research article p(3hb) based magnetic nanoc omposites...

Research ArticleP(3HB) Based Magnetic Nanocomposites Smart Materialsfor Bone Tissue Engineering

Everest Akaraonye1 Jan Filip2 Mirka Safarikova3 Vehid Salih45 Tajalli Keshavarz1

Jonathan C Knowles46 and Ipsita Roy1

1Applied Biotechnology Research Group Department of Life Sciences Faculty of Life Sciences University of WestminsterLondon W1W 6UW UK

2Regional Centre of Advanced Technologies and Materials Palacky University Slechtitelu 27 78371 Olomouc Czech Republic3Biology Centre AS CR ISB Department of Nanobiotechnology Na Sadkach 7 370 05 Ceske Budejovice Czech Republic4Department of Biomaterials and Tissue Engineering Eastman Dental Institute University College London London WC1X 8LD UK5Plymouth University Peninsula Schools of Medicine and Dentistry Portland Square Drake Circus Plymouth Devon PL4 8AA UK6WCU Research Centre of Nanobiomedical Science Dankook University San No 29 Anseo-dong Dongnam-guCheonan-si Chungnam Republic of Korea

Correspondence should be addressed to Ipsita Roy royiwminacuk

Received 12 January 2016 Revised 12 May 2016 Accepted 31 August 2016

Academic Editor Jean M Greneche

Copyright copy 2016 Everest Akaraonye et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The objective of this work was to investigate the potential application of Poly(3-hydroxybutyrate)magnetic nanoparticlesP(3HB)MNP and Poly(3-hydroxybutyrate)ferrofluid (P(3HB)FF) nanocomposites as a smart material for bone tissue repairThe composite films produced using conventional solvent casting technique exhibited a good uniform dispersion of magneticnanoparticles and ferrofluid and their aggregates within the P(3HB) matrix The result of the static test performed on the samplesshowed that there was a 277 and 327 increase in Youngrsquos modulus of the composite due to the incorporation of MNP andferrofluid respectively The storage modulus of the P(3HB)MNP and P(3HB)FF was found to have increased to 186 and 103respectively when compared to neat P(3HB) The introduction of MNP and ferrofluid positively increased the crystallinity ofthe composite scaffolds which has been suggested to be useful in bone regeneration The total amount of protein absorbed bythe P(3HB)MNP and P(3HB)FF composite scaffolds also increased by 91 and 83 respectively with respect to neat P(3HB)Cell attachment and proliferation were found to be optimal on the P(HB)MNP and P(3HB)FF composites compared to thetissue culture plate (TCP) and neat P(3HB) indicating a highly compatible surface for the adhesion and proliferation of the MG-63 cells Overall this work confirmed the potential of using P(3HB)MNP and P(3HB)FF composite scaffolds in bone tissueengineering

1 Introduction

Recently interest in tissue engineering and its solutions haveincreased significantly with bone and cartilage regenerationby autogenous cell delivery or tissue regeneration becomingone of the most promising modes of orthopaedic surgeryIn particular scaffolds have become fundamental tools inbone graft substitution and are used in combination with avariety of bioagents [1] Regenerative medicine benefits frombiocompatible scaffolds on which stem cells can grow and

differentiate either under preliminary ex vivo conditions forfurther grafting into the injured organ or as direct in vivoimplants Tissue engineering scaffolds offer microstructured2D or 3D surfaces for cell attachment differentiation andproliferation [2] In addition scaffolds provide biomechan-ical properties that are suitable for supporting novel tissuestructures and with no toxic effects However studies havedemonstrated the need for stimulators of cell attachmentas many polymeric materials used for scaffold fabricationinhibit cell attachment and proliferation In order to solve this

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2016 Article ID 3897592 14 pageshttpdxdoiorg10115520163897592

2 Journal of Nanomaterials

problem growth factors and cell ligands are incorporated asbiologically active molecules to facilitate cell attachment andexpansion

