con-centration of the collagen dispersion, the freezing rate andthe pH value since these factors are known to affect boththe nucleation and growth rate of the ice crystals [26]. Ahigher collagen concentration and higher freezing rate ofthe dispersion produced a lower porosity and smaller pore

Fig. 2. Effect of pH value on the Ca/P ratio of precipitates.

C. Liu et al. / Acta Biomaterialia 5 (2009) 661–669 663


size; higher porosity and larger pore sizes scaffolds could beobtained by a lower collagen concentration and low freez-ing rate [13].

The pore size and pore size distribution within the scaf-folds, as determined by micro-CT analysis, are shown inFig. 4(a). This figure illustrates that the pore size and poresize distribution do indeed vary. During precipitation, thenanocrystallites deposited onto collagen fibrils formed a col-lagen/nano-HA composite. The composite scaffold alsodemonstrated an interconnected network of pores, asrevealed by micro-CT examination and shown in Fig. 3(b).The pore size distribution of the resultant composite scaffoldis shown in Fig. 4(b). There was a peak at 64 lm, with a meanpore size of 59 ± 17.5 lm. Detailed analysis indicated that80% of pores within the resultant composite scaffold have apore size in the range of 50–80 lm.

Generally, it has been reported that a large pore size andporosity of the scaffold can allow effective nutrient supply,gas diffusion and metabolic waste removal, but leads to lowcell attachment and intracellular signalling; a small poresize or porosity can provide the opposite properties [30].Research has revealed that 70–120 lm pores are suitablefor chondrocyte ingrowth [31], 40–150 lm for fibroblastbinding [32] and 100–400 lm for bone regeneration,

depending on the porosity and the scaffold materials used[33,34]. It should be noted that the l-CT pore size analysisin this study was performed on dry collagen scaffold. Gen-erally, the collagen scaffold shrinks in the drying process.The scaffold will expand by 50–100% when wetted in cul-ture medium, so the pore size in the wet condition will belarger than that reported above. A previous study demon-strated the hMSCs can migrate into the scaffold and prolif-erate there [35].

SEM examination revealed that nanocrystallites weredeposited onto the collagen fibrils and filled part of voidspace of collagen matrix (Fig. 5(a)). This led to a decreasein the pore size of the resultant composite scaffold, asconfirmed by micro-CT examination. The precipitatednanocrystallites agglomerated to form flakes with a ‘‘petals”-like morphology (inset in Fig. 5(b)), with the ‘‘leaves” of theflakes becoming entangled together to form a densestructure.

The phase composition of the precipitated nanocrystal-lite was identified by XRD analysis as low-crystallinityHA, as shown in Fig. 6. The preferred orientation isobserved in natural bone, and the extent of such a pre-ferred orientation with respect to (002) or (211) can bedetermined from their intensity ratios. For the composite,

Fig. 3. Micro-CT 3D images of the scaffolds. (a) The 4% collagen scaffold has a porosity of 72%; (b) the nano-HA/collagen composite scaffold has aporosity of 45%.

Fig. 4. Pore size distributions of scaffolds as determined from micro-CT examination. (a) Collagen scaffold, mean pore size = 109.7 lm, SD = 5.6 lm; (b)nano-HA/collagen scaffold, mean pore size = 59.4 lm, SD = 17.5 lm.



the peak intensity ratio of I(002)/I(211) was calculated tobe 0.64 based on Gaussian curve fitting. This value is sim-ilar to the value of 0.46 of the untextured HA powder sam-ple and lower than the I(002)/I(211) ratio of 3.4 found inthe longitudinal direction of a rabbit ulna [15]. When thenanocrystallites precipitate onto the collagen fibrils, theorientation of the collagen fibrils and the structure of thecollagen matrix may exert an influence on the growth ori-entation of the HA nanocrystallites. Reports [36,37] haveshown that preferred orientations are observed whennano-HA is precipitated onto aligned collagen membranes.In this study, randomly aligned collagen fibrils would guideHA nanocrystallite precipitation and growth, resulting in anearly untextured structure, as revealed by XRD examina-tion. These results are in agreement with the TEM observa-tion of the sample. The bright field TEM examination andelectron diffraction pattern of the specimen are shown inFig. 7. As can be seen from the TEM bright field image,randomly orientated nano-HA crystallites have the typicalneedle-like prism-shaped morphology and a crystal particle

