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nCorresponding autE-mail address: c

Please cite this aJournal of the Me

Research Paper

Nanomechanical properties of hybrid coatings forbone tissue engineering

Amalia Skarmoutsoua, Georgios Lolasa, Costas A. Charitidisa,n,Maria Chatzinikolaidoub,c, Maria Vamvakakib,c, Maria Farsaric

aSchool of Chemical Engineering, National Technical University of Athens, 9 Heroon Polytechniou St.,Zographos, 15780 Athens, GreecebDepartment of Materials Science and Technology, University of Crete, GreececInstitute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH), Greece

a r t i c l e i n f o

Article history:

Received 6 November 2012

Received in revised form

29 April 2013

Accepted 3 May 2013

Keywords:

Nanomechanical properties

Hybrid coatings

Elastic–plastic behaviour

Wear resistance

Pile-up

Cell adhesion

Hardness correction

nt matter & 2013 Elsevie.1016/j.jmbbm.2013.05.003

hor. Tel.: +30 210 772 404haritidis@chemeng.ntua

rticle as: Skarmoutsou,chanical Behavior of B

a b s t r a c t

Bone tissue engineering has emerged as a promising alternative approach in the treatment

of bone injuries and defects arising from malformation, osteoporosis, and tumours. In this

approach, a temporary scaffold possessing mechanical properties resembling those of

natural bone is needed to serve as a substrate enhancing cell adhesion and growth, and a

physical support to guide the formation of the new bone. In this regard, the scaffold should

be biocompatible, biodegradable, malleable and mechanically strong. Herein, we investi-

gate the mechanical properties of three coatings of different chemical compositions onto

silanized glass substrates; a hybrid material consisting of methacryloxypropyl trimethox-

ysilane and zirconium propoxide, a type of a hybrid organic–inorganic material of the

above containing also 50 mol% 2-(dimethylamino)ethyl methacrylate (DMAEMA) moieties

and a pure organic material, based on PDMAEMA. This study investigates the variations in

the measured hardness and reduced modulus values, wear resistance and plastic

behaviour before and after samples' submersion in cell culture medium. Through this

analysis we aim to explain how hybrid materials behave under applied stresses (pile-up

formations), how water uptake changes this behaviour, and estimate how these materials

will react while interaction with cells in tissue engineering applications. Finally, we report

on the pre-osteoblastic cell adhesion and proliferation on three-dimensional structures of

the hybrid materials within the first hour and up to 7 days in culture. It was evident that

hybrid structure, consisting of 50 mol% organic–inorganic material, reveals good mechan-

ical behaviour, wear resistance and cell adhesion and proliferation, suggesting a possible

candidate in bone tissue engineering.

& 2013 Elsevier Ltd. All rights reserved.

r Ltd. All rights reserved.

6..gr (C.A. Charitidis).

A., et al., Nanomechanical properties of hybrid coatings for bone tissue engineering.iomedical Materials (2013), http://dx.doi.org/10.1016/j.jmbbm.2013.05.003

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1. Introduction

Bone tissue engineering has emerged as a promising alter-native approach to treat bone malfunctions. This approachinvolves the expansion of cells from a small biopsy, followedby the culturing of the cells in temporary three-dimensionalscaffolds to form the new bone tissue. A major challenge inthis approach is the design and fabrication of the scaffolds.Ideally scaffolds should meet several criteria such as (i) thesurface should permit cell adhesion and growth; (ii) thescaffolds should be biocompatible and biodegradable; (iii)the porosity should be high enough in order to provide cell–cell adhesion and cell–scaffold adhesion, extracellular matrixremodelling; and last but not least (iv) the material should bemechanically strong. Research has progressed to fabricatehybrid scaffolds in an effort to maximise the benefits of bothsynthetic and natural materials.

Hybrid materials are defined as composite materials, withorganic and inorganic components (Sanchez et al., 2010), andare characterized as homogenous or heterogenous andphase-seperated (Brinker and Scherer, 1990; Gómez-Romeroand Sanchez, 2004; Klein, 1988; Livage et al., 1988). The sol–gelprocess of hybrid materials fabrication provides many advan-tages in the production of low-cost organic–inorganic materi-als, since low temperature is employed and simpleequipment is required (Li et al., 1995). Additionally, throughthe sol–gel process, the synthesis of hybrid materials, withorganic and inorganic components in the nanoscale andvarying molar ratios, is achieved, owing to the versatility ofthe colloidal state and the use of metallo-organic precursors(Avnir et al., 1984; Corriu and Leclercq, 1996; Corriu, 1998; Loyand Shea, 1995; Sanchez et al., 2005, 2001; Sanchez and Ribot,1994). Direct Laser Writing (DLW) by multiphoton polymer-ization has been employed as a powerful tool for the fabrica-tion of readily-assembled, fully three-dimensional micro- andnano-structures for photonic applications (Juodkazis et al.,2009). In this study, the mechanical response of hybrid coat-ings onto glass substrates, which are proposed for bone tissueengineering applications, is discussed. The mechanical prop-erties of samples as synthesized and after submersion in acell culture medium are investigated via nanoindentation.A very important polymer characteristic, that plays a crucialrole in several applications, such as personal care products(Gourmand and Corpart, 1999), coatings, composite materials,membranes and sensors (Jensen and Hansen, 2001), andbiomedical products (Colombo, 1993; Dong and Hoffman,1991; Kuzma et al., 1996), is the material's moisture uptake.Material's water uptake changes its structure and results invariation of its mechanical properties. However, the totalabsorbed moisture is less dependent on the sample morphol-ogy, since the plasticizing effect of the absorbed water leadsto changes in the shape of the sample (Thijs et al., 2007). Themechanical properties are investigated in respect of theplastic deformation performed under very low applied loads,indicating the variations in their mechanical properties afterwater uptake. The nanoindentation test is a static methodthat can provide information about material's mechanicalbehaviour when it is subjected to sub-micron scale deforma-tion. The method developed by Oliver and Pharr (King, 1987;