Current and future applications of magnetic nanoparti-cles in biology and medicine have been largely dependent ontheir nanometer-size particles (3ndash10 nm in diameter) whichexhibit novel magnetic chemical and biomedical properties[3] Maghemite (120574-Fe2O3) magnetite (Fe3O4) and FeIIFeIIIoxides are technologically important compounds widely usedfor the production of magnetic materials and catalysts Asa confirmation of cytocompatibility of magnetic material intissue engineering an emerging tissue engineering strategymagnetic force-based tissue engineering (Mag-TE) employscells that have been magnetically labelled with magnetitecationic liposomes (MCLs) Such MCL-labelled cells can bemanipulated and organised by magnetic force and theirfunctionality is maintained The use of magnetic material tomanipulate cells has been tested in many cell lines includingmesenchymal stem cells cardiomyocytes [4] human umbil-ical vein endothelial cells [5] retinal pigment epithelial cells[6] and keratinocytes [7]

In this study polymeric P(3HB) based magnetic ironoxide (mainly magnetite) composite scaffolds were suc-cessfully developed using a simple and inexpensive com-pression mouldingparticulate leaching technique The twodifferent types of magnetic composite scaffolds producedincluded P(3HB)magnetic nanoparticles P(3HB)MNP andP(3HB)ferrofluid P(3HB)FFThe developed magnetic scaf-folds were characterized for their magnetic properties as wellas the effects of addition of magnetic materials (particles andfluid) on the thermomechanical properties of the compositescaffolds FTIR was employed to study the crystallographicproperties of the scaffolds In vitro degradation study of thecomposite scaffolds was performed in simulated body fluid(SBF) to understand the effect of magnetic materials on thebiodegradation of the composite scaffoldMagnetite was usedfor the manipulation of the nano- and microenvironmentson P(3HB) scaffolds in order to promote protein adsorptionand subsequently stimulate cell proliferation Cytocompat-ibility studies on the magnetic scaffolds were carried outusing the human osteosarcoma MG-63 cell line and totalprotein production and cell proliferation were assessed Thisstudy confirmed the promising potential application of thedeveloped composites in bone tissue engineering and boneregeneration therapy

2 Experimental Procedures

21 Bacterial Strain Cells and Culture Medium Poly(3-hydroxybutyrate) was produced following previously devel-oped biotechnological methods [8] The proliferation assayswere performed using the HOS cell lineMG-63 grown in lowglucose Dulbeccorsquos Modified Eagle Medium (DMEM) sup-plemented with 10 fetal calf serum and 1 (wv) penicillinand 1 (wv) streptomycin solution

22 Magnetic Nanoparticle Preparation Magnetic nanocrys-tals of Fe3O4 were prepared by the classical coprecipitation

method where a solution containing ferric and ferrouschlorides was introduced in an alkaline solution [9] Tothe mixture of 20 g FeCl2sdot4H2O in 5mL 2M HCl and54 g FeCl3 sdot6H2O in 20mL 2M HCl 50mL 07M NH3 wasdropped under mixing The magnetic particles were washedwith water and acetoneThese particles were used in the formof aggregates particle size ca 100 nm (1mL of centrifugedsample corresponded to 67mg of dry weight)

23 Preparation of Magnetic Fluid (Ferrofluid FF) Stabilizedin Chloroform To the mixture of 20 g FeCl2 sdot4H2O in 5mL2M HCl and 54 g FeCl3 sdot6 H2O in 20mL 2M HCl 50mL07M NH3 was dropped under mixing Then concentrated(25) NH3 was added to the total volume 66mL and pHca 10 After that 13mL oleic acid was added and thoroughlymixed for 1 h Then the mixture was heated in water bath to95∘C with temperature increasing 2∘Cs (45ndash60min) Aftercooling down 15HNO3 was added to pH 5The suspensionwas then washed with water and subsequently with acetone(both four times) Magnetite in acetone was heated for 15 hunder mixing in water bath at 35ndash50∘C until all acetone wasevaporated Then chloroform was added in small portionsuntil ferrofluid was formed To remove solid particles theferrofluid was repeatedly centrifuged (90min 9500 rpm)under the same conditions up to 4ndash6 days The relativemagnetic fluid concentration (25mgmL) was determined bya colorimetric method (Fe3O4 used as standard) pH was ca70 [10]