size range from 2 � 2 � 20 to 2 � 5 � 50 nm, similar tothose found in mammalian bone [38,39]. The electron dif-fraction pattern (Fig. 7(b)) exhibits a large number of ringstypical of a low crystalline structure. From the rings corre-sponding to the basal planes (002) and (300), the latticeparameters could be determined as a = 0.943 nm andc = 0.689 nm, respectively.

3.3. Chemical composition of the scaffolds

The chemical composition of the collagen/nano-HAcomposite was evaluated more specifically using FTIRspectroscopy. The spectra of collagen scaffold and compos-ite scaffold are shown in Fig. 8. For collagen scaffold, thespectrum exhibited typical amide bands derived from colla-gen. The peak at �1650 cm�1 is assigned to amide I derivedfrom the C@O stretch of the collagen, while those at �1550and �1236 cm�1 are assigned to amide II and amide III,respectively, derived from the N–H in-plane deformationplus the C–N stretch of collagen. Normally, the amide Iband is strong, the amide II band weak and the amideIII moderate. Typical bands, such as the N–H stretchingat �3326 cm�1 for the amide A and the C–H stretchingat �3074 cm�1, are also evident in the spectrum [13,16,28].

For the collagen/nano-HA composite scaffold, in addi-tion to the main peaks of collagen, namely amides I, IIand III, the spectrum also exhibited bands derived fromHA, and these bands formed the main peaks of the spec-trum. A broad phosphate band in the range from 1150 to950 cm�1 derives from the P–O asymmetric stretchingmode (m3) of the (PO4)3� group. The triple (m4) degeneratebending modes of the O–P–O bond were exhibited at 600and 572 cm�1 [14,15]. An XPS survey scan of the resultantcomposite scaffold is shown in Fig. 9. The XPS spectrademonstrated calcium, phosphorus, carbon, oxygen andnitrogen peaks. However, the concentrations of these ele-ments are different in the two sides of the scaffold disc.XPS elemental analysis results, as listed in Table 1, revealedthat the resultant scaffold has a Ca-rich side and a Ca-depleted side. On the Ca-rich side, the elements calcium

Fig. 5. SEM image of the collagen/nano-HA composite scaffold showing (a) nano-HA crystallites precipitated within the collagen scaffold on theCa-depleted side and (b) nano-HA crystallites with a ‘‘flower”-like morphology (inset) agglomerated on the HA-rich side.

Fig. 6. XRD spectrum of collagen/nano-HA composite scaffold.



and phosphorus, from the HA phase, comprise 34% of theatoms; in contrast, on the Ca-depleted side calcium andphosphorus only make up about 5% of the total atoms.Thus, there is much less precipitate on the Ca-depletedside.

4. Discussion

One reason for making a gradient composite scaffold isto mimic the natural extracellular matrix (ECM) of tissuesand organs with respect to the compositional and struc-

tural properties [40,41]. It is postulated that a gradientcomposite scaffold would provide an appropriate substrate

Fig. 7. TEM images of nano-HA/collagen composite scaffold. (a) Bright field micrograph demonstrating that needle-like crystallites (dark) existthroughout the cross-section; and (b) electron diffraction patterns of the composite confirming nano-HA crystallites (the Miller indices for eachcorresponding ring are labelled in brackets).

Fig. 8. FTIR spectrum of the nano-HA/collagen composite.

