J Mater Sci: Mater Med (2010) 21:2293–2298

DOI 10.1007/s10856-009-3964-1

Development and characterisation of a collagen

nano-hydroxyapatite composite scaffold for bone tissue
engineering
Gráinne M. Cunniffe • Glenn R. Dickson •

Sonia Partap • Kenneth T. Stanton •

Fergal J. O’Brien

Received: 31 August 2009 / Accepted: 2 December 2009 / Published online: 20 December 2009
Ó Springer Science+Business Media, LLC 2009

Abstract Bone regeneration requires scaffolds that pos- 1 Introduction

sess suitable mechanical and biological properties. This
study sought to develop a novel collagen-nHA biocomposite Tissue Engineering (TE) is an interdisciplinary field that
scaffold via two new methods. Firstly a stable nHA sus- applies the principles of engineering and life sciences
pension was produced and added to a collagen slurry (sus- towards the development of biological substitutes that
pension method), and secondly, porous collagen scaffolds can replace, restore, or improve tissue function [1]. One
were immersed in nHA suspension after freeze-drying approach of tissue engineering is to use 3-dimensional
(immersion method). Significantly stronger constructs were scaffolds to provide a suitable environment to induce tissue
produced using both methods compared to collagen only formation. Ideal scaffolds act as a guide supporting cell
scaffolds, with a high porosity maintained ([98.9%). It was growth and differentiation and ultimately the deposition of
found that Coll-nHA composite scaffolds produced by the regenerated tissue [2].
suspension method were up to 18 times stiffer than the col- In bone tissue engineering, the scaffold should be
lagen control (5.50 ± 1.70 kPa vs. 0.30 ± 0.09 kPa). The biocompatible with osteoconductive and osteoinductive
suspension method was also more reproducible, and the properties. The scaffold allows for cells to attach, prolif-
quantity of nHA incorporated could be varied with greater erate and form extracellular matrix (ECM). It should have
ease than with the immersion technique. In addition, an open and interconnected pore structure (with a porosity
Coll-nHA composites display excellent biological activity, [90%) that allows nutrients to penetrate into the scaffold
demonstrating their potential as bone graft substitutes in in vitro and then vascularisation to occur in vivo [3–5]. It
orthopaedic regenerative medicine. should also degrade at a suitable rate to match the rate of
tissue formation.
This paper focuses on the development of a novel
composite scaffold for bone regeneration using the two
G. M. Cunniffe  G. R. Dickson major constituents of bone; collagen type 1 and hydroxy-
Centre for Cancer Research and Cell Biology, apatite (HA; Ca10(PO4)6(OH)2) nanoparticles (\100 nm).
School of Medicine, Dentistry and Biomedical Sciences,
Collagen is used extensively as a scaffold biomaterial due
Queen’s University Belfast, Belfast, Northern Ireland, UK
to its biocompatible and biodegradable properties [6–10].
K. T. Stanton From an orthopaedic perspective however, collagen scaf-
UCD School of Electrical, Electronic and Mechanical folds are limited by their poor mechanical characteristics
Engineering, University College Dublin, Dublin, Ireland
and for this reason many studies, including research in our
G. M. Cunniffe  S. Partap  F. J. O’Brien (&) lab, have combined collagen with calcium phosphates to
Department of Anatomy, Royal College of Surgeons in Ireland, improve their mechanical properties [11–15]. However,
123 St. Stephen’s Green, Dublin 2, Ireland poor resorbability and brittle constructs are problems that
e-mail: [email protected]
occur when using micron-sized HA particles [16, 17].
S. Partap  F. J. O’Brien Therefore, the focus of this study was to develop a method
Centre for Bioengineering, Trinity College, Dublin, Ireland of incorporating nano-sized HA particles (that have

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recently been optimized in our lab [18]) into highly porous (I-low) and 0.01 M (I-high). The Ca/P ratio for all sus-
collagen scaffolds based on the lyophilization technique pensions was 1.67. All scaffolds were crosslinked and
[19, 20] to produce collagen-nHA (Coll-nHA) biocom- sterilized using a dehydrothermal treatment (0.05 bar at
posite scaffolds with improved resorbability and mechan- 105° for 24 h [19]).
ical characteristics.
2.3 Scaffold characterisation

