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doi:10.1016/j.bi

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Biomaterials 28 (2007) 2801–2809

www.elsevier.com/locate/biomaterials

Effects of polymer amount and processing conditions on the in vitrobehaviour of hybrid titanium dioxide/polycaprolactone composites

Roberto De Santisa,�, Michelina Cataurob, Lucy Di Silvioc, Luigi Mantoa, Maria G. Rauccia,Luigi Ambrosioa, Luigi Nicolaisd

aInstitute of Composite and Biomedical Materials IMCB-CNR, Piazzale Tecchio 80, Naples 80125, ItalybDepartment of Aerospace and Mechanical Engineering, Second University of Naples, Via Roma 21, Aversa 81031 (CE), Italy

cBiomaterials and Biomimetics Group, King’s College London, Strand, London WC2R 2LS, England, UKdDepartment of Engineering and Materials Production, University of Naples Federico II, Piazzale Tecchio 80, Naples 80125, Italy

Received 19 October 2006; accepted 10 February 2007

Available online 21 February 2007

Abstract

Titanium dioxide (TiO2) and TiO2 glasses containing poly(e-caprolactone) (PCL) up to 24% by weight were obtained by the sol–gel

process. Powder compaction was achieved providing heat and pressure. Properties were evaluated through compression and bending

tests assisted by X-ray micro-computed tomography imaging. The effects of compaction conditions (i.e. temperature, pressure and

duration) on mechanical properties of inorganic/organic composites were investigated. Biocompatibility tests on organic/inorganic

composites were carried out using human cells and the MTT assay to determine viability. Results indicated that the mechanical

properties (i.e. Young’s modulus and maximum strength), in both compression and bending, were a function of the compression

moulding conditions. Highest mechanical properties were measured using a compaction pressure of 1500MPa acting for 90min at a die

temperature of 100 1C. The results, however, also suggest that mechanical properties can be tailored by varying the amount of PCL to

TiO2. Strength and stiffness spanned between the properties of spongy and cortical bone. Young’s modulus in both compression and

bending were higher for PCL amounts of 6%. Instead, higher bending strength values were measured for PCL amounts of 12%. These

weight amounts of PCL also provide higher average density values, thus suggesting that the polymeric phase is effective in toughening

TiO2-based materials. The investigated materials also showed a very good cytocompatibility as indicated by the MTT assay results.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Titanium dioxide; Polycaprolactone; Mechanical properties; Microtomography; MTT assay; Bone

1. Introduction

Ceramics and hybrid dioxide-based material for therepairing of muscle–skeletal tissues are being increasinglyapplied over the last half century [1,2]. Orthopaedic andmaxillo–facial prosthesis provide evidence for the enhancedbiomechanical performance of titanium and its alloyamong metallic prosthetic components [3]. Moreover, thepassivation process, which forms a titanium dioxide (TiO2)layer on the surface, promote the osteointegration cap-ability of the applied permanent prostheses. On the otherhand, TiO2-based bioactive ceramic suggests that bone

e front matter r 2007 Elsevier Ltd. All rights reserved.

omaterials.2007.02.014

ing author. Tel.: +39081 2425936; fax: +39 081 2425932.

ess: rosantis@unina.it (R.D. Santis).

grafting is achieved by supporting the precipitation ofcalcium (Ca) and phosphorus (P) into a structure similar tothe mineral phase of bone. Accordingly, titanium is verypromising to develop biomedical materials and devicesdesigned as hard tissue substitute with improved interfaceproperties [1–4].The sol–gel technique, which is based on the hydrolysis

and polycondensation of metal alkoxides, is a veryattractive technique to synthesize TiO2. The main advan-tage is that the sintering process of this hybrid biomaterialcan be carried out at low temperature (e.g., roomtemperature) [5–8]. Unfortunately, the main drawback ofTiO2 bioceramics is high brittleness [9,10]. Therefore,considerable attention has been given to organic–inorganichybrid composites. A variety of polymers have been

ARTICLE IN PRESSR.D. Santis et al. / Biomaterials 28 (2007) 2801–28092802

investigated to overcome this problem. It has beensuggested that a polymeric phase can be convenientlyadded to bioceramics formulations in order to designcomposites with enhanced biocompatibility [9,11,12].Poly(e-caprolactone) (PCL) is a very attractive polymerto design composite materials with or without covalentbonds with the inorganic phase [13]. Although the mainfeature of this hydrophobic polyester is its bio-degradationcapability, PCL is also remarkable for mechanical proper-ties and easy miscibility with other materials [13,14].

