research article hydroxyapatite whiskers based resin...

Research ArticleHydroxyapatite Whiskers Based Resin Compositeversus Commercial Dental Composites Mechanical andBiocompatibility Characterization

L Calabrese1 F Fabiano12 M Currograve3 C Borsellino4 L M Bonaccorsi1

V Fabiano5 R Ientile3 and E Proverbio1

1Department of Electronic Engineering Chemistry and Industrial Engineering University of Messina Contrada di Dio98166 Messina Italy2Department of Experimental Specialized Medical-Surgical and Odontostomatological Sciences University of MessinaVia Consolare Valeria 1 98125 Messina Italy3Department of Biomedical Sciences and Morpho-Functional Imaging University of Messina Via Consolare Valeria 198125 Messina Italy4Department of Civil Engineering Computing Construction Environmental and Applied MathematicsUniversity of Messina Contrada di Dio 98166 Messina Italy5Department of Human Pathology University of Messina Via Consolare Valeria 1 98125 Messina Italy

Correspondence should be addressed to L Calabrese lcalabreseunimeit

Received 13 October 2015 Revised 6 January 2016 Accepted 12 January 2016

Academic Editor Jun Liu

Copyright copy 2016 L Calabrese et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A systematic evaluation of mechanical and biocompatibility properties of different volume fractions of hydroxyapatite whiskers incomparison with three commercial dental composites filled with micro- and nanosilica particles was carried out Six groups withdifferent hydroxyapatite whiskers mass fractions were taken into account in order to be compared with the performances of silicaparticles based composites group Flexural properties were evaluated via a universal testing machine (25 kN Zwick Line) with a2 kN load-cell (sensitivity 0001N) The test was replicated 10 times for the seven experimental groups to better identify staticallythe significance of the mechanical performances data MTT quantitative colorimetric assay was performed in order to evaluate themitochondrial activity of living cells exposed to different resin composites Data obtained show better interfacial interaction withfillermatrix until 20wt of hydroxyapatite whiskers partially replaced silica particles filler After this threshold the mechanicalperformances decrease dramatically due to both the hydroxyapatite agglomerates formation and the low degree of resin conversionIn addition biocompatibility test showed less cytotoxic effect with the addition of 20wt of hydroxyapatite in comparison withhigher rates

1 Introduction

Nowadays in order to avoid limits affecting long-termdurability of resin composites dentalmaterials companies arefocusing their attention on the improvement of the mechan-ical and biological properties of filler Thus working on fillerpacking optimization of filler content and development of anew innovative fillers is needed

Hydroxyapatite (HA) Ca10(PO4)6(OH)2 was introduced

since 1975 as filling material for intrabony defects [1ndash4]

As the main biomineral component in enamel and dentinehydroxyapatite is responsible for their higher mechanicalperformances In fact enamel shows an average of about90GPa for the elastic modulus and 480GPa for hardnesswhile dentin shows an average of 090GPa for hardness and20GPa for the elastic modulus [5]

For this reason in the recent five years hydroxyapatiteparticles and whiskers have been added as novel bioactiveand biocompatible reinforcing filler in dental restorations Infact it was shown that the incorporation of hydroxyapatite

Hindawi Publishing CorporationAdvances in Materials Science and EngineeringVolume 2016 Article ID 2172365 9 pageshttpdxdoiorg10115520162172365

2 Advances in Materials Science and Engineering

with whiskers morphology can provide larger load transferand favor toughening mechanisms thus increasing flexuralmodulus and fracture toughness of resins in comparisonwith conventional silica based fillers [6ndash11] In additionhydroxyapatite whiskers (wHA) have been considered thebest option as reinforcing filler in comparison with otherwhiskers based on carbon ceramic glass metal and polymerdue to the absence of cytotoxicity effects [12 13]

Due to its good cation exchange rate with metalsCa10(PO4)6(OH)2represents the most promising substrate

for potential release of antimicrobial molecule or ions (iesilver copper and zinc) This approach is promising in orderto prevent the major bacterial biofilm retention of resincomposite which is the main reason of secondary cariesinsurgence and then restoration failure [14]

To overcome the present lack in the literature abouthydroxyapatite based composite we proposed a systematicanalysis of the mechanical and biocompatibility perfor-mances of them in comparison with commercial dentalcomposite For wHA filled composite resins several effortsare still necessary to enhance the interphase combination andinvestigate the overall properties [8]

This information is of primary interest in order to achievea final consistency and specific formulation of hydroxyapatitewhiskers mixed with silica particles filler in resin compositeIn particular six different ratios of hydroxyapatitesilicaparticles were investigated

With this aim different mass fraction of hydroxyapatitebased composites and three commercial composites loadedwith silica particles were tested and compared in order toverify the effective enhancement in both biocompatibilityandmechanical properties In particular three-point bendingtest Vickers hardness morphological analysis and quantita-tive colorimetric assay to assess cell viability were performed

2 Materials and Methods

21Materials Camphorquinone (CQ) ethyl 4-dimethylami-nobenzoate (EDMAB) 22-bis[p-(21015840-hydroxy-31015840methacryl-oxypropoxy)phenyl]propane (Bis-GMA) triethylene gly-col dimethacrylate (TEGDMA) calcium nitrate tetrahy-drate (Ca(NO

3)2sdot4H2O) ammonium phosphate dibasic

((NH4)2HPO4) and urea ((NH

2)2CO) were purchased from

Sigma-Aldrich Company (Milano Italy) The inorganic fillerbased on 07 120583m size silica particles was provided by EsstechInc (Essington USA)

The human premonocytic cell line THP-1 was obtainedfrom DSMZ (Braunschweig Germany) RPMI-1640medium penicillinstreptomycin mixture L-glutamine 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)sodium pyruvate glucose fetal bovine serum (FBS) 3-(45-methylthiazol-2-yl)-25-diphenyl-tetrazolium bromide(MTT) and other chemicals of analytical grade werepurchased from Sigma Aldrich (Milano Italy)