24 Nanocomposite Film Preparation 1 g of P(3HB) wasdissolved in 10mL of CHCl3 at room temperature andstirred for 24 h The desired amount of magnetic nanoparti-clesferrofluid in CHCl3 was added to the P(3HB) solutionto give a final concentration of 14 27 and 54mgmL of eitherMNPor FF and themixturewas homogenized for 2minusinga homogenizer (Ultra-Turrax T25 basic Ika-Werke) Thesuspension was then degassed three times and poured intoa glass Petri-dish where the films were obtained by solventevaporation at room temperature Hence nanocompositefilms containing 14 27 and 54mgg of P(3HB)were prepared

25 Scanning Electron Microscopy (SEM) Scanning electronmicroscopy JEOL 5610LV instrument (JEOL USA) and anESEMFEIQuanta 200F was used to examine themicrostruc-ture of the test materials The samples were placed on 8mmdiameter aluminium stubs using a sticky tag to hold them Agold sputtering device (EMITECH-K550) was used to coatthe samples operating at a pressure of 7 times 10minus2 bar anddeposition current of 20mA for 2min Images were taken atvarious acceleration voltages (maximum of 20 kV) to avoidbeam damage on the polymer

26 SQUID Analysis A superconducting quantum inter-ference device magnetometer (SQUID MPMS XL-7 typeQuantum Design USA) was used for measurement of mag-netization of the solid samples The hysteresis loops of allstudied samples were collected at a temperature of 300Kunder an external magnetic field ranging from minus1 T to +1 T

Journal of Nanomaterials 3

(ie 10 kOe) For measurement a small sample (few mm3)of the scaffold was mounted onto the sample holder using aTeflon tape

27 Dynamic Mechanical Analysis (DMA) DMA experi-ments were carried out in tensile mode using a Perkin-Elmer Dynamic Mechanical Analyser (DMA 7e Perkin-Elmer Instruments USA) at room temperature Tensilestrength tests were conducted on flat specimens (width14mm length 7-8mm and thickness asymp100ndash120120583m) cut outfrom the solvent cast films To remove any trace of solventsamples were first heated from room temperature to 100∘Cat 10∘Cmin 1 Hz frequency and 001 of strain and thistemperature was kept for 10minThen dynamic cooling scanswere conducted from 100 to 30∘C at 2∘Cmin 1 Hz and 01of strain During the DMA experiment the static load waskept at 1mN and it was increased to 6000mN at a rate of200mNminminus1 Four repeat specimens were tested for eachsample and the average value was used

28 Infrared Spectroscopy (FTIR) Infrared spectra of driedtest samples were recorded using a FTS 6000 spectrometer(Portmann Instruments AG Biel-Benken Switzerland) Foreach sample the diamond crystal of an attenuated totalreflectance (ATR) accessory was brought into contact withthe area to be analysed The contact area was about 2mm2and a torque of 10 cNmwas used to ensure the same pressureon each sample All spectra were recorded between 4000 and400 cmminus1 with a resolution of 4 cmminus1 and 32 scans

29 Contact Angle Study In order to evaluate the wettabilityof the test materials static contact angle measurements werecarried out on each of the samples The experiment wascarried out on a KSV-Cam 200 optical contact angle meter(KSV Instruments Ltd Finland) An equal volume of water(20120583L) was placed on every sample by means of a gas tightmicrosyringe forming a meniscus Photos (frame intervals1 s number of frames 100) were taken to record the shapeof the meniscus The water contact angles on the specimenswere measured by analyzing the recorded drop images (fourrepeats for each sample) using theWindows based KSV-Camsoftware