2 Materials and methods 2.3.1 Mechanical testing

2.1 Nano-hydroxyapatite (nHA) synthesis Uniaxial compression testing was conducted to evaluate the
effect of nHA incorporation on the Young’s modulus of the
The nHA synthesis technique has recently been developed composite scaffolds. All testing was carried out using a
and optimized in our lab [18]. Briefly, nHA was produced mechanical testing machine (Z050, Zwick/Roell, Germany)
by adding a solution of 6 mM sodium phosphate tribasic fitted with a 5 N load cell. Samples were pre-hydrated in
dodecahydrate (Na3PO412H2O), 7.5 mM NaOH (Sigma, phosphate buffered saline (PBS) prior to, and during, test-
Dorset) and 0.1% Darvan 821A (R.T. Vanderbilt, Norwalk, ing. Compressive testing was performed at a strain rate of
CT) dispersant drop wise into an aqueous 10 mM solution 10% per minute. The compressive modulus was calculated
of calcium chloride dihydrate (Fisher Scientific, Pittsburg) from 2 to 5% of the stress–strain curve.
CaCl2. A Ca/P ratio of 1.67 was maintained. The resulting
precipitate (\100 nm) was washed and resuspended using 2.3.2 Fourier Transform Infra-Red Spectroscopy
10 mins of sonication.
Fourier Transform Infra-Red (FTIR) spectra were collected
2.2 Scaffold fabrication to analyse and compare the material characteristics of all
scaffold samples following nHA addition. FTIR analysis
2.2.1 Collagen control scaffolds was carried out using a Spectrum One FTIR (Perkin Elmer,
UK). Scaffolds were finely cut and mixed with potassium
Collagen control scaffolds were produced by freeze-drying bromide before being pressed into a transparent sample.
a collagen slurry (0.5% (w/v) containing type 1 bovine Spectra were collected between wavenumbers 4000 and
collagen (Integra Life Sciences, Plainsboro, NJ) in 0.05 M 400 cm-1.
glacial acetic acid. The slurry was freeze-dried in a stain-
less steel pan by cooling to -40°C at 0.9°C/min, followed 2.3.3 Scanning Electron Microscopy
by a sublimation step for 17 h at 0°C and 200 mtorr
(Advantage EL, Vir-Tis Co., Gardiner NY) [19]. Scanning Electron Microscopy (SEM) was used to examine
the microstructure of the scaffolds. Scaffold samples were
2.2.2 Composite scaffold fabrication cut using a punch and fixed to an adhesive carbon stub.
Imaging was carried out using a TableTop SEM (Hitachi
The Coll-nHA composites were synthesized using two High-Technologies Corp., Japan) operated at 15 kV.
different methods; (1) by adding the nHA particles to the
collagen slurry prior to lyophilization (suspension method), 2.3.4 Porosity
and (2) by soaking collagen scaffolds into a nHA suspen-
sion (immersion method). In the suspension method, nHA Porosity was calculated using the following equations:
particles suspended in water were added into the collagen Porosity ¼ ðqsample =qmaterial Þ  100
slurry during the blending stage, followed by lyophiliza-
tion. Two different concentrations of nHA suspensions where qsample is the density of the sample and qmaterial is the
were added (relative to weight of collagen used), 3.6 g density of the material the scaffold is made from. For
nHA yielding a 100 wt.% scaffold, (S-100) and 18 g nHA the composite scaffolds, qmaterial is worked out based on the
yielding a 500 wt.% scaffold (S-500). densities of collagen and nHA as follows:
In the immersion method, 9.5 mm 9 4 mm collagen qmaterial ¼ mcollagen þ mnHA =Vcollagen þ VnHA
scaffold discs were immersed in a nHA suspension for
4 days before being freeze-dried. The concentration of the where mcollagen and Vcollagen is the mass and volume of
calcium and phosphate solutions used were varied to gen- collagen in the slurry respectively, mnHA and VnHA is the
erate two scaffold variants; calcium concentration 0.001 M mass and volume of nHA in the slurry respectively.