Please cite this article as: Skarmoutsou, A., et al., NanomechanicJournal of the Mechanical Behavior of Biomedical Materials (201

Oliver and Pharr, 1992) allows determining the hardness (H)and the reduced modulus (Er) from the nanoindentation load–displacement data. Although, Oliver and Pharr model pro-vides accurate H and Er values for materials that do notperform any pile up around the indents, while measuring softmaterials and polymers with a Berkovich tip, piling up of thematerials is a frequent phenomenon. Consequently, in thisstudy a theoretical correction of H values is presented for thetested biomaterials, since the mechanical properties of thesubstrate affect the behaviour of cells' growth (Baker et al.,2009; Rohman et al., 2007). The evaluation of the elasticproperties of coatings is based on the precise measurementof the penetration depth of an indenter into the coating(Fabes and Oliver, 1990; Loubet et al., 1984). In order tocharacterize the mechanical properties of the coating, theindenter must have a well-defined geometry (Berkovich,1951). Additionally, when measuring the mechanical proper-ties of the coating the substrate effects should be avoided;which is achieved by limiting the investigated penetrationdepths to approximately 10% of the coating thickness. Thiscan be achieved through nanoindentation, namely, it ispossible to evaluate elastic properties (Young's modulus orEr) and the plastic behaviour which is directly related to thecoating's H (Doerner and Nix, 1986) at specific indentationdepths.

2. Materials and methods

2.1. Materials

All chemicals were obtained from Sigma-Aldrich (Germany)and were used as received unless otherwise stated. Thematerial used for the fabrication of the coatings is anorganic–inorganic composite, produced by the addition ofmethacryloxypropyl trimethoxysilane (MAPTMS, 99%) to zir-conium propoxide (ZPO, 70% in propanol). 2-(dimethylamino)ethyl methacrylate (DMAEMA, 499%) was also added whichwas copolymerized with MAPTMS upon photopolymeriza-tion. ZPO and the alkoxysilane groups of MAPTMS served asthe inorganic network forming moieties. 4,4-bis(diethyla-mino) benzophenone (BIS) was used as the photoinitiator.

2.2. Organic coating synthesis

The organic PDMAEMA film was prepared by spin coating aPDMAEMA solution in toluene onto purified glass slides. Thesolution was spin coated four times to prepare a thickpolymer film. Each layer was spin coated at 1000 rpm for30 s before the next layer was added on top.

2.3. Hybrid coatings synthesis

MAPTMS is first hydrolysed using HCl solution (0.1 M) at a1:0.1 ratio. After 5 min, the ZPO is slowly added to thehydrolysed MAPTMS at a 3:7 ZPO:MAPTMS molar ratio. Afterstirring for 15 min, DMAEMA is added at a DMAEMA/MAPTMSmolar ratio 5:5. Following another 30 min, water is added tothe mixture at a 2.5:5 MAPTMS:H2O molar ratio. Finally, thephotoinitiator, at a 1% w/w concentration with respect to

al properties of hybrid coatings for bone tissue engineering.3), http://dx.doi.org/10.1016/j.jmbbm.2013.05.003

Table 1 – Synthesised samples' composition.

Name Composition Thermalprocessing

Coatingthickness (nm)

1 Glass substrate 100 μm glass substrate – –

2 Organic coating (�500 nm) PDMAEMA – 20070.43 Hybrid 1 coating (�6.3 μm) MAPTMS+ZrO2 T¼50 1C,

t¼10 min630072

4 Hybrid 2 coating (50% organic–50%inorganic) (�2.4 μm)

Organic content: PDMAEMA+inorganic content:MAPTMS+ZrO2

T¼50 1C,t¼10 min

236572

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photopolymerizable methacrylate moieties is added to themixture. After stirring further, for 15 min, the composite isfiltered using a 0.22 μm syringe filter. A hybrid materialcomprising only MAPTMS and ZPO in the absence ofDMAEMA is also prepared following a procedure similar tothat described above.

The different coatings are prepared by drop-casting orspin-coating the aforementioned solutions, onto 100 μm-thick glass substrates, and they are dried on a hotplate at50 1C for 10 min before the photopolymerization (Table 1).The heating process leads to the condensation of the alkoxidegroups and the formation of the inorganic matrix. Next, themethacrylate moieties are polymerized using a UV lamp,operating at 248 nm, resulting in the formation of irreversibleand fully saturated aliphatic C–C covalent bonds that furtherincrease the connectivity of the material. Finally, the samplesare developed for 30 min in a 50:50 solution of n-propanol:isopropanol, and are further rinsed with isopropanol. Thesynthesis of the tested samples is analytically presented in aprevious work (Terzaki et al., 2013).

2.4. Fabrication of hybrid 2 material structures bydirect fs laser writing

The experimental set-up employed for 3D fabrication of blockand mesh structures used in the present work has beenpreviously described (Sakellari et al., 2012; Melissinaki et al.,2011). A Ti:Sapphire femtosecond laser (Femtolasers Fusion,800 nm, 75 MHz, o20 fs) is focused into the photopolymeriz-able composite using a microscope objective lens (20� , N.A.¼0.65 and 40� N.A.¼0.95, Zeiss, Plan Apochromat). Sam-ple movement is achieved using piezoelectric and linearstages, for fine and step movement, respectively (PhysikInstrumente GmbH, Germany). The whole DLW set-up iscomputer-controlled using the 3DPoli1 software. The averagepower used for the fabrication of the high-resolution struc-tures is 80 mW, measured before the objective, while theaverage transmission is 20%. The scanning speed is alwaysset at 2 mm/s.