210 Protein Adsorption Study Protein adsorption assay wascarried out on 2D (films) using fetal bovine serum (FBS)All measurements were carried out in triplicate per sampleThe samples were immersed with 200120583L of undiluted FBSin a 15mL Eppendorf and incubated at 37∘C for 24 hThe serum was then removed from the samples and thesamples were washed three times with phosphate buffersaline (PBS) The proteins adsorbed on the samples werecollected by incubating the samples with 1mL of 2 sodiumdodecyl sulphate (SDS) in PBS for 24 h at room temperatureand under vigorous shaking This was done to ensure theadsorbed proteins were dissolved in the PBS (with SDS)Amount of total protein adsorbed was measured using acommercial protein quantification kit (Qubit Protein AssayKits) Fluorescence was measured at 485590 nm against a

calibration curve using bovine serum albumin BSA (pro-vided in the kit)

211 Change in pH of Simulated Body Fluid SBF afterImmersion of Samples A total of nine samples from eachgroup of the test materials were immersed in SBF for 1 23 and 4 weeks and 20mL SBF was used for each sampleSBF was chosen because it has similar ionic concentrationto human blood plasma [11] The pH-values of SBF weremonitored every week by an electrolyte-type pHmeter (PHS-2C JingkeLeici Co Shanghai China) The values presentedwere the mean values of triplicate measurements

212 Cytocompatibility Studies In vitro cell culture stud-ies were performed using MG-63 osteoblast a humanosteosarcoma cell line Cells were cultured in DulbeccorsquosModified Eagles Medium (DMEM) from PAA Germanysupplemented with 10 (vv) fetal calf serum and 1 (vv)penicillin and streptomycin solution and incubated at 37∘C ina humidified atmosphere (5CO2 in 95 air) prior to its useThe samples were UV-sterilized for 30min and passivatedin DMEM culture medium for 12 h prior to seeding thecells The samples were placed in a polystyrene 24 well flatbottomed tissue culture plate with the film samples placedin the centre of the well Standard tissue culture plasticwas used as the control surface The specified amountsof cells (20000 cellssamples) were seeded using 50 120583L ofDMEM medium for initial attachment of the MG-63 on thesample surface The plates were incubated in a humidifiedenvironment (37∘C 5 CO2) for a period of up to 2 h andthen samples transferred to a new well-plate and replacedwith 1mL of DMEM medium The cells were then allowedto grow on the films (119899 = 3) for a period of up to 7 dayswith the medium changed every second day At specific timeintervals the cell proliferation measurements were carriedout using the Alamar blue (AbD Serotec UK) assay and thefluorescence of the samples was measured at 560 nm (119860560)and 590nm (119860590) Films were also examined under SEMJEOL 5610LV instrument (JEOL USA) after day 7 to assessthe cell spreading and attachment

3 Results

31 Microstructural Analysis of Neat P(3HB) P(3HB)MNPand P(3HB)FF Composites Microstructural analysis of theneat P(3HB) and the composite test materials (P(3HB)MNPand P(3HB)FF) were carried out using SEM Film samplescontaining 54mgmL of either MNPs or FF were shown toillustrate the differences in the surface morphology of thetested samples

The surface morphology of the films produced by solventcasting as observed from the SEM imaging is shown inFigure 1 The SEM image revealed micronanosurfaces onthe test materials Nanomicroscale crystals and sparselydistributed aggregates of nanoscale crystals were observed onthe surface of the P(3HB)MNP composite These nanoscalecrystals were more visible at a higher magnification (times500)image As expected the surfaces of the composite materialswere slightly rougher than that of neat P(3HB)


100120583m 100120583m

100120583m

300120583m

60120583m 40120583m

Figure 1 SEM image of (A1) neat P(3HB) (B1) P(3HB)MNP) (54mgmL) (C1) P(3HB)FF (54mgmL) at lower magnification (times150) andhigher magnification (A2) neat (P(3HB) (B2) P(3HB)MNP (54mgmL) (C2) P(3HB)FF (54mgmL) The arrows show the uniformlydispersed magnetic particles

MNP

P(3HB)

(a)

P(3HB)FF

(b)