Fig. 9. XPS survey scan revealing a gradient HA concentration within thecomposite specimen, forming an HA-rich side and an HA-depleted side ofthe composite scaffold.

Table 1Elemental composition of composite determined by XPS analysis (inat.%).

Side C O N P Ca Cl Ca/P

Ca-rich 31.7 35.7 0 13.7 18.9 0 1.38Ca-depleted 60.2 22.4 12.2 2.2 2.7 0.3 1.23

Centrea 62.1 16.0 21.9 1.36

a Determined by FIB fitted with EDX.



to facilitate the formation of tissue for regions of tissuesthat are attached to each other, where each region differsin terms of its resident cell type and composition [20].

Yaylaoglu et al. [42] developed a gradient scaffold,prepared by stepwise formation of calcium phosphatecrystals within a collagen matrix, and in vitro evaluationdemonstrated it could be used for the tissue engineeringof an osteochondral implant. The study of the co-cultureof chondrocytes and osteogenic cells on a gradient com-posite scaffold demonstrated the generation of osteochon-dral tissue with a well-defined cartilage–bone interfacewhich is tissue culture duration dependent [43]. Apartfrom the composition gradient, the porosity and poresize gradient also influence cell growth and migration,and ingrowth and tissue regeneration [34]. One in vitroand in vivo study using a pore size gradient scaffoldrevealed that different cells and bone tissues have differ-ent pore size ranges for effective cell growth and tissueregeneration [30]. Kikuchi et al. [11] reported a self-orga-nized HA/collagen bone-like nanocomposite scaffold thatdemonstrated biocompatibility and biointegrative equiva-lent to autogenous bone, and their in vitro and in vivostudy revealed that such a composite scaffold could beused in tissue regeneration.

When the phosphate solution is brought into contactwith calcium solution (separated by the porous collagenscaffold, as shown in Fig. 1), diffusion through the collagenscaffold occurs under the effect of the concentration gradi-ent. Calcium ions diffuse towards the phosphate side whilephosphate ions diffuse in the reverse direction, towards thecalcium side.

For a given scaffold with set thickness and porosity, thediffusion dynamics of the ions through the porous scaffoldcould be described using the following equations [44]:

qoci

ot¼ �rJ i ð2Þ

with

J i ¼ �Di rci þ ziF

RTciru

� �ð3Þ

where q is the porosity of the scaffold, c is the concentra-tion of the ionic species i, J is the flux of the ionic species,D is the diffusion coefficient, z is the charge number, F isthe Faraday constant, R is the gas constant, T is the tem-perature and u is the electrical potential created betweenthe different ionic species in the pore solution.

Apparently, the flux of ionic species J is dependent onthe diffusion coefficient D and the concentration gradientrc. Factors that might affect D include a charge gradientin the scaffold or differences in ionic radii of the diffusingspecies.

Considering first the ionic radius, the diffusion coeffi-cient can be expressed as:

D ¼ RT6pgr

ð4Þ

where g is the viscosity of the medium and r is the hydro-dynamic (Stokes) radius. The Stokes radius takes into ac-count all the water molecules the ion carries in itshydration sphere. The data of Nightingale [45] show thatthe dynamic radii of PO4

3� ions and Ca2+ ions are similar.Thus the diffusion coefficients are likely to be similar forthe two ions and hence the diffusion layer thickness willbe the same for both ions [36]. It has been found that col-lagen can in fact behave as a cation-selective membrane,but only at low values of ionic strength – much lower thanthose used in this study [36]. At the values used here themembrane does not determine the diffusion rates of thetwo main ions.