2.5. Ellipsometry

A variable angle spectroscopic ellipsometer (model VASE,J.A. Woollam Co., Inc.) is used to determine the thickness andthe refractive index of the polymeric and hybrid films.The measurements are performed at three different angles

13DPoli@gmail.com.

Please cite this article as: Skarmoutsou, A., et al., NanomechanicJournal of the Mechanical Behavior of Biomedical Materials (201

of incidence 651, 701 and 751 in the wavelength range 450–1200 nm. Results on coatings thickness are presented inTable 1.

2.6. Nanoindentation tests

The indentation tests are performed at a Hysitron TriboLabs

nanomechanical test instrument. Indentation tests havebeen conducted using a two-dimensional force–displacementtransducer. The transducer has the ability to perform loads inthe range of 1–10,000 μN with a high load resolution of 1 nN,while the maximum penetration depth that can be recordedis 3000 nm (3 μm) with a resolution of 0.04 nm. A three-sidedpyramidal Berkovich diamond indenter (120 nm radius ofcurvature) is used for the measurement of H and Er values.The calibration process is conducted on fused silica, (prior toany experimental procedure) which is a standard material(Bei et al., 2005), in order to calculate the tip radius (tip areafunction). Experiments are performed in a clean area envir-onment with ∼45% humidity and 23 1C ambient temperature(Charitidis, 2010). Based on the half-space elastic deformationtheory, H and Er values can be extracted from the experi-mental data (load–displacement curves) using the Oliver andPharr method (King, 1987; Oliver and Pharr, 1992), where thederived expressions for calculating the elastic modulus fromindentation experiments are based on Sneddon's elastic contacttheory (Sneddon, 1948).

Indentation tests are performed on 100 μm silanized glasssubstrates, organic and hybrid coatings. Moreover, the hybridcoatings were submerged for 2 h in a cell culture medium(MEM alpha modification, pH 7.4, composition: amino acids,vitamins, sugars, inorganic salts, 1% foetal bovine serum) at24 1C. After submersion, samples are rinsed with H2O andtheir nanomechanical properties and wear resistance aremeasured. Tests are performed at several applied loads,ranging from 2 to 6000 μN following a trapezoidal loadfunction, for all the samples mentioned above, with loadingand unloading time fixed at 10 s each. All coatings had beentested with different holding time segments (10, 5 and 1 s).For the hybrid coatings no difference in the measured valueswas observed, since no creep phenomenon or nose effect atthe unloading curve was observed; consequently in this workwe present the obtained values of only 1 s hold time. How-ever, creep at the maximum applied load, while testing theorganic coating, was observed. As a result, the performedtests for the organic coating have loading and unloadingsegments of 2 s each, and hold time of 10 s, in order toeliminate creep phenomenon and avoid the nose effect in the

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upper part of the unloading curve. Each experimental resultpresented here is an average of 8–10 tests, with the corre-sponding error bars. Neighbouring indentation sites abstainmore than 6 μm, in order to avoid the lateral size of theplastic zone around the largest indents and exclude themutual influence of individual indents (Rabkin et al., 2010).The instrument, employed in this study, is also equippedwith a Scanning Probe Microscope (SPM), in which the probetip is moved in a raster scan pattern across sample surfaceusing a three-axis piezo positioner.

2.7. Cell culture

Early passages of the pre-osteoblastic cell line MC3T3-E1 ofmurine calvaria origin are grown in cell culture flasks usingalpha-MEM, supplemented with glutamine (2 mM), penicillin(50 IU/mL), streptomycin (50 g/mL) and 10 vol% foetal bovineserum (FBS) in a humidified atmosphere and 5% CO2 at 37 1Cin a cell incubator (Thermo Scientific). Confluent cells arewashed with phosphate buffer saline (PBS) and passagedafter trypsination (0.25% trypsin in 1 mM EDTA), seeded at80% confluence and cultured before the next passage(Chatzinikolaidou et al., 2010). For cell adhesion experimentson the composite material-coated specimens, cells are cul-tured in alpha-MEM with only 1 vol% FBS, in order to reducethe effect of serum on cell attachment and growth. Cellculture media and reagents are purchased by Invitrogen.

2.8. Scanning electron microscopy

2�104 cells in alpha-MEM with 1% FBS were seeded on the 3Dscaffolds consisting of the material hybrid 2 (50 mol%MAPTMS+ZPO+50 mol% DMAEMA) and were placed in thecell culture incubator at 37 1C for 2 h. Specimens were thenremoved from the incubator and rinsed three times with PBS,fixed with 2% paraformaldehyde for 1 h, post-fixed with 1%osmium tetroxide, and dehydrated in increasing concentra-tions (from 30% to 100%) of ethanol. The specimens werethen dried in a critical point drier (Baltec CPD 030), sputter-coated with a 15 nm thick layer of gold (Baltec SCD 050) andobserved under a scanning electron microscope (JEOL JSM-6390 LV) at an accelerating voltage of 15 kV.