Figure 2 Electron diffraction patterns of (a) P(3HB)MNP and (b) P(3HB)FF composite films

32 Morphology and Structure of Magnetic Materials inthe Composite Scaffolds Electron diffraction patterns ofthe test materials are presented in Figure 2 The electrondiffraction patterns show a randomly oriented Fe3O4 in theP(3HB)MNP and P(3HB)FF composite scaffolds

33 Magnetic Properties of P(3HB)MNP and P(3HB)FFComposite Material Magnetic properties of the biodegra-dable nanocomposite scaffolds were investigated usingsuperconducting quantum interference device (SQUID) asdescribed in Section 26 For each sample the magnetizationat 300K was measured over a range of applied fields betweenminus10000 and +10000Oe Figure 3(a) shows the neat P(3HB)having a very low maximum magnetization 119872max+ at 1 T(0004 emug) while Figure 3(b) shows that near plusmn1 themagnetization reached a saturation value (0004 06182 and08707 emug) roughly proportional to the MNP content(14 27 and 54mg) in the scaffolds Figure 4(a) featuresthe hysteresis loops of the composite scaffold produced byaddition of ferrofluid (FF) (54mg ca = 10ndash15 nm) whileFigure 4(b) features the hysteresis loops of the scaffold

produced by the incorporation of magnetic nanoparticles(MNP) (54mgmL) Figures 4(a) and 4(b) show that thehysteresis loops of P(3HB)FF and P(3HB)MNP compositesalmost saturate at plusmn1 Tesla without coercivity or remnantmagnetization (ie ascending and descending curves arealmost the same)

34 Mechanical andThermomechanical Properties of P(3HB)MNP and P(3HB)FF Composite Dynamic mechanical anal-ysis DMA has been demonstrated to be useful in evaluatingthe viscoelastic properties of polymers [12 13] For polymericmaterials the following equations hold [14]

119864 =120590

120576= 1198641015840 + 11986410158401015840

tan 120575 = 1198641015840

11986410158401015840

(1)

where 119864 is the dynamic modulus 1198641015840 is termed storage modu-lus 11986410158401015840 is the loss modulus 120590 (sigma) is stress 120576 (epsilon) ispercentage change in strain and tan 120575 (tan delta) is the ratio


P(3HB)T = 300K

minus20

minus15

minus10

minus5

0

5

10

15

20M

agne

tizat

ionM

(10minus3

emu

g)

minus8 minus6 minus4 minus2 0 2 4 6 8 10minus10

Intensity of magnetic field H (kOe)

(a)

T = 300K

minus08

minus06

minus04

minus02

00

02

04

06

08

Mag

netiz

atio

nM

(em

ug)


Intensity of magnetic field H (kOe)54mg27mg

14mg

minus 06

minus 04

minus 02

00

02

04

06

M(e

mu

g)

minus05 00 10minus10 05

H (kOe)

(b)

Figure 3 Hysteresis loops of SQUID measurement of (a) neat P(3HB) and (b) P(3HB)MNP composite film containing different amountsof MNP (14 27 and 54mg)

T = 300K

minus05 00 05 10minus10

H (kOe)

minus 08

minus 06

minus 04

minus 02

00

02

04

06

08

M(e

mu

g)



minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

Mag

netiz

atio

nM

(em

ug)

(a)

T = 300K



minus12

minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

12

Mag

netiz

atio

nM

(em

ug)

minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

M(e

mu

g)


H (kOe)

(b)

Figure 4 Hysteresis loops of SQUID measurement of (a) P(3HB)FF composite film (54mgmL average particle size ca = 10ndash15 nm) andP(3HB)MNP composite (54mg ca = 100 nm)

of the energy dissipated per cycle to the energy stored duringthe cycle

Table 1 shows Youngrsquos modulus of the P(3HB)MNP andP(3HB)FF composites which were significantly influencedby the incorporation of magnetic oxides either as nanopar-ticles or as ferrofluid Youngrsquos modulus of the neat P(3HB)measured was 022 plusmn 004GPa This value increased to 083

plusmn 02 and 094 plusmn 04GPa on addition of 14mg of MNP and14mg of ferrofluid respectively to 1 g of the P(3HB) matrixto form composites (Figure 5) The result of the static testthus showed that there was a 277 and 327 increase inYoungrsquos modulus of the composite due to the incorporationof 14mg of either MNP or ferrofluid respectively Statisticallysignificant differences (119901 lt 005) were observed between