22 Synthesis of HAP Whiskers Hydroxyapatite whiskers(wHA) were prepared by hydrothermal homogeneous pre-cipitation at 180∘C (in autogenous pressure) for 10 h Anaqueous solution containing stoichiometric amounts of

Figure 1 SEM image of synthesized whiskers

Ca(NO3)2sdot4H2O (10ndash334mmol Lminus1) and (NH

4)2HPO4(6ndash

200mmol Lminus1) with a final molar ratio CaP of 167 wasprepared The solution pH was adjusted to 3 by adding urea(CO(NH

2)2) The resulting mixture was then poured into a

PTFE container inside a stainless steel autoclave and put in anoven at 180∘C for 10 hoursThe final solution was centrifugedto recover the precipitate powder which was washed withdistilled water and dried at 80∘C overnight

Figure 1 shows SEM micrograph of synthesized HAwhiskers Whiskers are well defined and mainly monodis-perse although some ones that fused each other can beidentified

The aspect ratios (ie lengthdiameter) varied in therange 2 plusmn 10 with average whisker diameter and length 35 nmand 200 nm respectively

23 Dental Composite Preparation Dental resin matrix wasobtained mixing 495 wt Bis-GMA and 495 wt dilu-ent comonomer TEGDMA Camphorquinone and ethyl 4-dimethylaminobenzoate were added as the initiator andcoinitiator in the ratio of 05 wt respectively Variousvolume fractions of HA whiskers were added into organicmatrix replacing the silica particles in particular from 0 volto 100 vol HA whiskers as reported in Table 1

Furthermore three commercial resin composites werechosen as mechanical and biocompatibility evaluation refer-ence in this work

(i) Quadrant universal LC is a light-cured fluoridereleasing radiopaque microglass composite for uni-versal application It is based on a Bis-GMA matrixand contains 60 filler by volume or 72 by weightwhich is formed by Ba-Al-F-silicate glass (002ndash2 120583m)and highly dispersed silicon dioxide (002ndash007120583m)

(ii) Durafill is a microfilled light-cured composite fillingmaterial with a total amount of 66 of inorganic andorganic fillers by volume The filler contains highlydisperse silicon dioxide (002ndash007 120583m) and splinterpolymer (lt20120583m)

(iii) Renamel microfill is a microfilled light-cured com-posite filling material which consists of multifunc-tional acrylic resins and fillers of 004ndash02 micronsized particles of inorganic and prepolymerized com-posite (70 by weight and 60 by volume)

Advances in Materials Science and Engineering 3

Table 1 Different volume fraction of HA whiskers and inorganic filler in resin based composite formulation

CODE HA density gcm3 Silica density gcm3 Vol HA (wt) Vol Silica (wt) HASilica Vol ratio Vol resinHA-0 31 24 00 (00) 300 (462) 00 300HA-20 31 24 60 (116) 240 (36) 200 300HA-40 31 24 120 (226) 180 (263) 400 300HA-60 31 24 180 (331) 120 (171) 600 300HA-80 31 24 240 (431) 60 (83) 800 300HA-100 31 24 300 (525) 00 (00) 1000 700

24 Mechanical Test Flexural properties were evaluatedusing rectangular-shaped specimens with dimensions25mm times 2mm times 2mm To make the samples a customizedstainless steel mold was used where the lateral sides are madeby two glass slides with a polyester film interposed betweenthe glass and the mold Such mold was filled with theuncured resin composite then the glass slide was removedand the exit window of a visible light unit (Optilux-501 KerrCT USA) with a wavelength of 400ndash505 nm and output of1000mWcm2 was positioned at the center of the specimenagainst the glass slide so that the specimen was irradiatedfor polymerization time of 60 s After the photoactivationof the specimenrsquos center the exit window was moved to thesection next to the center overlapping the previous sectionby half the diameter of the exit window the same for thesection on the other side of the center After that both sideswere irradiated and the specimens were stored in distilledwater at 37∘C for 24 h before testing according to ISO 4049standard

Three-point bending test was performed at room temper-ature using a universal testing machine (25 kN Zwick Line)2 kN load-cell (sensitivity 0001N)The cross-head speed was075mmmin

Microhardness tests were performed by using a Future-Tech Microhardness Tester FM-300 (Vickers indenter andcompressive load 100 g) Surface microstructure of hydrox-yapatite whiskers and resin composite was examined byfocused ion beam scanning electron microscopy (FIB-SEMCrossbeam Zeiss)

25 Cell Culture and Viability Test THP-1 cells weremaintained in RPMI 1640 supplemented with L-glutamine(2mM) HEPES (10mM) sodium pyruvate (1mM) glucose(25 gl) 2-mercaptoethanol (005mM) 10 heat-inactivatedfetal bovine serum (FBS) and 1 penicillinstreptomycin at37∘C in a 5 CO

295 air humidified atmosphere Medium

was renewed every 2 days and split performed when cellsreached maximum density (1 times 106 cellsml) In our experi-mental conditions THP-1 cells were seeded at a density of 5times 105 cellsml into culture plates in RPMI complete mediumplus 10 FBS and incubated at 37∘C for 48 h with variouscomposite powders containing different rate of HAwhiskersat concentrations ranging between 005 and 1mgml

To assess the effects of different composites on cellviability we evaluated the mitochondrial activity of livingcells by a MTT quantitative colorimetric assay After treat-ment THP-1 cells were harvested by centrifugation and

incubated in 96-well culture plates with fresh red-phenol-free medium containing MTT (05mgmL) at 37∘C for 4 hsThen insoluble formazan crystals were dissolved in 100 120583L ofa 004NHClisopropanol solution for 1 hThe optical densityin each well was determined at 570 nm using a microplatereader (Tecan Italia Cologno Monzese Italy)

26 Statistical Analysis Results were expressed as mean plusmnstandard error of the mean for each group of specimensOne-way analysis of variance (ANOVA) tests were performedfor the dependent variables such as elastic modulus flexuralstrength and cell viability In particular flexural tests wereperformed in 10 replicas for each group while biocompatibil-ity test was replicated five times for each experimental groupStudent-Newman-Keuls post hoc test was used for multiplecomparison and statistical significance was considered at 119901 lt005