We turn now to the concentration gradient. Thisinvolves calculating the proportion of the total calciumand phosphate concentrations that are present as free ions.The important equation governing the free calcium ionconcentration is:

CaOHþ () Ca2þ þOH� ð5ÞAt the values of pH used in this experiment the equation ispushed far to the right and practically all the calcium ispresent as free calcium ions [46]. The phosphate equilib-rium is more complicated, but it has been shown that atthe pH range used here, HPO4

2� and H2PO4� ions domi-nate over PO4

3� ions, which have a lower concentrationby a factor of approximately 5 [36,47]. Thus, overall, theflux of phosphate ions is much lower than that of Ca2+ ionsdue to the lower phosphate concentration gradient. Theconcentration of phosphate ions is only high enough toprovide a supersaturated calcium phosphate solution onthe phosphate side of the scaffold and so this is where pre-cipitation occurs. The differences in Ca:P ratio across thescaffold can also be explained in a similar manner. TheHPO4

2� ions diffuse more rapidly than the PO43� ions be-

cause of their greater concentration and so give rise to pre-cipitates containing lower Ca:P ratios because the HPO4

3�

ions will have substituted for the PO43� ions on the HA lat-

tice, and Ca2+ vacancies will have formed to compensate.The initially formed nano-HA crystallites are deposited

on the collagen fibrils and fill some of the voids; this leadsto a decrease in the porosity q of the scaffold. Because ofthis decrease in porosity, the diffusion rates of ions throughthe scaffold also decrease. This results in precipitation ofnano-HA inside the scaffold, with a reduction in the con-centration of HA toward the calcium side. The examina-tion by the FIB fitted with EDX confirmed that Ca2+

and PO43� diffuse, react and form HA inside the scaffold,

as demonstrated in Fig. 10. This slow diffusion processresults in a different morphology (Fig. 5(a)) of the nano-HA formed within the scaffold from the more rapidly pre-cipitated nano-HA formed at the initial precipitation stage(Fig. 5(b)), as demonstrated in the SEM examination.

Previous studies have demonstrated that the mechanicalproperties and biodegradation of collagen scaffold dependon the crosslink treatment [35]. Dynamic mechanical analy-sis has demonstrated that DHT-treated scaffolds show a



‘‘soft” and ‘‘flexible” behaviour. By contrast, crosslink-trea-ted scaffold, using lysine as the crosslinking agent, exhibiteda more ‘‘rigid” behaviour. The in vitro biodegradation eval-uation using a collagenase assay revealed that crosslinktreatment can improve the biodegradation resistancegreatly, with only 33% degradation being measured after2 h of collagenase incubation. In vivo results suggested the50% collagen remained even after 4 weeks of culture.

5. Conclusions

A gradient collagen/nano-HA composite scaffold, with aCa-rich side and a Ca-depleted side, has been developed by abiomimetic diffusion–precipitate method. This method isable to precipitate nano-HA crystallites in the interior of acollagen scaffold to form a compositional and structural gra-dient composite scaffold by careful control of the precipita-tion parameters, such as the concentration of ions and theporosity of the collagen matrix. Both TEM and XRD exam-inations have confirmed the formation of needle-like prism-shaped nano-HA crystallites. The pore size and porosity ofthe composite scaffold are dependent on the template scaf-fold used, which is controllable by adjusting the collagenconcentration and freezing rate. Such a gradient compositescaffold may be an appropriate substrate that facilitates theformation of tissue for regions of tissues that are attachedto each other, where each region differs in terms of its residentcell type and composition.

Acknowledgements

This work was funded by the Wellcome Trust throughthe University Translation Award (Contract No. 074486).

Professor Grovenor is thanked for the provision of labora-tory facilities. Past and present members of the Biomateri-als Group at Oxford University are thanked for helpfuldiscussions. The Healthcare Engineering Research Groupand the Institute of Polymer Technology and MaterialsEngineering (IPTME) at Loughborough are thanked forthe provision of laboratory facilities.

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Fig. 10. FIB fitted with EDAX examination revealing the porous interiorstructure of the nano-HA/collagen composite and showing that nano-HAcrystallites were also formed inside the composite scaffold.



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Gradient collagen/nanohydroxyapatite composite scaffold: Development and characterization