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2.4 In vitro analysis 3 Results

2.4.1 Static culture Collagen-nHA (Coll-nHA) composite scaffolds were suc-

cessfully synthesised by using the suspension and immer-
Scaffold samples (/ 12.7 mm) were seeded with 2 9 106 sion methods. Significant increases in modulus were
MC3T3-E1 cells and cultured in media (DMEM supple- achieved using both methods (Fig. 1), however both
mented with 10% foetal bovine serum (FBS), 2% penicil- methods displayed a concentration effect i.e. higher moduli
lin/streptomycin and 1% L-Glutamine (Sigma-Aldrich were seen when higher concentrations of nHA were
Ireland, Dublin)) to assess the behaviour of the composite incorporated. The S-100 scaffold (100% nHA added via the
scaffold following nHA addition (S-100) compared to the suspension method) showed no significant improvement
collagen only control. versus the collagen control, whereas the S-500 scaffold
showed an 18 fold increase (5.50 ± 1.70 kPa vs. 0.30 ±
2.4.2 DNA quantification 0.09 kPa) compared to it. Scaffolds made via the immer-
sion method also showed improved compressive modulus
A DNA Hoechst 33258 assay (Sigma-Aldrich, Germany) values compared to the collagen control; at lower nHA
was used to evaluate cell number on the scaffolds follow- concentrations a 2.5 fold increase was observed (I-low),
ing 7 days in culture by fluorescently labelling double- and this was further improved (12 fold increase) when
stranded DNA. Constructs were digested and homogenised higher concentrations of nHA were used (I-high). This
in QIAzol lysis reagent (Qiagen Sciences, Maryland 20874, demonstrates the ability to tailor the mechanical properties
USA). Hoechst dye solution was then added to the digested of Coll-nHA scaffolds by varying the amount of nHA
samples and fluorescence was measured (excitation: added using each method.
355 nm, emission: 460 nm) using a fluorescence spectro- Fourier Transform Infra-Red (FTIR) spectra in Fig. 2
photometer (Wallac Victor2, PerkinElmer Life Sciences). show the presence of nHA in the Coll-nHA composite
Readings were converted to cell number using a standard scaffolds made by both methods (S-100 and I-High).
curve. Characteristic peaks for hydroxyapatite are located in the
region of 500–600 cm-1, the asymmetric bending and the
2.4.3 Histological analysis stretching band of the (PO4)3- group is found at
1063 cm-1. In addition, characteristic peaks for collagen
Wax embedded scaffolds were sliced (10 lm) and stained are seen at 2800–2950 cm-1 (C–H stretching), 1652 cm-1
using Haematoxylin and Eosin (H&E) to reveal the dis- (C=O group), and 3420 cm-1 (N–H stretching).
tribution of cells throughout the construct. Figure 3 shows the pore structure of the collagen control
(Fig. 3a) and Coll-nHA composites made by the suspen-
2.5 Statistical analysis sion (Fig. 3b) and immersion techniques (Fig. 3c) using
Scanning Electron Microscopy. The images show an open
All data was analysed for significance (P B 0.05) using and interconnected porous structure with homogeneous
one-way analysis of variance (ANOVA) tests to compare pores for both the collagen control scaffolds and Coll-nHA
group means. Post hoc tests to determine significant dif- composites. All scaffolds were found to be highly porous,
ferences between group means were performed using the retaining porosities above 98.9% (collagen control: 99.5%,
Tukey test. S-100 scaffold: 99.4%, I-high scaffold: 98.9%).

Fig. 1 Compressive strength of

Coll-nHA composite scaffolds.
Both methods show improved
compressive moduli when
compared to the collagen only
control (* and ** denote
significance P \ 0.05)

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Fig. 2 Fourier Transform Infra-

Red (FTIR) spectra of collagen
control scaffold, and Coll-nHA
composites made by the
immersion and suspension
methods (S-100 and I-high)
revealing the presence of nHA
peaks in both composite
scaffolds

Fig. 3 Scanning Electron Microscopy (SEM) images of a collagen control scaffold, b S-100 and c I-high composite scaffolds demonstrating the
highly porous, interconnected structure of the new scaffolds comparable with the collagen control

In vitro analysis was performed to assess the effect of to the collagen control (Fig. 4). Haematoxylin and Eosin
the addition of nHA on biocompatibility. Upon addition of (H&E) stained sections of the collagen control and (S-100)
100% wt nHA via the suspension method (S-100) there was constructs revealed deep penetration of the MC3T3-E1
no significant difference observed in cell number compared cells after 7 days in both constructs (Fig. 5). This verifies
that the addition of nHA to collagen scaffolds does not
have a detrimental effect on cell behaviour.