Therefore, TiO2/PCL composites are promising materi-als to manufacture hard tissue substitutes (i.e., prostheticdevices and scaffolds for the muscle–skeletal system). Theaim of this investigation was to evaluate, in vitro, the effectsof processing conditions (i.e., pressure and temperature)and polymer amount on the mechanical properties incompression and bending of TiO2-based materials. Theinvestigated composites ability to perform with an appro-priate host response in a specific application [15], wasassessed using the MTT assay. This indirect in vitro methodallows the evaluation of cell viability and proliferation[16–18], and it is one of the preferred screening tests usedby the National Cancer Institute [19,20].

2. Materials and methods

Hybrid inorganic/organic composites were sintered by means of the

sol–gel process described elsewhere [5]. Reagents (Sigma-Aldrich, Milano,

Italy) comprised titanium butoxide (TBT) as precursor and PCL

(Mw ¼ 65000) stirred in a chloroform, ethanol and water mixture. The

time of gelification was 10min, hence gels were dried in air environment at

50 1C for 24 h to remove the residual solvent. It was previously shown that

Fig. 1. Tools used to prepare compression and bending specimens: (a) comp

opened die; (b) mounted compression specimen mould; (c) compression specim

specimen mould tools; (f) bending specimen, unit bar is cm.

PCL was incorporated into the network by hydrogen bonds between the

carboxylic groups of the polymer and the hydroxyl groups of the inorganic

matrix [5]. TiO2-based glasses were milled in a rotating ball milling

machine. Fine TiO2/PCL powders (diametero0.125mm) were classified

according to the weight amount of PCL. Accordingly, TiO2/PCL 94/6,

88/12 and 76/24 samples distinguished inorganic/organic composite

containing 6%, 12% and 24% by weight of PCL, respectively. TiO2

indicated the control group hybrid obtained via the sol–gel process

without the incorporation of PCL. Powders were heat compressed in

stainless-steel moulds. The effects of compression moulding conditions on

the mechanical properties of the materials were investigated by varying

temperature (between 25 and 250 1C), pressure (between 500 and

1500MPa) and duration of the process (between 25 and 90min).

A hollow mould characterised by a cylindrical die (Figs. 1a and b)

was used to realise compression specimens (Fig. 1c) according to the

geometry recommended by ASTMD-695. Compression was provided

by a force controlled hydraulic actuator of 200 kN (Instron 8800,

Bucks, UK) while heat was generated by a thermal chamber (Instron

3119, Bucks, UK). A prismatic die (Figs. 1d and e) was used to realise

bending specimens (Fig. 1f) according to the ASTMD-790. Heat and

compression to manufacture rectangular cross-section bending specimens

(25� 50� 1mm3) was provided by the hydraulic press Collin P300P

(GmbH, Aichach, Germany).

2.1. Mechanical testing

Mechanical tests were carried out at room temperature using the

electromechanical dynamometer Instron 4204 (Bucks, UK). A load cell of

5 kN was used for compression tests of specimens (diameter of 5mm and

height of 10mm) at a cross-head speed of 1mm/min, while a load cell of

1 kN was used for 3-point bending tests at a speed of 1mm/min. The

dynamometer compliance was considered to calculate compressive

properties of each composite formulation. Mechanical data were acquired

at a rate of 20 pt/s. Young’s modulus and maximum stress values were

derived from stress–strain diagrams, and statistical differences among the

groups were assessed using the one-way ANOVA through the Origin 6.0

(Microcol, Northampton, USA) software.

ression specimens mould tools consisting of piston, cylindrical frame and

en, unit bar is cm; (d) bending specimen mould tools; (e) mounted bending

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Fig. 2. Effect of processing conditions on mechanical properties. (a) Effect

of temperature on the compressive strength of TiO2 specimens sintered at

500MPa. Bars represent the standard deviation (n ¼ 5). (b) Effect of

sintering duration on the Young’s modulus in compression of TiO2

specimens sintered at 100 1C.