3 Results and Discussion

In Figure 2 three-point flexural test for a HA-20 resincomposite samples is shown as reference because of show-ing the better mechanical performances between HA-basedexperimental groups Stress (on the main axis) and modulus(on the secondary axis) are plotted at increasing the strain

Analyzing the figure three significant regions can beidentified

At first the initial trend of stress-strain curve is relatedwith themechanical adjustment phases of the set-up configu-rationThis section of the curve is usually quite disturbed andit is not indicative of the mechanical behavior of the sample

After the stabilization phase the stress increases quitelinearly with the strain In this phase the relation betweenstress and strain can be considered linear (Figure 2) and themodulus 119864 is quite constant (119864max about 3500MPa) In thisphase the material has a pure linear elastic behavior withlimited strain values (up to 0003)

On the contrary at higher deformations the relationshipbetween stress and strain becomes nonlinear and the stiffnessprogressively decreases This phase is influenced by theviscous behavior of the matrix In this region the interfacialfiller-matrix strength plays an important role on themechan-ical performance of the composite paste influencing trendslope and threshold interfacial stresses

Finally a mainly brittle fracture occurred at 003 strainThe maximum stress observed at failure was about 55MPa


0

10

20

30

40

50

60

0 0005 001 0015 002 0025

Nonlinearzone

Linearzone

Linearzone

Failurezone

Stabilizationzone

Stabilizationzone

003 0035

Mod

ulus

(MPa

)

Modulus

Stre

ss (M

Pa)

Stress

Strain

Figure 2 Youngrsquos modulus (continuous line) and flexural strength(dotted line) in function of deformation for a HA-20 referencecomposite sample

0

10

20

30

40

50

60

0 001

HA content

HA-100HA-80

HA-60HA-40

HA-0HA-20

002 003 004 005

120590(M

pa)

120576

Figure 3 Stress-strain curves of samples at different mass fractionof hydroxyapatite whiskers filled resin composite

In order to evaluate the effect on the mechanical perfor-mances of the hydroxyapatite addiction in the filler mixingcomposition of the composite paste the stress-strain curveevolution at increasing HA amount is shown in Figure 3The mechanical performances of the composite materialare influenced by the addiction of HA whiskers At first aslight increase of failure stress can be observed for HA-20sample Afterwards a progressive reduction of maximumstress and modulus (slope of stress-strain curve) can beidentified at increasing HA amount (with the consequentreduction of silica filler content) Furthermore the strainfailure progressively decreases at increasing HA amountSamples with a large amount of hydroxyapatite evidenced asignificant reduction of mechanical performances

Analyzing in detail stress-strain curves reported in Fig-ure 3 0 HASilica and 20 HASilica samples are ableto carry high stresses and strains combining high stiffnessand strength In fact they can reach high deformationwithout the insurgence of critical failure conditions These

Table 2 Vickers hardness of different mass fraction of hydroxyap-atite based composites

Samples Vickers hardness (HV)a

HA-0 58 (9)HA-20 77 (6)HA-40 69 (12)HA-60 57 (7)HA-80 55 (10)HA-100 53 (8)aMean value (standard deviation)

samples evidenced a slight nonlinear curve with a first elasticregion until medium deformation (about 0005) Afterwardsat increasing deflections the stress-strain curve exhibits aprogressive moderate deviation from linear trend Anywaya mainly linear elastic behavior can be observed for thesesamples (HA-0 and HA-20) These samples maintain anelastic regime also at high strain evidencing an elastic-brittle behavior typical for well cross-linked composite resin[15 16] Thus the interfacial interaction between filler andmatrix which participate positively in stress transfer betweencomposite constituents becomes the driving force in thestrengthening and toughening effects on the particular com-posite [17]

At increasing hydroxyapatite content a progressiveincrease of viscous contribution in the mechanical behaviorof the composite paste can be observed HA-80 and HA-100samples exhibited an elastoplastic behavior as confirmed bythe nonlinear trend of the stress-strain curveThe low perfor-mances observed for large amount of HA could be due to areduction of cohesive strength at the matrix-filler interphasethat favors low failure stressed At the same time the hydrox-yapatite induces a shielding effect on the photoactivation ofthe resin on the bulk [18] consequently a large amount of HAfiller could act as inhibitor of the photoactivation of the resininducing nonoptimal crosslinking [19] This is confirmed bythe Vickers hardness data reported in Table 2 The degree ofconversion is defined as the percentage of reactedC=Cdoublebonds [20] Hardness has been shown to be a good indicatorof conversion of double bonds and was therefore used inthe present study as indirect measurements of conversionThe unreacted compounds characterized by high mobilityinfluence significantly the plastic deformation inducing lowstrength and stiffness on the sample In fact if the deepestlayers of composite restorations are not adequately curedthe elastic modulus at the bottom will be lower than thatat the surface this can increase the material strain undermasticatory forces [21] At the same time nonhomogenousreticulation of the composite paste stimulates differentialpolymerization shrinkage stresses inducing tensile stresseson the core of resin composite Furthermore the prematurefailure at both low stress and strain values could indicate areduced cohesive strength of the dental restorative paste dueto a nonoptimal interfacial interaction of the composite fillerswith the matrix

The variation of flexural strength against wHA fillerloading is shown in Figure 4 Each datum in the plots provides


Table 3 Statistically significant differences on flexural strength values were evaluated by one-way ANOVA 119875 lt 005

Source SS df MS 119865

120572 5119865critical 119875 value

Between 6209998 5 1242000 61322 2386 0000 RejectWithin (error) 1093703 54 20254Total 7303701 59

01500

2500

3500

4500

HA (vol)

Mod

ulus

(MPa

)

10 20 30 40 50 60 70 80 90 100

Figure 4 Failure strength and Youngrsquos modulus values at increasingHASilica filler ratio Errors bars are standard deviation

the mean value of 10 measurements with standard deviationreported as 119910 error bar Mechanical performances decreasedincreasing wHA volume fraction In fact at low amount ofhydroxyapatite whiskers (until 20wt of HA) the flexuralstrength of the composite paste is quite constant maintaining119904 stable maximum stress and modulus of about respectively55MPa and 3500MPa