3. Results and discussion

3.1. Nanoindentation results

The glass substrate reveals elastoplastic behaviour at allapplied loads, and the measured H and Er values are0.7570.06 GPa and 1.7470.53 GPa, respectively, for the bulksubstrate. Fig. 1 presents load–unload curves of the synthe-sized coatings before and after submersion of the samples inthe cell culture medium at ∼100 μN (inset graphs) and at∼4000 μN. Hybrid samples are submerged in 12 mL of cellculture medium at 24 1C, with the exception of the organiccoating, because PDMAEMA is soluble in water and thus, thepolymer film would be solubilized in the medium (Vamvakakiet al., 2001). It is observed that the organic coating (Fig. 1a) isthe softest material of all tested samples, revealing high

Please cite this article as: Skarmoutsou, A., et al., NanomechanicJournal of the Mechanical Behavior of Biomedical Materials (201

indentation depths at the lowest applied loads. In Fig. 1a, aload–unload curve at 10% of the organic coating thickness ispresented. The hold time at the maximum load has beenextracted. The hybrid 1 coating (Fig. 1b) exhibits elasticbehaviour at low applied loads and elastoplastic behaviourat higher applied loads before submersion in the medium.After submersion into the cell culture medium, softening andmore plastic behaviour of the hybrid 1 coating is observed,which is attributed to the non-condensed –OH groups influ-ence and possible H2O adsorption. The remained non-condensed –OH groups indicate an incomplete formation ofthe inorganic network, which reorient towards the surface ofthe hybrid film after submersion in the aqueous medium(Soppera et al., 2005). The hybrid 2 coating reveals plasticbehaviour at low penetration depths and elastoplastic beha-viour at higher depths. The plastic behaviour observed at thefirst surface layers can be attributed to the strong influence ofthe presence of ZPO on the surface properties of the hybrid 2coating (Soppera et al., 2005). The composition of that coat-ing, which contains a significant content of organic matter,affects its elastic properties and its behaviour resembles thatof a polymer (Etienne-Calas et al., 2004). However, the hybrid2 coating (Fig. 1c) reveals hardening and higher resistance toall applied loads compared to hybrid 1 coating (Fig. 1b).Hardening of hybrid 2 coating compared to hybrid 1 aftersubmersion is attributed to the higher conversion of thecondensation reaction. This is due to the presence of theliquid DMAEMA monomer, which reduces the viscosity of thehybrid material in the spin-coated film, allowing a higherflexibility of the inorganic network formed during the con-densation reaction upon heating. After submersion in the cellculture medium, all samples reveal discontinuities in theirloading curve, which are attributed to higher interactionsbetween the tip and the surface of the coating.

Using the Oliver and Pharr model the H and Er values forall coatings before and after submersion in the cell culturemedium are calculated and presented in Fig. 2. Fig. 2a pre-sents the H values for all tested samples before and aftersubmersion. It is observed that the organic and hybrid 1coating exhibit the lowest and the highest H value, respec-tively. The hybrid 2 coating present H values between thosemeasured for the organic and hybrid 1 coatings. The H valuespresented in Fig. 2a for the hybrid 1 and 2 coatings are closeto those reported in literature (Bautista et al., 2011; Etienne-Calas et al., 2004). The difference in the H and Er valuesobserved for the hybrid 1 coating is attributed to the absenceof ZPO in the coating mentioned in the literature (Bautistaet al., 2011). Fig. 2b shows the Er values of the tested samples.It is observed that the hybrid 1 and 2 coatings beforesubmersion exhibit similar Er values; the lowest value isfound for the organic coating. The difference in the Er value,measured for the unsubmerged hybrid 1 coating compared tosilica (69 GPa), is attributed to the effect of the organic partwhich leads to a decrease in the inorganic reticulation andconsequently, an increase in network flexibility (Etienne-Calas et al., 2004). Mechanical properties of hybrid materialsare greatly influenced by the presence of organic groups,because they decrease the inorganic network connectivity(Etienne et al., 1998) and form softer regions in the hybridmaterials.

al properties of hybrid coatings for bone tissue engineering.3), http://dx.doi.org/10.1016/j.jmbbm.2013.05.003

Fig. 2 – (a) Hardness (H) and (b) reduced modulus (Er) values at a 10% of the coating thickness.

Fig. 1 – Load–unload curves at (a) ∼10% of the organic coating thickness, (b, c) ∼4000 μN applied load for hybrid 1 and 2 coatings.In the inset of each graph, load–unload curves at ∼100 μN applied load are presented.

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Please cite this article as: Skarmoutsou, A., et al., Nanomechanical properties of hybrid coatings for bone tissue engineering.Journal of the Mechanical Behavior of Biomedical Materials (2013), http://dx.doi.org/10.1016/j.jmbbm.2013.05.003

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After submersion of the samples in the cell culturemedium, hybrid 2 coating presents an increase in H and Ervalues, as it was expected from the load–unload curvespresented in Fig. 1c. This increase cannot be attributed to adecrease in the coating thickness after its submersion in thecell culture medium, since the samples were rinsed thor-oughly in alcohol and water before the measurements, and itis possible evidence of H2O absorption. The decrease in the Hand Er values for the hybrid 1 coating can be attributed to theinteraction of the cell culture medium with remaining –OHgroups, due to an incomplete reaction of the inorganic net-work formation (Brinker and Scherer, 1990). This interactionwill probably cause differences in the network stability of theMAPTMS/ZPO coating (Varma et al., 2010).

Fig. 3 presents SPM images of the tested areas for allsynthesized coatings before and after submersion in themedium. No cracks around or underneath the indents areobserved for any of the coatings, before and after submersionof the samples in the cell culture medium revealing goodadhesion to the substrate for the organic coating and lowbrittle behaviour of the inorganic and hybrid coatings(Fournier et al., 2007). Crack initiation is also correlated withpop-in observations on the loading curve (Alvarado-Riveraet al., 2011; Fournier et al., 2007). As a result, since thesamples do not exhibit any pop-in phenomenon, it is evidentthat cracks are not formed around the indents. However, pile-ups around the performed indents are observed for all coat-ings, apart from the hybrid 1 coating. Pile-ups are denotedwith circles in Fig. 3. The pile-up phenomenon is oftenexcessive in situations of a soft coating onto a hard substrate,

Fig. 3 – SPM images of tested areas for the glass substrate, the orcircles around the performed indents.