R.D. Santis et al. / Biomaterials 28 (2007) 2801–2809 2803

2.2. Morphological analysis

X-ray micro-computed tomography (X-ray mCT) was used to analyse

the structure of TiO2-based materials through the SkyScan 1072

(Aartselaar, Belgium) 3D scanner, operating at 56 kV. Serial bitmap

images of 1024� 1024, each pixel cross-section size being 16mm, were

analysed in the SkyScan’s Tview software. The grey-scale imaging

attributed a pixel value of 100% to whiter pixels and a 0% value to the

darker pixel. Hence, low pixel values also identify regions characterised by

higher density.

2.3. In vitro cell culture and elution materials

The investigated materials were processed in a cylindrical dye in a similar

fashion as the process used to produce bending specimens. Discs with a

diameter of 13mm and a thickness of 2mm were obtained and sterilised in

an autoclave for 20min at 120 1C. A human osteosarcoma cell line was used

(HOS TE 85 EACC No.87070202), and cells were cultured in Dulbecco’s

modified Eagle’s medium (DMEM), supplemented with 10% foetal

calf serum (FCS), 1% non-essential amino acids (MEM), L-ascorbic acid

(150g/ml), 0.02M L-glutamine, 1M HEPES, 100units/ml penicillin and

100mg/ml streptomycin. The cells were expanded prior to use and

maintained in culture until confluent. Elution studies were performed in

DMEM, by adding 2.5ml of solution to each 0.1 g of test material. These

were placed on a rotating mixer for a period of 24 and 72h. The elution

fluid was removed and 100ml pipetted into each well in a sterile 96-well cell

culture plates (Falcon USA) that had been pre-seeded 24h earlier with

1.5� 105/ml HOS TE85 prior to exposure to the eluted supernatant. The

negative (non-toxic) control was DMEM while positive toxic control was

10% ethanol. The plates were further incubated at 37 1C, 5% CO2 and 95%

air humidity for 48h exposure. MTT (5mgml�1 in DMEM without phenol

red) was added to the wells in an amount equivalent to 10% of the culture

medium. Following 4h incubation at 37 1C, the medium was aspirated and

the insoluble formazan produced was dissolved in dimethyl sulfoxide

(DMSO). The plates were gently agitated for 5min to ensure complete

crystal dissolution and optical densities were measured at 570nm,

subtracting background absorbance determined at 620nm. For the live/

dead stain, 100ml of eluant from the sterilized materials, after 24 and 72h

immersion in complete human osteoblast media, was pipetted into a sterile

96-well cell culture plates (Falcon USA), containing 1.5� 104 pre-incubated

HOS cells per well and incubated for 24 and 72h. At each respective time

point, the eluted media was removed and replaced with 100ml of a solution

containing 1ml of phenol red free complete DMEM with 1ml calcein AM

and 1ml ethidium homodimer-1 (EthD-1). Tissue culture plastic and a 10%

ethanol were used as negative and positive controls, respectively. The plates

were incubated under normal cellular conditions for 1 h and observed using

an Olympus IX51 fluorescent microscope. The live cells stained green and

the dead cells red. Calcein AM is a frequently used cell viability stain due to

its superior cell retention and the relative insensitivity of its fluorescence to

pH in the physiological range. Calcein, the hydrolysis product of calcein

AM, is a polyanionic fluorescein derivative that has about six negative

charges and two positive charges at pH 7. EthD-1 was used as a dead-cell

indicator for cell viability and cytotoxicity testing in combination with

calcein AM. The high affinity of EthD-1 permitted the use of very low

concentrations to stain dead cells, thus avoiding the use of large quantities

of the potentially hazardous ethidium bromide or propidium iodide. With

its high affinity for DNA and low membrane permeability, EthD-1 is often

the preferred red-fluorescent dead-cell indicator.

3. Results

Fig. 1 shows the tools used to prepare specimens forcompression and bending tests. Fig. 2a shows the effectof temperature on the maximum compressive stress ofTiO2 sample compressed with a load 10 kN (pressure of500MPa) for 25min. A maximum stress of 30MPa

(73MPa) was obtained for specimens sintered at 100 1C.This compressive strength value was statistically higherthan those measured for samples sintered using othertemperatures (po0.01). A slight increase of the compres-sive strength from 30 to 34MPa was observed as thesintering pressure was increased from 10 kN (500MPa)to 30 kN (1500MPa). The effect of the duration of the