The mechanical performances progressively decrease atincreasing HA amount The 60 HASilica samples show areduction of about 241 of Young modulus and 218 ofmaximum stress compared to 0 HASilica samples Thisbehavior could be explained considering that the compositereinforced with HA is characterized by a reduced cohesivestrength with higher heterogeneity than the commercialcomposite which favors a premature fracture at lower stresslevel Finally above 70 of HA the mechanical propertiesof the composite materials are very low A further increaseof HA amount does not induce significant variation offlexural strength observing a plateau at about 32MPa ofstress and 1950MPaofYoungmodulusThese lowmechanicalperformances can be related to the untreatment with silaneof hydroxyapatite fillers as reported also by Santos et al [9]that is less relevant until 20wt of HAwhich permits a bettercontact with the polymer matrix

The results summarized in Figure 4 evidence that afford-able mechanical performances can be obtained with theaddiction of reduced content of HA filler (under 40)evidencing an effective interaction between silica fillerhydroxyapatite whiskers and Bis-GMA based matrix Thepercentage of HA of 40 can be considered a threshold valueabove witch the mechanical properties are not suitable for

restoration applications In fact the Youngmodulus at 20wtof wHA was 349782MPa while at 0 wt was 356065MPawith a nonsignificant decrease of 17 Instead the differencebetween 20wt and 80wtofwHAfiller highlights a furtherdecrease of 43 despite the composite resin with 20wt ofHA The Young modulus for HA-100 points out a decreaseof about 50 in comparison with data obtained with theresin composite filled with the 20wt of wHA confirmingthat with large amount of HA particles the mechanicalperformances are compromised

However the addition of filler to the matrix may increasethe adhesive interaction to a tooth substrate [22] and decreasethe degradation of the material over time [23] without prej-udicing its mechanical performances The group with 20HA showed very affordable mechanical performances withno significant difference compared to the control group HA-0 (a slight enhancement of average mechanical performancescan be observed although the spread data indicate thatdifferences are not statistically significant) Consequentlyaccording to [21] the addition of low amount of HA filler toa commercial hybrid layer could maintain stable mechanicalproperties of this layer and increase the bond strengthInstead samples with higher amounts of HA filler evidencedlow mechanical properties with subsequent lower bondstrength when applied for restorative dental applications

The influence of the HA content factor on modulus andfailure stress of the composite resin has been evaluated byANOVA (for 119875 = 005) and performed by Minitabreg softwareThe results are summarized in Tables 3 and 4

Considering that from the ANOVA the 119875 value is zero(119875 = 0000 lt 005) for both analyses then it is unlikely thatthe differences observed are due to random sampling

This rejects the null hypothesis and concludes that not allof population means are equal Consequently the hypothesisthat the populations of samples at varying HA content havestatistically identical means can be rejected This implies thatmodulus and stress are influenced by HA addiction on thefiller composition and consequently ldquoHA contentrdquo is statis-tically significant factor for the mechanical performances ofcomposite resin

Interesting consideration can be extrapolated better ana-lyzing the filler-matrix interaction for 80HASilica samplesas shown in the SEM micrograph reported in Figure 5This analysis allows us to evaluate the morphology of thecomposite material and to study its fracture mechanics Thestructure of the HA-80 composite paste consists of a centralcore of Bis-GMA based resin surrounded by whiskers of HAhaving micrometric dimensions (length asymp 4-5 120583m)

This morphology is due to nonoptimal mixing betweenthe resin and the filler due to the high viscosity induced by


Table 4 Statistically significant differences on flexural modulus values were evaluated by one-way ANOVA 119875 lt 005


120572 5119865critical 119875 value


HA-whiskers

Resin

Figure 5 SEM micrograph at high magnification of the compositeresin reinforced with hydroxyapatite whiskers

the addition of large amount of hydroxyapatite [21] Since thevolume percentage of filler of the composite is high the resinis not sufficient towet the fillers andmicrodomains consistingof resin particles coated ceramic filler are created

As in all composite systems filler reinforcement efficacydepends substantially on the quality and typology of theinterface via matrix links

In order to increase the threshold filler content Zhangand Darvell [8] explored the effect of silanization of wHAand filler loading on properties of the composite resinand compared the results with hydroxyapatite particle fillerdemonstrating the better reinforcing ability and bioactivity ofwhisker

However the influence of silanization was not deeplyinvestigated and voids between whiskers and matrix lead toundesirable low adhesion strength favoring premature failureat low stress level

The morphology seen in Figure 5 may be representedas a micelle structure (Figure 6) The core is made of Bis-GMA resin surrounded by the needle-like HA filler arrangedradially in space with respect to the nucleus assuming ashape similar to a hedgehogThe whiskers are not distributedhomogeneously around the core of the resin but they areanchored on the surface assuming random orientations anddistributing the filler not uniformly across the surface ofthe core Consequently the micelle is characterized by acombination of dense and voids areas

The nonoptimal interaction between the filler and thematrix implies that regions with hydroxyapatite not impreg-nated with resin are created in particular that occurswhen the HA whiskers are aggregated and the resin cannot

VoidsResin

HA

HA

HA

HA-Bis-GMA resin micelle

Interphase of the micelle

Figure 6 Schematization of hydroxyapatite whiskersmatrix inter-action Three fundamental components can be highlighted in thecomposite the resin which consists predominantly of Bis-GMAwhich is shown in blue the filler of hydroxyapatite (HA) withneedle-like morphology (whiskers) and micrometric size and thevoids in white

penetrate into the interstices Hydroxyapatite is anchored tothe resin core only in correspondence of a small portion butalmost all of its length is not wet the radial arrangementof the whiskers of HA hinders the permeation of resin inthe spaces between the various lamellae which then remainsempty (magnification shown in Figure 5) consequently thevoids formation is favored