Table 1 Mean and standard deviation of storage modulus (1198641015840) and loss modulus (11986410158401015840) for neat P(3HB) P(3HB)MNP and P(3HB)FFcomposites at different temperatures

Measurement Material Temperature (∘C)minus20 0 20 50

Storage modulus (GPa)Neat P(3HB) 098 plusmn 07 086 plusmn 04 074 plusmn 06 061 plusmn 04P(3HB)MNP 277 plusmn 07 228 plusmn 05 187 plusmn 030 136 plusmn 05P(3HB)FF 197 plusmn 05 159 plusmn 01 130 plusmn 03 095 plusmn 01

Loss modulus (GPa)Neat P(3HB) 009 plusmn 002 008 plusmn 002 006 plusmn 001 006 plusmn 004P(3HB)MNP 076 plusmn 03 068 plusmn 01 006 plusmn 001 005 plusmn 001P(3HB)FF 084 plusmn 02 067 plusmn 01 049 plusmn 01 034 plusmn 03

tan 120575Neat P(3HB) 009 plusmn 002 009 plusmn 004 008 plusmn 003 010 plusmn 007P(3HB)MNP 027 plusmn 004 029 plusmn 009 026 plusmn 002 037 plusmn 002P(3HB)FF 043 plusmn 002 042 plusmn 001 038 plusmn 003 036 plusmn 001

MNP (54mgmL) FF (54mgmL) and tan 120575 (tan delta) (119899 = 3 error = plusmnSD)

00

02

04

06

08

10

12

Youn

grsquos m

odul

us (G

Pa)

P(3HB)MNP P(3HB)FFNeat P(3HB)

Figure 5 Youngrsquos modulus (119864) measurement for neat P(3HB)P(3HB)MNP and P(3HB)FF composite containing 14mg of eitherMNP or ferrofluid solution in 1 g of P(3HB) (Error bars = plusmnSD)

the elastic moduli of the P(3HB)MNP and P(3HB)FF com-posites

Figures 6(a) and 6(b) highlight the result of the dynamicmodulimeasured onboth the neat P(3HB) andP(3HB)MNPand P(3HB)FF composites The dynamic storage and lossmodulus were obtained in the temperature range of minus20∘Cand 130∘C and a typical profile of the data obtained is shownin Figures 6(a) and 6(b) A detailed set of data with standarddeviations on the three sets of analyses performed is shown inTable 1 The result shows that the storage modulus increaseddue to the incorporation of either magnetic nanoparticlesor ferrofluid to the polymeric matrix However the storagemodulus of the P(3HB)MNP composite was found to begreater than the storage modulus measured for both neatP(3HB) and P(3HB)FF composite At minus20∘C the storagemodulus was found to be 097 plusmn 07 277 plusmn 07 and 197 plusmn05GPa for the neat P(3HB) P(3HB)MNP and P(3HB)FFcomposites respectivelyMoreover both the storagemodulusand loss modulus were found to decrease with increase intemperature Apart from dynamic storage and loss modulustan 120575 (which is the ratio of the energy dissipated per cycle to

the energy stored during the cycle) was used to quantify theinternal friction existing in the material The test performedclearly revealed that tan 120575 increased with the addition ofeither magnetic nanoparticles or ferrofluid to the polymericmaterial Also tan 120575 decreased with increase in temperaturein both P(3HB)MNP and P(3HB)FF composite materialsbut increased with increase in temperature above 20∘C forneat P(3HB) (Table 1)