Please cite this article as: Skarmoutsou, A., et al., NanomechanicJournal of the Mechanical Behavior of Biomedical Materials (201

because the indenter often penetrates well into the substrate,or reaches the coating-substrate interface. The pile-up phe-nomenon is not preferred for the exact measurement of thematerials' hardness. When pile-up occurs, the real contactarea of the indenter is larger than the one calculated by theOliver and Pharr model. Consequently, the Oliver and Pharrmodel calculates lower contact depth, hc, values, resulting inthe overestimation of the H values (Field et al., 2003; Saha andNix, 2002, 2001). It should be noted that the organic coatingexhibits pile-ups with higher plastic deformation of the piled-up lobes. Furthermore, it is observed that after submersion inthe cell culture medium, the surface of the hybrid coatings issmoother and the observed indents are larger in width.Additionally, the observed pile-ups after submersion in thecell culture medium for hybrid 2 coating are lower in height,at the same applied loads.

3.2. Elastic–plastic behaviour

The elastic behaviour of the materials can describe theirsurface properties. The H/Er ratio describes the “elastic strainto failure” (Halling, 1982; Matthews, 1984; Oberle, 1951) of amaterial; namely, the wear resistance of a surface can beexplained through the plasticity index (Oberle, 1951). Fig. 4apresents the obtained H/Er values of the tested samples atvarious penetration depths, expressed as percentage coatingthickness. It is observed that the H/Er ratio of the organiccoating is the lowest of all samples and remains constant atall indentation depths, i.e. all applied loads. On the contrary,the ratio for the hybrid samples increases with increasing

ganic and hybrid 1 and 2 coatings. Pile-ups are denoted with

al properties of hybrid coatings for bone tissue engineering.3), http://dx.doi.org/10.1016/j.jmbbm.2013.05.003

Fig. 4 – (a) H/Er and (b) H3/Er2 values calculated at different coatings thickness (%) for all tested samples.

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indentation depth, reaching a steady value. Hybrid 1 coatingreveals higher H/Er values than hybrid 2 coating, indicatingbetter wear resistance in applied loads. However, the H/Ervalues of hybrid 1 coating present higher decrease compareto hybrid 2 coating, after submersion to the cell culturemedium, indicating that hybrid 1 coating is greater affectedby H2O absorption. It is also observed that the transition fromthe elastic area to the plastic deformation of the tested hybridmaterials (before and after submersion) occurs at the 10% ofcoatings thickness, revealing the substrate effect on the hybridcoatings.

Additionally, the elastic behaviour of the coatings wasanalysed based on a contact analysis for solids of revolution(Oliver and Pharr, 1992), using the Tresca yield criterion andTabor's relation (Tabor, 1951), according to which, the load atthe onset of yielding is proportional to H3/Er

2 (Oliver andPharr, 1992). This ratio is indicative of the material's elasti-city, i.e. high (low) values of H3/Er

2 reflect a predominantlyelastic (plastic) behaviour (Charitidis et al., 1999; Durand-Drouhin et al., 2002; Johnson, 1985; Lungscheider et al., 1998).Fig. 4b presents the H3/Er

2 values of the tested samples atvarious penetration depths. It is observed that the H3/Er

2 ratiopresents approximately the same behaviour as the H/Er ratiofor all samples. The lowest values are calculated for theorganic coating, revealing plastic behaviour and low adhesionof the coating with the glass substrate. Hybrid coatings revealelastic behaviour, present sufficient adhesion with the sub-strate and more plastic behaviour after submersion in the cellculture medium. However, hybrid 1coating after submersionin the cell culture medium present enormous decrease in theH3/Er

2 ratio, revealing that when this synthesis is subjected tohigh humidity lacks of elastic properties compared to hybrid2 coating, due to H2O absorption.

The low wear resistance values of the hybrid 2 coatingafter submersion in the cell culture medium (Fig. 4a), inconjunction with its sufficient elastic behaviour (Fig. 4b),compared to the decrease in the H3/Er

2 ratio calculated forhybrid 1 coating after submersion, suggest that this hybridcoating could provide a suitable substrate for cell adhesion.Considering also the low wear resistance, it is indicated that

Please cite this article as: Skarmoutsou, A., et al., NanomechanicJournal of the Mechanical Behavior of Biomedical Materials (201

the adherence of cells onto a coating of the synthesis ofhybrid 2 coating will be preferable. Furthermore, no delami-nation or extensive plastic deformation is observed, revealingsuccessful coating adherence. Data on cell morphology andproliferation on hybrid 2 structures are presented below.

3.3. Cell adhesion on hybrid 2 structures

Scanning electron microscopy images (Fig. 5) demonstratepre-osteoblastic cells that adhere on hybrid 2 structures andextend protrusions in the materials structures. Figs. 5a and bindicate a strong initial attachment of cells adhered after 2 hon a 3D fabricated mesh scaffold with bar distances of 20 μm.Cells spread out and their flattened morphology signals goodadhesion within the first 2 h after seeding on the material.After 24 h cells present a distinct pseudopodia formation thatenhances their spread morphology embracing the structuresof the mesh scaffolds as depicted in Fig. 5c and d. Cells grownfor 3 and 7 days on the hybrid 2 block structures (Fig. 5e and f)demonstrate a densely-packed cell sheet, reflecting a prolif-eration increase within the mentioned time periods andvalidating the biocompatibility of the hybrid 2 material.