Based on these considerations deduced analyzing Fig-ure 5 it is possible to schematize a three-dimensional struc-ture of the HA-Bis-GMA composite (Figure 6) it can be seenas a set of micelles having a shape like a hedgehog arrangedin a disordered manner in a bulk of Bis-GMA resin

The amount of voids is remarkable because each micellehas an intrinsic amount of dry regions related to poor wettingand compatibility between the HA filler and the polymericBis-GMAmatrix

Within the framework of the HA-Bis-GMA composite(Figure 7) we can distinguish three different zones In zone1 the presence of voids (air bubbles) is observed They couldact as defects inducing an increase of stress-intensity factorIn 2 and 3 zones the rigid HA whiskers of the micellesare in contact with each other favoring the transfer of thestresses that lead to a stiffening of the composite Thiscould explain the slight increase of elastic modulus E of thecomposite reinforced with lower amount of HA comparedto commercial composite Instead for high amount of HAwhiskers the large presence of voids due to nonoptimal filler


HA

3

1

1

2

Bis-GMAVoids

Figure 7 Three-dimensional structure of the HA-Bis-GMA com-posite

HA bridges

Resin Resin

Mechanical linksResin

Resin

Adhesive links

Figure 8 The mechanisms of transfer of the stresses betweenmicelles

distribution obstacles the stress transfer at the resinfillerinterface Thus low mechanical performances are observed

Themechanisms of transfer of the stresses in 2 and 3 zonescould bemainly two (Figure 8) the first can be called adhesivelinks and it is characterized by the formation of bridges of HAbetween two neighboring micelles The second is named asmechanical link and it is mainly due to friction between themicelles that are in contact

With regard to the stiffening of the composite adhesivelinks are better than the mechanical links In the formerbridges of HA resist better to the tensile stress applieddespite the mechanical links (this structural configuration ischaracterized by a low mechanical resistance to shear)

For the sake of comparison we also tested some com-mercial composites for which the mean values of flexuralmodulus and standard deviations (SD) are summarized inTable 5

In Figure 9 the comparison between three commercialdental restorative materials and 20 HASilica samples isreported

Table 5 Results ofmechanical properties of four evaluated compos-ites

MaterialsYoungrsquos

modulus 119864(MPa)a

Flexuralstrength 120590(MPa)a

Strain 120576max

Durafill VS 2294 (85)a 5437 (5)a 002069Renamel 4000 (109) 8029 (11) 001376Quadrant LC 3800 (92) 7015 (3) 001184aMean value (standard deviation)

Renamel QuadrantDurafill VSLC

HAglass(20wt)

050010001500200025003000350040004500

Flex

ural

mod

ulus

(MPa

)

0102030405060708090

100

Flex

ural

stre

ngth

(MPa

)Figure 9 Flexural strength (dark grey) and elastic modulus (lightgrey) of four-resin based composite

Renamel composite showed much higher mechanicalperformances with a maximum flexural stress of 8029MPa20HASilica andDurafill VS samples showed no significantdifference in flexural strength with a mean value that isaround 545MPa Renamel and Quadrant LC showed nosignificant difference with a flexural strength respectively of80MPa and 70MPa

ANOVA revealed that Renamel showed higher modulus(4000MPa) which was statistically significant in comparisonwith Durafill VS (119875 lt 005) Durafill had the lowestflexural modulus (2294MPa) while between Renamel andQuadrant LC (3800MPa) there was no significant differencein addition also Quadrant LC and 20 HASilica composite(3500MPa) do not show any significant difference (119875 gt 005)

Renamel composite outlines a stiff and brittle behaviorwhich reflects the mean values detected for natural teethOn the contrary Durafill and 20 HASilica do not showeffective stiffness but possess good ductility which is usefulin the presence of internal stresses or dynamic loads

The SEM analysis of the HA-Bis-GMA composite hasshown adhesion problems between filler and polymer matrixthat it could be solved by using of surfactants or silanemodified coupling agents Their addiction allows improvingthe interaction between the constituents of the dental pasteThe addition of plasticizers with low viscosity could also beused to improve the workability of the composite by reducingthe number of voids Such adhesion problems are not presentin the commercial composite [24 25]

31 Biocompatibility Tests The biocompatibility of theseinnovative formulations with different rate of HA whiskerswas assessed by evaluating the toxicity in an immortalized


CCHA 20HA 40

HA 60HA 80HA 100

50

60

70

80

90

100

110

Cell

via

bilit

y (

of c

ontro

l cel

ls)

005 010 015 020 025 030 035 040 045 050000(mgmL)

Figure 10 Effects of various composite powders modification(exchange of silica filer with HA whiskers ) on THP-1 cellviability THP-1 cells were treated with different concentrations ofHA or CC (005ndash05mgml) for 48 h and then cell viability wasassessed by the MTT assay Results are expressed as percentages ofuntreated cultures Data are the mean plusmn SEM from five separateexperiments Statistically significant differences were evaluated byone-way ANOVA followed by Newman-Keuls post hoc test lowast119901 lt005 lowastlowast119901 lt 001 and 119901 lt 0001 significant differences incomparison to untreated cells

human monocyte THP-1 cell line We also compared theeffects of HA whiskers with commercial composite

The incubation of THP-1 cells with different concen-trations of various composite powders for 48 hs caused adose-dependent decrease in cell viability measured by MTTassay (Figure 10) In particular the resin composites withhighest HA content (80ndash100) at all concentrations exertedcytotoxic effects on THP-1 cell culture reducing cell viabilityup to 45 at the highest concentration in comparison tocontrol cells (119875 lt 0001) Composites with 40ndash60 HAcontent were also cytotoxicThe cell viability was significantlyreduced (119875 lt 005) in a concentration range of 025ndash05mgml while the incubation with 005 and 01mgmlof HA 40ndash60 produced no significant reduction of cellviability by 10ndash15 in comparison to untreated cells (119875 gt005) This was mainly due to the shielding effect of thehydroxyapatite fillers that at high content reduce the degreeof conversion of the matrix consequently increasing theavailability of cytotoxic uncuredmonomers and initiator [21]