4 Discussion

The aim of this study was to develop and evaluate different

methods of incorporating resorbable nHA particles
(\100 nm) into collagen scaffolds for use in bone tissue
engineering. Two novel methods were applied to produce
composite scaffolds (a suspension method and an immer-
sion method). The results show that the mechanical prop-
Fig. 4 Graph showing cell number on collagen only control and erties of the composites synthesized by each of the two
Coll-nHA composite scaffolds (S-100) following 7 days in culture.
methods could be tailored by varying the nHA content. The
There is no significant difference in cell number between the two
groups, demonstrating that the high biological activity of the greatest increase in compressive modulus (18 fold vs.
constructs is maintained following nHA addition collagen control) was obtained by adding 500 wt.% nHA

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Fig. 5 Haematoxylin and Eosin stained transverse sections showing cell distribution after 7 days in the a collagen only control scaffold and
b Coll-nHA composite scaffold (S-100) where cells have infiltrated into the centre of both constructs

using the suspension method. This result highlights the the immersed scaffolds were shown to also have a signif-
important mechanical role of the reinforcing ceramic icant level of NaCl present, which is due to the synthesis
material. Previous difficulties have been reported with technique as this was not observed in the suspension
increasing the filler content in composite constructs, often scaffolds. For these reasons, scaffolds produced using the
leading to irregular distribution of the ceramic and a suspension method, (S-100), were selected to investigate
reduction of the mechanical properties of the resultant the biological effect of the addition nHA to the biocom-
scaffold [15, 21]. These problems have been overcome by patible collagen scaffold.
using nano-sized particles and the novel approaches doc- H&E staining showed the widespread distribution of
umented in this study, with up to 500 wt.% nHA being MC3T3-E1 cells throughout the scaffolds. The images
incorporated homogeneously. showed that cells had migrated into the centre of the collagen
The variation of nHA content was more controllable control and Coll-nHA constructs (S-100) by 7 days, while
using the suspension method, and higher quantities could the DNA assay revealed high cell numbers (ca. 2 9 106).
be incorporated. There is an upper limit to the concentra- This confirmed that the incorporation of nHA had no
tion of the nHA suspension used in the immersion tech- detrimental effect on osteoblasts, as scaffolds made by the
nique, and hence to the amount of nHA that can be added suspension method displayed the same biological charac-
in this way. This is due to larger particles forming during teristics as the collagen only control. In addition, an ala-
the synthesis at higher initial calcium and phosphate pre- marBlueÒ assay which measures cell metabolic activity
cursor concentrations, and these aggregated particles can- demonstrated that cells remained viable on both constructs
not penetrate into the porous collagen sponge during the up to day 7 (data not shown). Therefore, the resorbable Coll-
immersion as required [18]. nHA scaffold demonstrated the same high biological activity
FTIR spectroscopy confirmed the incorporation of nHA as the collagen control scaffold, with considerably improved
into the scaffolds via both methods, while SEM demon- mechanical properties, validating its potential for bone tissue
strated that all composites displayed an open and inter- regeneration.
connected pore structure, similar to the collagen only
control scaffold. Porosities were maintained above 98.9%
regardless of which method was used and the amount of 5 Conclusion
nHA added. Higher magnification SEM images showed the
formation of nHA aggregates (1–2 lm) in the scaffold This study has led to the successful development of two
immersed in higher concentration (I-high), which may be novel techniques for the synthesis of resorbable Coll-nHA
due to the long soaking period (4 days). Energy dispersive composite scaffolds with high biological activity. The
X-Ray analysis (EDX) substantiated the FTIR finding, suspension and immersion methods created significantly
confirming the presence of nHA in the Coll-nHA, with stiffer scaffolds with high degrees of porosity (\98.9%).
EDX results also demonstrating that a homogeneous dis- Scaffolds made by the suspension method are more
tribution is achieved throughout the constructs. However, reproducible, less time consuming and the quantity of nHA