3.4. Plasticity observations

The % Plasticity values of the samples at different applied loadswere calculated by integrating the areas under the loading andthe unloading curves using the following equation:

% Plasticity¼ Etotal−Eunloading

Etotal100 ð1Þ

In Fig. 6a and b, the % Plasticity values of all samples arepresented as a function of applied load. It is observed that the% Plasticity values for all samples increase with increasingapplied loads, reaching a maximum. Further increase in loadleads to a small decrease and finally, a constant % Plasticityvalue for all samples. Additionally, in Fig. 6c and d compar-ison of the plastic deformation at the near surface and at 10%of the coating thickness, respectively, is presented for all

al properties of hybrid coatings for bone tissue engineering.3), http://dx.doi.org/10.1016/j.jmbbm.2013.05.003

Fig. 5 – Scanning electron microscopy images of MC3T3-E1 pre-osteoblastic cells adhered on various 3D structures of the hybrid2 material (50 mol% MAPTMS+ZPO+50 mol% DMAEMA) at (a,b) 2 h, (c,d) 24 h, and proliferated (e) 72 h and (f) 7 days post-seeding. Cells adhere on the mesh scaffolds and extend protrusions in the material within the first 2 h, and proliferate forming adense cell sheet after 3 and 7 days which completely covers the block structures.

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samples. It is observed that the highest values are attributedto the organic coating. In addition, plasticity of the hybridsamples at the near surface (Fig. 6c) is found to be highercompared to the 10% coating thickness (Fig. 6d), indicatingthat thermal treatment during the fabrication process inducesurface hardening, leading to the plastic behaviour. Ferchichiet al. reported that during thermal treatment shrinkage dueto the condensation of the siloxane bonds is involved, whichleads to higher residual stress values (Ferchichi et al., 2009).However, as the indenter further penetrates into the samples'structure, elastoplastic behaviour (viscoelastic) is observed.Adequately, the submerged hybrid 1 and 2 coatings at thenear surface present higher % Plasticity values, indicating amore plastic behaviour, which was also evident in the SPMimages presented in Fig. 3. However, after submersion in thecell culture medium hybrid 2 coating reveals lower increasein % Plasticity values at the near surface compared to hybrid 1coating. This indicates that when hybrid 2 coating interactswith cells it will not deform significantly, thus changing thesurface where the cells are attached.

3.5. Pile-up/sink-in deformation

The contact area is influenced by the formation of pile-upsand sink-ins during the indentation process. Accurate mea-surement of the indentation contact area requires the

Please cite this article as: Skarmoutsou, A., et al., NanomechanicJournal of the Mechanical Behavior of Biomedical Materials (201

calculation of the pile-up or sink-in deformations. The pre-sence of creep (time/rate dependent property of materials)during nanoindentation has an effect on pile-up, whichresults in incorrect measurement of the material properties(Fischer-Cripps, 2004). More specifically, the contact area issignificantly underestimated when the indentation techni-ques are applied to soft materials that exhibit extensivepiling-up, resulting in overestimation of materials' hardnessand elastic modulus (Bolshakov et al., 1996; Lim et al., 1999;McElhaney et al., 1998; Tsui et al., 1996). It has been pre-viously reported that materials' behaviour, i.e. pile-up or sink-in, mainly depends on the ratio of the yield stress sy to theYoung's modulus E and the strain-hardening exponent naccording to the geometry of the indenter (Cheng and Cheng,1998; Mata et al., 2002; Xu and Rowcliffe, 2002). It was necessaryto express experimental parameters directly determined byload–unload curves, which are obtained by the indentationtechnique. Consequently, there are some ratios that can char-acterize the pile-up or sink-in behaviour of the materials. Thefirst ratio is that of the elastic recovery depth, hf, or the residualdepth, heff, to the total penetration depth, hmax (Bolshakov et al.,1996; Giannakopoulos and Suresh, 1999; Xu and Rowcliffe, 2002).It is suggested that when hf/hmaxo0.125 (or heff/hmax40.875) pile-up occurs (Giannakopoulos and Suresh, 1999). The second ratioindicating the formation of pile-up or sink-in is that of contactdepth, hc, to the total penetration depth, hmax (Biwa and

al properties of hybrid coatings for bone tissue engineering.3), http://dx.doi.org/10.1016/j.jmbbm.2013.05.003

Fig. 6 – % Plasticity values evolution as a function of applied load for the (a) organic coating and (b) rest of the samples. (c), (d).Plastic deformation comparative graphs for all samples at the near surface and at 10% coating's thickness, respectively.

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Storåkers, 1995; Cheng and Cheng, 1998; Hill et al., 1989). Pile-ups are observed when hc/hmax values exceed 0.7–0.88 (Khanet al., 2010). In this study an analysis based on hc/hmax ispresented, although, the aforementioned ratios are widelyused for the characterization of hard materials, i.e. metals andceramics.

In Fig. 7a and b, the ratio hc/hmax is plotted as a function ofhmax and normalised hardness H/Er, respectively, for allsamples. In Fig. 7a, it is observed that for the soft materialsstudied herein, the hc/hmax40.5 value indicate materials thatpile-up, with the exception of the submerged hybrid 1 coat-ing. In Fig. 3 it was already presented that all samples, apartfrom the unsubmerged and submerged hybrid 1 coating,exhibit pile-ups. Consequently, it is essential to carefullyevaluate the hc/hmax ratio in conjunction with the SPMimages, when applied to soft materials, in order to reportthe hc/hmax ratio threshold for piling-up. Generally, it is shownthat high values of H/Er, i.e. values characterizing hard

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materials, indicate sink-in, regardless of work hardeningand strain rate sensitivity; whereas low H/Er values, i.e. softmaterials, indicate materials' pile-up and depends on thedegree of work hardening (Elmustafa, 2007). When H/Ervalues are high, higher stresses are expected, and high stressconcentrations are developed towards the indenter tip. Onthe other hand, for low H/Er values, the stresses are lower andare distributed more evenly across the cross-section of thematerial (Gleiter, 2000). It is reported that when the hc/hmvalue approaches unity for small H/Er ratios, deformation isdominated by pile-up (Hill et al., 1989). Additionally, whenhc/hm-0, for high H/Er values elastic deformation and sink-inphenomenon is dominant, as prescribed by the Hertziancontact mechanics (Cheng and Cheng, 1998).