Among allHA compositesHA-20 demonstrated very lowcytotoxic effects In agreement with other results [26] it ispossible to speculate that slight but no significant increasein MTT values occurring in the presence of HA-20 may beascribed to a partial effect on cell proliferation

After incubation of THP-1 cells with HA 20 at con-centrations ranging between 005 and 025mgml we didnot observe significant changes in cell viability in compar-ison with control cells Similarly commercial composite in

the same concentration range was not cytotoxic Howeverat highest concentration (005mgml) both HA 20 andcommercial compounds showed a significant increase incell death reducing cell viability by 25 in comparison tountreated cells (119875 lt 005)

4 Conclusion

The study on a new hybrid composite conducted thus farshows howmechanical properties can be compared with thatof commercial composites by the addition of a 20 volumeratio of hydroxyapatite with whisker morphology on conven-tional silica filler Until this range the links between micellesprovide a good interaction at matrixfiller interface Biocom-patibility assessment confirms the better response of THP-1cell culture to 20 HA based composite in comparison withboth commercial composites to higher wHA concentrationsconfirming promising results achieved by the replacementof silica filler with small amount of hydroxyapatite (up to20) The effect of adding hydroxyapatite whiskers filler onthe bond strength with dentine must be further investigated

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] F Liu R Wang Y Cheng X Jiang Q Zhang and MZhu ldquoPolymer grafted hydroxyapatite whisker as a filler fordental composite resin with enhanced physical and mechanicalpropertiesrdquo Materials Science and Engineering C vol 33 no 8pp 4994ndash5000 2013

[2] N Kantharia S Naik S Apte M Kheur S Kheur and B KaleldquoNano-hydroxyapatite and its contemporary applicationsrdquo Jour-nal of Dental Research and Scientific Development vol 1 pp 15ndash19 2014

[3] C Domingo R W Arcıs A Lopez-Macipe et al ldquoDental com-posites reinforced with hydroxyapatite mechanical behaviorand absorptionelution characteristicsrdquo Journal of BiomedicalMaterials Research vol 56 no 2 pp 297ndash305 2001

[4] H Zhang and B W Darvell ldquoMechanical properties of hydrox-yapatite whisker-reinforced bis-GMA-based resin compositesrdquoDental Materials vol 28 no 8 pp 824ndash830 2012

[5] Y-R Zhang W Du X-D Zhou and H-Y Yu ldquoReview ofresearch on the mechanical properties of the human toothrdquoInternational Journal of Oral Science vol 6 no 2 pp 61ndash692014

[6] J M Correa M Mori H L Sanches A D da Cruz E PoiateJr and I A V Pola Poiate ldquoSilver nanoparticles in dentalbiomaterialsrdquo International Journal of Biomaterials vol 2015Article ID 485275 9 pages 2015

[7] CDomingo RWArcıs E Osorio et al ldquoHydrolytic stability ofexperimental hydroxyapatite-filled dental compositematerialsrdquoDental Materials vol 19 no 6 pp 478ndash486 2003

[8] H Zhang and B W Darvell ldquoFailure and behavior in waterof hydroxyapatite whisker-reinforced bis-GMA-based resincompositesrdquo Journal of the Mechanical Behavior of BiomedicalMaterials vol 10 pp 39ndash47 2012


[9] C Santos R L Clarke M Braden F Guitian and K W MDavy ldquoWater absorption characteristics of dental compositesincorporating hydroxyapatite fillerrdquo Biomaterials vol 23 no 8pp 1897ndash1904 2002

[10] M Lezaja D N Veljovic B M Jokic I Cvijovic-Alagic MM Zrilic and V Miletic ldquoEffect of hydroxyapatite sphereswhiskers and nanoparticles on mechanical properties of amodel BisGMATEGDMA composite initially and after stor-agerdquo Journal of Biomedical Materials Research Part B AppliedBiomaterials vol 101 no 8 pp 1469ndash1476 2013

[11] R W Arcıs A Lopez-Macipe M Toledano et al ldquoMechanicalproperties of visible light-cured resins reinforced with hydrox-yapatite for dental restorationrdquo Dental Materials vol 18 no 1pp 49ndash57 2002

[12] A Tuncel A K Ozdemir Z Sumer F Hurmuzlu and ZPolat ldquoCytotoxicity evaluation of two different compositeswithwithout fibers and one nanohybrid compositerdquo DentalMaterials Journal vol 25 no 2 pp 267ndash271 2006

[13] H Yilmaz C Aydin A Caglar and A Yasar ldquoThe effect of glassfiber reinforcement on the residual monomer content of twodenture base resinsrdquo Quintessence International vol 34 no 2pp 148ndash153 2003

[14] V Stanic S Dimitrijevic J Antic-Stankovic et al ldquoSynthesischaracterization and antimicrobial activity of copper and zinc-doped hydroxyapatite nanopowdersrdquo Applied Surface Sciencevol 256 no 20 pp 6083ndash6089 2010

[15] A U J Yap and S H Teoh ldquoComparison of flexural propertiesof composite restoratives using the ISO andmini-flexural testsrdquoJournal of Oral Rehabilitation vol 30 no 2 pp 171ndash177 2003

[16] L M Bonaccorsi C Borsellino L Calabrese et al ldquoPerfor-mances evaluation of a Bis-GMA resin-based composite fordental restorationrdquoActaMedicaMediterranea vol 28 no 2 pp163ndash166 2012

[17] V MittalNanocomposites with Biodegradable Polymers Synthe-sis Properties and Future Perspectives Oxford University PressOxford UK 2011

[18] A Yasukawa S Ruike K Gotoh and K Kandori ldquoUltra-violet shielding properties of cotton fabric supported bycerium-calcium hydroxyapatite solid solution particlesrdquo TextileResearch Journal vol 84 no 15 pp 1578ndash1585 2014

[19] A Apicella M Simeone R Aversa A Lanza and D ApicellaldquoLight shielding effect of overlaying resin composite on thephotopolymerization cure kinetics of a resin composite and adentin adhesiverdquo Dental Materials vol 21 no 10 pp 954ndash9612005