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added can be varied with greater ease than those produced 10. Visser R, Arrabal PM, Becerra J, Rinas U, Cifuentes M. The
using the immersion method, establishing them as potential effect of an rhBMP-2 absorbable collagen sponge-targeted sys-
tem on bone formation in vivo. Biomaterials. 2009;30(11):2032.
bone graft substitutes in regenerative medicine. 11. Rodrigues CVM, et al. Characterization of a bovine collagen-
hydroxyapatite composite scaffold for bone tissue engineering.
Acknowledgements We would like to acknowledge the financial Biomaterials. 2003;24(27):4987.
support for this study from Science Foundation Ireland (SFI); 12. Al-Munajjed AA, et al. Development of a biomimetic collagen-
Research Frontiers Programme (06/RFP/ENM012) and President of hydroxyapatite scaffold for bone tissue engineering using a SBF
Ireland Young Researcher Award (04/Yl1/B531). We would also like immersion technique. J Biomed Mater Res B Appl Biomater.
to thank Eilis McGrath, School of Electrical, Electronic and 2009;90B(2):584–91.
Mechanical Engineering, UCD for help with using SEM. 13. Al-Munajjed AA, O’Brien FJ. Influence of a novel calcium-
phosphate coating on the mechanical properties of highly porous
collagen scaffolds for bone repair. J Mech Behav Biomed Mater.
References 2009;2(2):138.
14. Wahl D, Czernuszka J. Collagen-hydroxyapatite composites for
hard tissue repair. Eur Cells Mater. 2006;11:43–56.
1. Langer R, Vacanti JP. Tissue engineering. Science. 1993; 15. Song J-H, Kim H-E, Kim H-W. Electrospun fibrous web of
260(5110):920–6. collagen–apatite precipitated nanocomposite for bone regenera-
2. O’Brien FJ, Harley BA, Waller MA, Yannas IV, Gibson LJ, tion. J Mater Sci Mater Med. 2008;19(8):2925.
Prendergast PJ. The effect of pore size on permeability and cell 16. Heo SJ, et al. Fabrication and characterization of novel nano- and
attachment in collagen scaffolds for tissue engineering. Technol micro-HA/PCL composite scaffolds using a modified rapid pro-
Health Care. 2007;15(1):3–17. totyping process. J Biomed Mater Res A. 2009;89(1):108–16.
3. O’Brien FJ, Harley BA, Yannas IV, Gibson LJ. The effect of pore 17. Wei G, Ma PX. Structure and properties of nano-hydroxyapatite/
size on cell adhesion in collagen-GAG scaffolds. Biomaterials. polymer composite scaffolds for bone tissue engineering. Bio-
2005;26(4):433–41. materials. 2004;25(19):4749.
4. Harley BA, Leung JH, Silva EC, Gibson LJ. Mechanical char- 18. Cunniffe GM, O’Brien FJ, Partap S, Levingstone T, Stanton KT,
acterization of collagen-glycosaminoglycan scaffolds. Acta Bio- Dickson G. The synthesis and characterisation of nanophase
mater. 2007;3(4):463–74. hydroxyapatite using a novel dispersant-aided precipitation
5. Hutmacher DW. Scaffolds in tissue engineering bone and carti- method. J Biomed Mater Res Part A. 2009 (under revision).
lage. Biomaterials. 2000;21(24):2529. 19. Haugh MG, Jaasma MJ, O’Brien FJ. The effect of dehydrother-
6. Tierney C, Jaasma M, O’Brien FJ. Osteoblast activity on colla- mal treatment on the mechanical and structural properties of
gen-GAG scaffolds is affected by collagen and GAG concentra- collagen-GAG scaffolds. J Biomed Mater Res Part A. 2009;
tions. J Biomed Mater Res Part A. 2009;91A(1):92–101. 89A(2):363–9.
7. Harley BA, Kim HD, Zaman MH, Yannas IV, Lauffenburger DA, 20. O’Brien FJ, Harley BA, Yannas IV, Gibson L. Influence of
Gibson LJ. Microarchitecture of three-dimensional scaffolds freezing rate on pore structure in freeze-dried collagen-GAG
influences cell migration behavior via junction interactions. scaffolds. Biomaterials. 2004;25(6):1077–86.
Biophys J. 2008;95(8):4013–24. 21. Juhasz JA, et al. Mechanical properties of glass-ceramic A-W-
8. Sun X-D, Jeng L, Bolliet C, Olsen BR, Spector M. Non-viral polyethylene composites: effect of filler content and particle size.
endostatin plasmid transfection of mesenchymal stem cells via Biomaterials. 2004;25(6):949.
collagen scaffolds. Biomaterials. 2009;30(6):1222.
9. Holladay C, Keeney M, Greiser U, Murphy M, O’Brien T, Pandit
A. A matrix reservoir for improved control of non-viral gene
delivery. J Control Release. 2009;136(3):220.

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