ARTICLE IN PRESSR.D. Santis et al. / Biomaterials 28 (2007) 2801–28092804

compression moulding process on the mechanical proper-ties of TiO2 specimens is depicted in Fig. 2b. Both theYoung’s modulus and the compressive strength increasedas the duration of the sintering process was increased. Theprocess extended for 90min provided values of 3.4GPa(70.5GPa) and 33MPa (73.5MPa) for the Young’smodulus and maximum stress, respectively. These valueswere statistically higher than those measured for sinteringdurations shorter than 75min (po0.01). Non-destructiveimaging through X-ray mCT of radial sections of TiO2/PCL94/6, TiO2/PCL 88/12 and TiO2/PCL 76/24 samples aredepicted in Figs. 3a, b and c, respectively. The markeddecrease of X-ray absorption in the radial directionsuggests that density is higher in the left side of eachimage. This is the region corresponding to the wall of thecompaction die, where large friction forces developedduring compression moulding. Table 1 reports mechanicalproperties in compression of TiO2, TiO2/PCL 94/6, TiO2/PCL 88/12 and TiO2/PCL 76/24 samples compacted at100 1C for 90min using a pressure of 1500MPa. Bendingproperties for samples sintered using the same processingconditions are reported in Table 2. In particular, the 6%

Fig. 3. Non-destructive X-ray mCT imaging of radial sections of organic/inorga

Left side of each image is the peripheral region of each specimen corresponding

all composite samples in the radial direction.

PCL (TiO2/PCL 94/6) provided the highest value forYoung’s modulus in both tension and compression.Mechanical properties of TiO2 and TiO2/PCL 76/24samples (Tables 1 and 2) were statistically lower thanTiO2/PCL 94/6, TiO2/PCL 88/12 samples (po0.01).Amounts of 12% of PCL provided the highest values ofbending strength (Table 2). Table 3 reports the averagedpixel value according to the sample region. Lower valuescorrespond to darker pixels, hence also to the region withhigher density. Higher density is observed close to theperiphery of each sample, thus suggesting that higherpressure developed in this region during compaction. Theminimum and the average pixel values in the peripheryincreased as the PCL amount increased. The opposite trendwas derived for density. Instead, the averaged values in thecentre region, and also on a line going from the peripheryto the centre, suggests that TiO2/PCL 94/6 sample has adensity higher than TiO2 sample. Interestingly, the max-imum pixel value in each investigated regions was almostconstant between samples.The results of MTT assay after 48 h of incubation are

shown in Fig. 4. Optical density values measured for cells

nic samples: (a) TiO2/PCL 94/6; (b) TiO2/PCL 88/12; (c) TiO2/PCL 76/24.

to the wall of the compaction die. A marked density gradient is evident for

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Table 1

Compressive properties of TiO2, TiO2/PCL 94/6, TiO2/PCL 88/12 and TiO2/PCL 76/24 samples

Max. strain (mm/mm %) Max. stress (MPa) Young modulus (GPa)

TiO2 1.0270.18 33.3173.5 3.470.70

TiO2/PCL 94/6 1.1070.21 88.9579.2 9.671.38

TiO2/PCL 88/12 1.0870.21 40.8073.6 5.871.13

TiO2/PCL 76/24 1.1670.20 20.7872.2 2.070.32

Mean values and standard deviations are reported. The Young’s modulus and strength of TiO2/PCL 94/6 are statistically higher than the other samples

(po0.01).

Table 2

Bending properties of TiO2, TiO2/PCL 94/6, TiO2/PCL 88/12 and TiO2/PCL 76/24 samples

Max. strain (mm/mm %) Max. stress (MPa) Young modulus (GPa)

TiO2 0.1170.02 4.9370.50 4.170.6

TiO2/PCL 94/6 0.1070.01 9.3471.00 12.471.4

TiO2/PCL 88/12 0.1770.02 10.2271.10 9.271.3

TiO2/PCL 76/24 0.1770.02 3.9470.45 2.370.4

Mean values and standard deviations are reported. The Young’s modulus and strength of TiO2/PCL 94/6 are statistically higher than the other samples

(po0.01).

Table 3

Averaged pixel values according to the region of each sample

Periphery Centre Periphery to centre

Average % Min/max % Average % Min/max % Average % Min/max %

TiO2 27 9/49 52 48/60 50 10/62

TiO2/PCL 94/6 32 16/52 44 27/65 43 15/70

TiO2/PCL 88/12 37 20/50 54 35/63 49 24/68

TiO2/PCL 76/24 42 30/53 62 50/72 56 32/69

Periphery denotates the region closer to the die wall during compression moulding. The value of 100% corresponds to the whiter pixel.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

negative

TiO 2

TiO 2/PCL94/6

TiO 2/PCL88/12

TiO 2/PCL76/24

positive

o.d

. at

570 n

m

Fig. 4. MTT indirect, elution study: cells exposed for 48 h in 24 and 72 h

eluants of materials sterilized by autoclave (20min at 120 1C).