[20] J R David O M Gomes J C Gomes A D Loguercioand A Reis ldquoEffect of exposure time on curing efficiencyof polymerizing units equipped with light-emitting diodesrdquoJournal of Oral Science vol 49 no 1 pp 19ndash24 2007

[21] V C B Leitune F M Collares R M Trommer D G AndrioliC P Bergmann and S M W Samuel ldquoThe addition ofnanostructured hydroxyapatite to an experimental adhesiveresinrdquo Journal of Dentistry vol 41 no 4 pp 321ndash327 2013

[22] K L Van Landuyt J Snauwaert J De Munck et al ldquoSystematicreview of the chemical composition of contemporary dentaladhesivesrdquo Biomaterials vol 28 no 26 pp 3757ndash3785 2007

[23] S Kalachandra ldquoInfluence of fillers on the water sorption ofcompositesrdquo Dental Materials vol 5 no 4 pp 283ndash288 1989

[24] F Fabiano C Borsellino L M Bonaccorsi L Calabrese VFabiano andGMavilia ldquoInfluence of irradiation exposure timeon the depth cure of restorative resin compositerdquo Atti della

Accademia Peloritana dei PericolantimdashClasse di Scienze FisicheMatematiche e Naturali vol 92 supplement 1 pp A1ndashA8 2014

[25] L Calabrese F Fabiano L M Bonaccorsi V Fabiano and CBorsellino ldquoEvaluation of the clinical impact of ISO 4049 incomparisonwithminiflexural test onmechanical performancesof resin based compositerdquo International Journal of Biomaterialsvol 2015 Article ID 149798 7 pages 2015

[26] LHernandez J Parra B Vazquez et al ldquoInjectable acrylic bonecements for vertebroplasty based on a radiopaque hydroxya-patite Bioactivity and biocompatibilityrdquo Journal of BiomedicalMaterials Research Part B Applied Biomaterials vol 88 no 1pp 103ndash114 2009

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


with whiskers morphology can provide larger load transferand favor toughening mechanisms thus increasing flexuralmodulus and fracture toughness of resins in comparisonwith conventional silica based fillers [6ndash11] In additionhydroxyapatite whiskers (wHA) have been considered thebest option as reinforcing filler in comparison with otherwhiskers based on carbon ceramic glass metal and polymerdue to the absence of cytotoxicity effects [12 13]

Due to its good cation exchange rate with metalsCa10(PO4)6(OH)2represents the most promising substrate

for potential release of antimicrobial molecule or ions (iesilver copper and zinc) This approach is promising in orderto prevent the major bacterial biofilm retention of resincomposite which is the main reason of secondary cariesinsurgence and then restoration failure [14]

To overcome the present lack in the literature abouthydroxyapatite based composite we proposed a systematicanalysis of the mechanical and biocompatibility perfor-mances of them in comparison with commercial dentalcomposite For wHA filled composite resins several effortsare still necessary to enhance the interphase combination andinvestigate the overall properties [8]

This information is of primary interest in order to achievea final consistency and specific formulation of hydroxyapatitewhiskers mixed with silica particles filler in resin compositeIn particular six different ratios of hydroxyapatitesilicaparticles were investigated

With this aim different mass fraction of hydroxyapatitebased composites and three commercial composites loadedwith silica particles were tested and compared in order toverify the effective enhancement in both biocompatibilityandmechanical properties In particular three-point bendingtest Vickers hardness morphological analysis and quantita-tive colorimetric assay to assess cell viability were performed

2 Materials and Methods

21Materials Camphorquinone (CQ) ethyl 4-dimethylami-nobenzoate (EDMAB) 22-bis[p-(21015840-hydroxy-31015840methacryl-oxypropoxy)phenyl]propane (Bis-GMA) triethylene gly-col dimethacrylate (TEGDMA) calcium nitrate tetrahy-drate (Ca(NO

3)2sdot4H2O) ammonium phosphate dibasic

((NH4)2HPO4) and urea ((NH

2)2CO) were purchased from

Sigma-Aldrich Company (Milano Italy) The inorganic fillerbased on 07 120583m size silica particles was provided by EsstechInc (Essington USA)

The human premonocytic cell line THP-1 was obtainedfrom DSMZ (Braunschweig Germany) RPMI-1640medium penicillinstreptomycin mixture L-glutamine 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)sodium pyruvate glucose fetal bovine serum (FBS) 3-(45-methylthiazol-2-yl)-25-diphenyl-tetrazolium bromide(MTT) and other chemicals of analytical grade werepurchased from Sigma Aldrich (Milano Italy)

22 Synthesis of HAP Whiskers Hydroxyapatite whiskers(wHA) were prepared by hydrothermal homogeneous pre-cipitation at 180∘C (in autogenous pressure) for 10 h Anaqueous solution containing stoichiometric amounts of

Figure 1 SEM image of synthesized whiskers

Ca(NO3)2sdot4H2O (10ndash334mmol Lminus1) and (NH

4)2HPO4(6ndash

200mmol Lminus1) with a final molar ratio CaP of 167 wasprepared The solution pH was adjusted to 3 by adding urea(CO(NH

2)2) The resulting mixture was then poured into a

PTFE container inside a stainless steel autoclave and put in anoven at 180∘C for 10 hoursThe final solution was centrifugedto recover the precipitate powder which was washed withdistilled water and dried at 80∘C overnight

Figure 1 shows SEM micrograph of synthesized HAwhiskers Whiskers are well defined and mainly monodis-perse although some ones that fused each other can beidentified

The aspect ratios (ie lengthdiameter) varied in therange 2 plusmn 10 with average whisker diameter and length 35 nmand 200 nm respectively

23 Dental Composite Preparation Dental resin matrix wasobtained mixing 495 wt Bis-GMA and 495 wt dilu-ent comonomer TEGDMA Camphorquinone and ethyl 4-dimethylaminobenzoate were added as the initiator andcoinitiator in the ratio of 05 wt respectively Variousvolume fractions of HA whiskers were added into organicmatrix replacing the silica particles in particular from 0 volto 100 vol HA whiskers as reported in Table 1