However, in Fig. 7b it is presented that the aforementionedobservations are not in conjunction with the experimentalcalculations. The soft materials, i.e. polymer and polymer-like (hybrid) materials, studied herein, do not reveal sink-in

al properties of hybrid coatings for bone tissue engineering.3), http://dx.doi.org/10.1016/j.jmbbm.2013.05.003

Fig. 7 – Experimental pile-up or sink-in expression hc/hmax as a function of (a) hmax and (b) H/Er ratio. (c) A comparative graph ofthe hc/hmax and pile-up height values at 10% coating thickness.

Fig. 8 – Indentation profiles of the organic coating, indicatingpile-up features.

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when the H/Er value increases. Although, in Fig. 7b, adecrease in the hc/hmax values is observed with the increaseof the H/Er values, this does not denote sink-in, since, asalready discussed above, samples with hc/hmax40.5 exhibitpile-ups. This conclusion is clearly observed in Fig. 7c, whereit is shown that samples with hc/hmax40.55 reveal pile-ups,and the values of the pile-ups height are provided. Pile-upheights were measured through indentation analysis of theSPM images. The above analysis has been mostly conductedin the literature in mathematical simulated materials orcrystalline materials. It has been reported that changes inthe Poisson's ratio or strain rates slightly deviate from thepile-up or sink-in observations (Elmustafa, 2007); however,the crystalline order effect is not yet well studied/analysed.Additionally, higher pile-up heights are observed for theorganic coating, namely, the one that presents the moreviscous behaviour due to the effect of DMAEMA. However, itshould be noted that the pile-up height measured for theorganic coating is also affected by the different holdingtime (10 s), due to the presence of creep upon indentation(Rar et al., 2005).

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3.6. Pile-up, indent analysis and hardness corrections

Fig. 8 presents the indentation profiles of the organic coating,for the nanoindentation test performed at ∼10% of the

al properties of hybrid coatings for bone tissue engineering.3), http://dx.doi.org/10.1016/j.jmbbm.2013.05.003

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coating thickness, whereas the hc/hmax and H/Er ratios are alsodisplayed. In Fig. 8 the H values obtained by three differentmethods (a new geometrical calculation of pile-up area, andtwo models previously reported in the literature (Kese et al.,2005; Zhou et al., 2008)) are compared with the H valueobtained from the nanoindentation test performed in thiswork. The models by Kese et al. (2005) and Zhou et al. (2008)proposed are based on the triangular impression of theindenter on the samples surface. However, they developeda method using the geometrical features of a perfect Berko-vich indenter, in order to avoid the direct measurement viaoptical analysis (AFM/SPM/SEM) of the indentation impres-sion and pile-up around the indent (Kese et al., 2005; Zhouet al., 2008). Kese et al. (2005) calculated the pile-up contactarea by assuming that the pile-up is a semi-ellipse of a majoraxis b and minor axis ai. Axis b denotes the length of thetriangle side, imprinted on the sample surface, due to theBerkovich indenter, and depends on the applied load (or hc).Axis ai is the pile-up contact width (indicated in Fig. 8),namely, it is referred to the distance of the three pile-uplobes occurring after indentation (Kese et al., 2005). They alsoassumed that ai distances are not equal (Kese et al., 2005).Kese et al. proposed that the impression of the Berkovichindenter on the surface of a sample is an equilateral triangleof side b, and the area is given by

Aeq ¼tan 601

4b2 ¼

ffiffiffi3

p

4b2 ¼ 0:433b2 ð2Þ

Then they assumed that Aeq equals to Ac (simplification ofAðhcÞ ¼ 24;5h2 þ a1hþ a1=2h

1=2 þ :::þ a1=16h1=16 which defines

the area function for a perfect Berkovich indenter), in orderto correlate b with hc, and they resulted that

b¼ hc

ffiffiffiffiffiffiffiffiffiffiffiffi24:50:433

r¼ 7:531hc ð3Þ

The contact area of a semi-elliptical pile-up is defined asπ4(bai). Therefore, the total contact area of the pile-ups is

APU ¼ πb4∑ai -

Eq: ð2ÞAPU ¼ 5:915hc∑ai ð4Þ

The second model proposed by Zhou et al. (2008) differscompared to the aforementioned model in the calculation ofthe pile-up contact area, where they assume that the pile-upis an arc, and the arc's centre is located at the opposite cornerof the triangular impression of the indent (Zhou et al., 2008).Therefore, the contact area of the pile-up is given by

Ap−u ¼ 3πR2

6−

ffiffiffi3

p

4a2

!≈

2π−3ffiffiffi3

p

4

!α2 ð5Þ

where R is the arc radius, which is approximately equal to a,and a is the triangular side of the indenter's impression. Kese

Table 2 – Corrected hardness expressions after the pile-up cont

Total contact area Pile-up conta

Ac ¼AO−P –

AKese et al: ¼AO−P þ APU APU ¼ 5:915hc

AZhou et al: ¼AO−P þ 3Ap−u Ap−u ¼ 15:368

ANEW ¼AO−P þ APile−up APile−up ¼∑ πp

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et al. (2005) and Zhou et al. (2008) replaced parameter a, withhc, with respect to the geometrical features of the perfectindent residue by a Berkovich indenter and they obtained thefollowing equation:

AOP≈Atriangle ¼ 3ffiffiffi3

ph2c tan 65:31 -

Eq: ð5Þa¼ 2

ffiffiffi3

ptan 65:31 ð6Þ

Replacement of a in Eq. (5) gives the contact area of onepile-up

Ap−u ¼ 15:368hc ð7Þ

These models predict well the contact area of the pile-upsfor materials that tend to form small in width and heightpile-ups, hence, it was essential to further develop the modelthat Kese et al. (2005) proposed. In this work we assume thatthe pile-up is a semi-elliptical area, with major axis bi (Fig. 8),which is the length of the three triangle sides, and minor axisβi. The lengths bi, significantly differ and we cannot assume ofan equilateral triangle. The feature βi is the hypotenuse of aright triangle with right sides ai (as defined earlier) and hi,which defines the total height of the pile-up (Fig. 8). Wedetermined the features bi from the SPM images, althoughthe error is significant; however, it is observed that throughEqs. (3) and (6) the triangle side is overestimated. Therefore,the contact area of the pile-ups is given by

APile−up ¼∑πβibi4

¼∑πffiffiffiffiffiffiffiffiaihi

pbi

4ð8Þ

In Table 2 the final equations used in order to determinethe piled-up areas and the non-overestimated values of H arepresented.