R.D. Santis et al. / Biomaterials 28 (2007) 2801–2809 2805

exposed to the eluants for 24 and 72 h suggested nosignificant difference between control, TiO2 and TiO2PCL94/6. The lower values measured for TiO2PCL 88/12 and

TiO2PCL 76/24 are probably due to some leacheablesfrom trapped solvent used to prepare the discs. However,the samples with higher amount of PCL show an increasein cell metabolic activity after 72 h exposure time (Fig. 4),and this is consistent with light microscope images (Fig. 5).This indicates negligible leachable toxicity, and normalmetabolic cell activity prevailed. The normal cell viabilitywas restored and this was confirmed by the live–deadcell images reported in Fig. 6. As expected, positivecontrol supernatant resulted in severe cell death. In fact,Figs. 6i and j clearly show the toxic condition for thecells; the cellular response with rounded live cells andlarger number of dead cells suggests that cell death wasapparent.

4. Discussions

Bioactivity features of TiO2/PCL composites are welldocumented [1–9]. Moreover, it has been also shown thatthese materials can incorporate high doses of antibiotics(e.g., sodium ampicillin), thus supplying a controlled drugrelease as the material is applied in the body environment[5]. Therefore, organic/inorganic composites have been

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Fig. 5. Light micrographs of cells incubated for 48 h in the 72 h eluant: (a) negative control tissue culture plastic; (b) positive control, cells incubated in

10% ethanol; (c) TiO2; (d) TiO2PCL (6%wt); (e) TiO2PCL (12%wt); (f) TiO2PCL (24%wt).

R.D. Santis et al. / Biomaterials 28 (2007) 2801–28092806

explored by synthesizing TiO2-based powders containingrelatively high amounts of PCL. Biocompatibility in vitro

tests suggest adequate cellular response for all investigatedcomposites. Similarly to oxide glasses, the main drawback ofTiO2-based composites is the brittleness [10]. By analysingthe effects of processing condition (i.e., temperature,pressure and duration) on the mechanical properties ofTiO2 dioxide sample, the results clearly indicated thatYoung’s modulus and strength, in both compression andbending, can be conveniently modified by selecting theprocess conditions (Fig. 2). The Young’s modulus andstrength of spongy bone mainly depend on density distribu-tion, and they vary in the range of 0.1–1GPa and 1–10MPa,respectively [21,22]. Higher Young’s modulus (10–20GPa)and strength (150–250MPa) are instead measured forcortical bone, and variations are mainly related to osteonorientation [23,24]. Therefore, Young’s modulus andstrength of the sinterd TiO2 dioxide (Fig. 2) are in the rangeof spongy bone properties. As a consequence of brittleness,ultimate strain of TiO2 specimens in bending (Table 2) areabout one order of magnitude lower than bone.

A polymeric phase is often used to design composites inorder to reduce brittlness of TiO2-based materials [9,13].

Results presented in Tables 1 and 2 clearly show that smallamounts of PCL produce an increase of stiffness andstrength, thus toughening the structure. Moreover, theeffect of polymer amount on compressive properties(Table 1) is stronger than the effects related to processingconditions (Fig. 2). Therefore, the use of a polymeric phaseis also a more powerful tool to tailor mechanical propertiesof inorganic/organic composites in order to matchmechanical properties of hard tissues. It is interesting toobserve that high amounts of PCL provide mechanicalproperties lower than the TiO2 sample (po0.01) in bothtension and compression. However, mechanical propertiesof TiO2/PCL 76/24 sample (Tables 1 and 2) are stillremarkable if compared to spongy bone strength andmodulus. Moreover, the higher compliance of this sampleis also supported by higher strain at fracture in bothcompression and bending, thus suggesting a plasticisingeffect of PCL. The sintering of glass powders through heatand compression treatments is a common process to obtainTiO2-based materials characterised by adequate mechan-ical properties [25,26]. Mechanical results of TiO2/PCL94/6 sample, compacted at a temperature of 100 1C for90min at a load of 30 kN, give attractive strength values in

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Fig. 6. Live–dead assay: Olympus IX51 fluorescent microscope. Assay of cells incubated for 48 h in the 72 h eluant; the live cells were stained green and the

dead cells red. (a and b) Negative control tissue culture plastic; (c and d) TiO2; (e and f) TiO2PCL (6%wt); (g and h) TiO2PCL (24%wt); (i and j) positive

control, cells incubated in 10% ethanol.