Furthermore three commercial resin composites werechosen as mechanical and biocompatibility evaluation refer-ence in this work

(i) Quadrant universal LC is a light-cured fluoridereleasing radiopaque microglass composite for uni-versal application It is based on a Bis-GMA matrixand contains 60 filler by volume or 72 by weightwhich is formed by Ba-Al-F-silicate glass (002ndash2 120583m)and highly dispersed silicon dioxide (002ndash007120583m)

(ii) Durafill is a microfilled light-cured composite fillingmaterial with a total amount of 66 of inorganic andorganic fillers by volume The filler contains highlydisperse silicon dioxide (002ndash007 120583m) and splinterpolymer (lt20120583m)

(iii) Renamel microfill is a microfilled light-cured com-posite filling material which consists of multifunc-tional acrylic resins and fillers of 004ndash02 micronsized particles of inorganic and prepolymerized com-posite (70 by weight and 60 by volume)





















0

10

20

30

40

50

60

0 0005 001 0015 002 0025

Nonlinearzone

Linearzone

Linearzone

Failurezone

Stabilizationzone

Stabilizationzone

003 0035

Mod

ulus

(MPa

)

Modulus

Stre

ss (M

Pa)

Stress

Strain


0

10

20

30

40

50

60

0 001

HA content

HA-100HA-80

HA-60HA-40

HA-0HA-20

002 003 004 005

120590(M

pa)

120576













120572 5119865critical 119875 value


01500

2500

3500

4500

HA (vol)

Mod

ulus

(MPa

)

10 20 30 40 50 60 70 80 90 100















120572 5119865critical 119875 value


HA-whiskers

Resin








VoidsResin

HA

HA

HA









HA

3

1

1

2

Bis-GMAVoids


HA bridges

Resin Resin


Resin

Adhesive links











Strain 120576max



HAglass(20wt)

050010001500200025003000350040004500

Flex

ural

mod

ulus

(MPa

)

0102030405060708090

100

Flex

ural

stre

ngth

(MPa








CCHA 20HA 40

HA 60HA 80HA 100

50

60

70

80

90

100

110

Cell

via

bilit

y (

of c

ontro

l cel

ls)

005 010 015 020 025 030 035 040 045 050000(mgmL)







4 Conclusion




References




































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials























0

10

20

30

40

50

60

0 0005 001 0015 002 0025

Nonlinearzone

Linearzone

Linearzone

Failurezone

Stabilizationzone

Stabilizationzone

003 0035

Mod

ulus

(MPa

)

Modulus

Stre

ss (M

Pa)

Stress

Strain


0

10

20

30

40

50

60

0 001

HA content

HA-100HA-80

HA-60HA-40

HA-0HA-20

002 003 004 005

120590(M

pa)

120576













120572 5119865critical 119875 value


01500

2500

3500

4500

HA (vol)

Mod

ulus

(MPa

)

10 20 30 40 50 60 70 80 90 100















120572 5119865critical 119875 value


HA-whiskers

Resin








VoidsResin

HA

HA

HA









HA

3

1

1

2

Bis-GMAVoids


HA bridges

Resin Resin


Resin

Adhesive links











Strain 120576max



HAglass(20wt)

050010001500200025003000350040004500

Flex

ural

mod

ulus

(MPa

)

0102030405060708090

100

Flex

ural

stre

ngth

(MPa








CCHA 20HA 40

HA 60HA 80HA 100

50

60

70

80

90

100

110

Cell

via

bilit

y (

of c

ontro

l cel

ls)

005 010 015 020 025 030 035 040 045 050000(mgmL)







4 Conclusion




References




































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






120572 5119865critical 119875 value


01500

2500

3500

4500

HA (vol)

Mod

ulus

(MPa

)

10 20 30 40 50 60 70 80 90 100















120572 5119865critical 119875 value


HA-whiskers

Resin








VoidsResin

HA

HA

HA









HA

3

1

1

2

Bis-GMAVoids


HA bridges

Resin Resin


Resin

Adhesive links











Strain 120576max



HAglass(20wt)

050010001500200025003000350040004500

Flex

ural

mod

ulus

(MPa

)

0102030405060708090

100

Flex

ural

stre

ngth

(MPa








CCHA 20HA 40

HA 60HA 80HA 100

50

60

70

80

90

100

110

Cell

via

bilit

y (

of c

ontro

l cel

ls)

005 010 015 020 025 030 035 040 045 050000(mgmL)







4 Conclusion




References




































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






120572 5119865critical 119875 value


HA-whiskers

Resin








VoidsResin

HA

HA

HA









HA

3

1

1

2

Bis-GMAVoids


HA bridges

Resin Resin


Resin

Adhesive links











Strain 120576max



HAglass(20wt)

050010001500200025003000350040004500

Flex

ural

mod

ulus

(MPa

)

0102030405060708090

100

Flex

ural

stre

ngth

(MPa








CCHA 20HA 40

HA 60HA 80HA 100

50

60

70

80

90

100

110

Cell

via

bilit

y (

of c

ontro

l cel

ls)

005 010 015 020 025 030 035 040 045 050000(mgmL)







4 Conclusion




References




































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




HA

3

1

1

2

Bis-GMAVoids


HA bridges

Resin Resin


Resin

Adhesive links











Strain 120576max



HAglass(20wt)

050010001500200025003000350040004500

Flex

ural

mod

ulus

(MPa

)

0102030405060708090

100

Flex

ural

stre

ngth

(MPa








CCHA 20HA 40

HA 60HA 80HA 100

50

60

70

80

90

100

110

Cell

via

bilit

y (

of c

ontro

l cel

ls)

005 010 015 020 025 030 035 040 045 050000(mgmL)







4 Conclusion




References




































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




CCHA 20HA 40

HA 60HA 80HA 100

50

60

70

80

90

100

110

Cell

via

bilit

y (

of c

ontro

l cel

ls)

005 010 015 020 025 030 035 040 045 050000(mgmL)







4 Conclusion




References




































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



research article hydroxyapatite whiskers based resin...

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