These methods provide data for the contact area of thepiled-up material. The new values of the contact area of thepiled-up material are subtracted from the contact area valuesobtained by Oliver and Pharr model and the respective Hvalues were calculated through the equations presented inTable 2. These H values are displayed in Fig. 8 around theindent performed at ∼10% of the coating thickness for theorganic coating. It is observed that all three models provide Hvalues which are four order of magnitude lower than the Hvalues measured by the indentation tests; revealing that theOliver and Pharr model overestimates by �100% the H valueswhen very soft materials (polymer and polymer-like) pile-up.It is noted that the H values obtained by these three modelsdiffer significantly. The models proposed in this study (HNEW)and the one by Kese et al. (2005) (HKese et al.) consider thegeometrical features of pile-ups. The first considers theheight of the pile-up and the length of every triangle sideleft from the Berkovich indenter impression. The one pro-posed by Kese et al. takes into account the width of the pile-up (ai) (Kese et al., 2005). However, the model proposed by

act area calculation.

ct area Hardness

Hmeasured ¼ Pmax=Ac

∑ai HKese et al: ¼ Pmax=AKese et al:

hc HZhou et al: ¼ Pmax=AZhou et al:ffiffiffiffiffiffiaihibi4

HNEW ¼ Pmax=ANEW

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Zhou et al. (2008) (HZhou et al.) depends on the hc values of eachindentation test; consequently, when a material performspile-up with enhanced width, the model of Zhou et al. doesnot provide accurate H values.

In the SPM images pile-up or sink-in formations are notalways evident; hence, we attempted to estimate the H valuesof the tested samples if they were bulk. Fig. 9a and b show thesquare of indentation hardness H2 versus the reciprocal ofthe indentation depth 1/hmax, for all samples. It can be seenthat a linear relation is closely followed for all samples at verylow values of 1/hmax, in agreement with the literature(Rodriguez and Gutierrez, 2003). The classical indentationhardness (for very large indentation depth) is determined bythe intercept with the vertical axis. In the inset graph ofFig. 9a and b, the enlargement of the large indentation depths(very small 1/hmax values) is presented, in order to calculatethe extrapolated hardness, Hextrapolated, from the intercept withthe vertical axis. Hardness data assessed to low indentationdepths have been excluded from this calculation, for tworeasons. First, uncertainties in the contact area are higherand second, the indenter's shape is characterised as non-self-similar at low indentation depths (Nix and Gao, 1998).

Fig. 9 – (a, b) Square of the nanohardness value against the inveat 10% coating thickness and the extrapolated hardness.

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In Fig. 9c the H values at 10% coating thickness are presentedwith Hextrapolated. It is observed that the measured H values arehigher than Hextrapolated for all samples. The difference in themeasured and calculated H values may be attributed to thestrong dependence of the measured H value on the indenta-tion depth and the material behaviour (observed pile-uparound the indents). Polymers and hybrid materials areinfluenced by the conformation of the macromolecules andthe structure of the formed network, respectively, as well asthe energy of the interactions between the macromoleculesand the tip.

4. Conclusions

The nanomechanical properties of novel organic–inorganichybrid coatings of different chemical composition onto glasssubstrates before and after submersion in cell culture med-ium have been investigated. The mechanical properties of thematerials are affected by their chemical composition, synth-esis and thickness, influencing their response in appliedloads and resulting in plastic deformation. However, no

rse of the depth and (c) comparison of the hardness

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delamination of any sample is observed, indicating goodadhesion on the glass substrate. It is evident that moistureuptake influence the H and Er values of the samples, resultingin sample softening with the exception of hybrid 2 (50%organic and 50% inorganic) coating. The observed hardeningresults and the low wear resistance values (H/Er) of hybrid 2material indicate sufficient plastic behaviour and cell adhe-sion with spread cell morphology from the first hour ofobservation up to several days, together with cell prolifera-tion after 3 and 7 days. These observations adequately reflectthe biocompatibility of the hybrid 2 coating. In addition, acorrelation of the observed deformation (pile-up) and theexperimental results is presented, revealing that soft materi-als present pile-ups at lower hc/hmax values. The pile-upformation motivated us to estimate the real contact area ofthe Berkovich indenter with the tested samples and the real Hvalues, revealing that the Oliver and Pharr model cannotaccurately estimate the H and Er values when the materialspile-up. Finally, correlation of the H values of all samples at10% coating thickness with the extrapolated H value leads tothe observation that the higher H values measured by theOliver and Pharr model are attributed to their strong depen-dence on the indentation depth and the material behaviour(observed pile-up around the indents).

Acknowledgements

The authors A. Skarmoutsou and C.A. Charitidis kindlyacknowledge the financial support of the European Union(European Social Fund—ESF) and Greek national fundsthrough the Operational Program “Education and LifelongLearning” of the National Strategic Reference Framework(NSRF)—Research Funding Program: Heracleitus II. Investingin knowledge society through the European Social Fund.

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