R.D. Santis et al. / Biomaterials 28 (2007) 2801–2809 2807

both compression and bending (about 80 and 10MPa,respectively), thus suggesting a significant improvement ininteraction between powder grains.

A marked density distribution for each compositesample can be depicted from Fig. 3 and Table 3. Densitywas higher in the region closer to the die wall where higher

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pressure is developed during the compression mouldingprocess. Hence, density gradients are due to a non-uniformpressure distribution caused by die wall friction andinterparticles friction during compression moulding pro-cess. The average pixel values along a line going from theperiphery to the centre of TiO2 94/6 and TiO2/PCL 88/12samples (Table 3) is lower than TiO2. Thus, low amounts ofPCL leads to composites with a porosity fraction lowerthan TiO2 sample, and this is consistent with themechanical performance of TiO2 94/6 and TiO2/PCL 88/12(Tables 1 and 2).

Overall, mechanical properties of the investigatedcomposites are in between those of spongy and corticalbone. These materials can be suitable for several applica-tions related to bone surgery. Bone defects of vertebralbody, long bones and mandible, can be restored bymanufacturing suitable TiO2/PCL inlays through theheat-compression moulding technique described in thisstudy, thus reproducing the bone cavity to be filled; it hasbeen shown that TiO2 enhances the bonding of prostheticdevices to bone [3]. Another interesting application isrelated to bioactive intervertebral disc spacers; fusion ofadjacent vertebrae being promoted by TiO2 [27]. Bycombining bioactive and bioresorbable capabilities, TiO2/PCL composites are also becoming increasingly importantas partially bioresorbable scaffolds for hard tissue en-gineering [11,12].

5. Conclusions

Temperature, pressure and duration of the sinteringprocess of TiO2/PCL composites can be opportunelyselected in order to obtain organic/inorganic compositescharacterised by suitable mechanical and biological proper-ties. PCL is effective in reducing brittleness of TiO2 basedmaterials. Highest mechanical properties (i.e. Young’smodulus and maximum strength) are measured with acompaction pressure of 1500MPa acting for 90min at atemperature of about 100 1C. The amount of PCL furthercontrols the mechanical properties of TiO2/PCL compo-sites. Young’s modulus and strength, in both compressionand bending, are higher for 6% composition. Weightamounts of PCL of 12% provided the highest values ofbending strength. The materials investigated also showed aremarkable biological response.

Acknowledgements

The financial support of CRdC of Regione Campania,Work Package 1C ‘‘Innovazione nelle tecnologie deibiomateriali e ricostruzione dei tessuti’’, is gratefullyacknowledged.

The authors also wish to thank Mr. Rodolfo Morra forthe mechanical testing.

References

[1] Hench LL. Bioceramics: from concept to clinic. J Am Ceram Soc

1991;74:1487–510.

[2] Li P, de Groot K, Kokubo T. Bioactive Ca10(PO4)6(OH)2–TiO2

composite coating prepared by sol–gel process. J Sol–gel Sci Technol

1996;7:27–34.

[3] Kitsugi T, Nakamura T, Oka M, Yan WQ, Goto T, Shibuya T, et al.

Bone bonding behavior of titanium and its alloys when coated with

titanium oxide (TiO2) and titanium silicate (Ti5Si3). J Biomed Mater

Res 1996;32:149–56.

[4] Coreno J, Coreno O. Evaluation of calcium titanate as apatite growth

promoter. J Biomed Mater Res A 2005;75:478–84.

[5] Catauro M, Raucci MG, de Marco D, Ambrosio L. Release kinetics

of ampicillin, characterization and bioactivity of TiO2/PCL hybrid

materials synthesized by sol–gel processing. J Biomed Mater Res A

2006;77:340–50.

[6] Areva S, Paldan H, Peltola T, Narhi T, Jokinen M, Linden M. Use of

sol–gel-derived titania coating for direct soft tissue attachment. J

Biomed Mater Res A 2004;70:169–78.

[7] Chant JM, Harris B, Russell-Floyd R, Cooke RG, Scott VD,

Bleay SM. Mechanical properties and microstructures of

sol–gel derived ceramic–matrix composites. J Mater Sci 1995;30:

2769–84.

[8] Jokinen M, Patsi M, Rahiala H, Peltola T, Ritala M, Rosenholm JB.

Influence of sol and surface properties on in vitro bioactivity of

sol–gel-derived TiO2 and TiO2–SiO2 films deposited by dip-coating

method. J Biomed Mater Res 1998;42:295–302.

[9] Chen QZ, Blaker JJ, Boccaccini AR. Bioactive and mechanically

strong bioglass–poly(D,L-lactic acid) composite coatings on

surgical sutures. J Biomed Mater Res B Appl Biomater 2006;76:

354–63.

[10] Donald IW. Methods for improving the mechanical properties of

oxide glasses. J Mater Sci 1989;24:4177–208.

[11] Pena J, Vallet-Regi M, San Roman J. TiO2–polymer composites for

biomedical applications. J Biomed Mater Res 1997;35:129–34.

[12] Boccaccini AR, Blaker JJ. Bioactive composite materials for

tissue engineering scaffolds. Expert Rev Med Devices 2005;2:

303–17.

[13] Wu CS. In situ polymerization of titanium isopropoxide in

polycaprolactone: properties and characterization of the hybrid

nanocomposites. J Appl Polym Sci 2004;92:1749–57.

[14] Davis PA, Nicolais L, Ambrosio L, Huang SJ. Poly(2-hydroxyethyl-

metacrylate)/poly(caprolactone) semi-interpenetrating network. J Bio

Comp Polym 1988;3:205–18.

[15] Williams DF. Definitions in biomaterials: proceedings of a consensus

conference of the European Society of Biomaterials, 3–5 March,

Chester, UK. In: Williams DF, editor. Progress in biomedical

engineering, vol. 4. Amsterdam: Elsevier; 1987. p. 54.

[16] Kirkpatrick CJ, Dekker A. Quantitative evaluation of cell interaction

with biomaterials in vitro. Adv Biomater 1992;10:31–41.

[17] Kirkpatrick CJ. A critical view of current and proposed methodol-

ogies for biocompatibility testing: cytotoxicity in vitro. Regul Affairs

1992;4:13–32.

[18] Dekker A, Panfil C, Valdor M, Pennartz G, Richter H, Mittermayer

CH, et al. Quantitative methods for in vitro cytotoxicity testing of

biomaterials. Cells Mater 1994;4:101–12.

[19] Carmichael J, deGraff WG, Gazdar AF, Minna JD, Mitchell JB.

Evaluation of a tetrazolium-based semiautomated colorimetric

assay: assessment of chemosensitivity testing. Cancer Res 1987;47:

936–42.

[20] Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ,

Fine DL, et al. Feasibility of drug screening with panels of human

tumor cell lines using a microculture tetrazolium assay. Cancer Res

1988;48:589–601.

[21] Goldstein SA, Wilson DL, Sonstegard DA, Matthews LS. The

mechanical properties of human tibial trabecular bone as a function

of metaphyseal location. J Biomech 1983;12:965–9.

ARTICLE IN PRESSR.D. Santis et al. / Biomaterials 28 (2007) 2801–2809 2809

[22] Keaveny TM, Hayes WC. A 20-year perspective on the mechanical

properties of trabecular bone. J Biomech Eng 1993;115:534–42.

[23] Reilly DT, Burstein AH, Frankel VH. The elastic modulus for bone. J

Biomech 1974;7:271–5.

[24] Bonfield W, Grynpas MD. Anisotropy of the Young’s modulus of

bone. Nature 1977;270:453–4.

[25] Korinth MC, Hero T, Pandorf T, Zell D. Application of a

stand-alone interbody fusion cage based on a novel porous

TiO2/glass ceramic: biomechanical evaluation after implanta-

tion in the sheep cervical spine. Biomed Tech (Berl) 2005;50:

111–8.

[26] Sung YM. Mechanical properties of alfa-cordierite and beta-

spodumene glass-ceramics prepared by sintering and crystallization

heat treatments. Ceram Intern 1997;23:401–8.

[27] Ishizaki K, Nanko M. A hot isostatic process for fabricating porous

materials. J Porous Mater 1995;1:19–27.