35 Fourier Transform-Infrared FTIR Analysis Fouriertransform infrared (FTIR) spectroscopy was used to identifyfunctional groups and obtain structural information of neatP(3HB) and the composites In any miscible compositespolymers containing the carbonyl group usually are involvedin some interaction such as hydrogen bonds hence a shift ofthe adsorption wavelength corresponding to the C=O groupis usually observed

The FTIR spectra of P(3HB) P(3HB)MNP and P(3HB)FF composites are shown in Figure 7 The strong and sharptransmittance band at 1720 cmminus1 can be assigned to the C=Ostretching mode in both the neat P(3HB) and the compo-site specimens The peaks at 1275 cmminus1 and 1226 cmminus1 canbe assigned to the C-O-C stretching modes in the com-posites The peak at 1180 cmminus1 is attributed to the C-O-Cstretching band corresponding to the amorphous state [15]Generally the FTIR spectra of the composite specimens showmodified bands in shape and intensity when compared tothose characteristics of neat P(3HB) Most of the P(3HB)bands that are sensitive to the crystallinity of the samplebecame sharp and increased in intensity with the additionof either magnetic nanoparticles or ferrofluid Furthermorethe intensity of these bands increased more with the additionof ferrofluid than on addition of MNP For instance theintensity of the peaks at positions 2978 1720 1378 1275 11291050 909 and 592 cmminus1 increased in the order P(3HB) ltP(3HB)MNP lt P(3HB)FF Also conspicuous was the shiftin bands with the addition of the fillers The 591 cmminus1 peak inthe neat P(3HB) spectrum shifted to 592 cmminus1 in P(3HB)FFand 593 cmminus1 in P(3HB)MNP while the band at position


Neat P(3HB)P(3HB)FFP(3HB)MNP

Temperature (∘C)150100500

000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

175e + 9

200e + 9

225e + 9

250e + 9

275e + 9St

orag

e mod

ulus

(GPa

)

(a)



minus500e + 8

minus250e + 8

000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

Loss

mod

ulus

(GPa

)

(b)

Figure 6 Typical plot of (a) storage modulus (solid lines) and (b) loss modulus (dotted lines) of neat P(3HB) P(3HB)FF and P(3HB)MNPcomposites

2978

1720

1378

1275 1050

591

P(3HB)

P(3HB)FFP(3HB)MNP

minus04

minus02

00

02

04

06

08

Tran

smitt

ance

()

2500 10003000 15002000 500Wave number (cmminus1)

Figure 7 FTIR spectra of neat P(3HB) (blue line) P(3HB)MNP(54mgmL) (black line) and P(3HB)FF (54mgmL) (red line)

1451 cmminus1 in the neat P(3HB) shifted to 1454 cmminus1 on addi-tion of either magnetic nanoparticles or ferrofluid Besidesadditional bands were also found at position 511 cmminus1 in theP(3HB)MNP composite and 1556 cmminus1 in both compositespecimens (P(3HB)MNP and P(3HB)FF) which were dueto the presence of the fillers

36 HydrophilicityDetermination Thewater contact angle ofthe composites differed significantly from that of the neatP(3HB) as seen in Figure 8 While the water contact anglemeasured on the neat P(3HB) specimen was 681 plusmn 7 thosemeasured on the P(3HB)MNP and P(3HB)FF were 597 plusmn41 and 632 plusmn 64 respectively Hence the hydrophilicity of

P(3HB)MNP P(3HB)FFP(3HB)0

20

40

60

80M

ean

cont

act a

ngle

(deg

)

Figure 8 Water contact angles for neat P(3HB) P(3HB)MNP(54mgmL) and P(3HB)FF (54mgmL) composite films (119899 = 3error = plusmnSD)

the composite specimens increased by 12 and 7 on addi-tion of magnetic nanoparticles and ferrofluid respectively

37 Total Protein Adsorption on the P(3HB)MNP andP(3HB)FF Composite Films The total amount of proteinadsorbed onto the composite disc in 120583gcm2 is shown inFigure 9 For all the specimens the total amount of adsorbedprotein increased significantly with the addition of eithermagnetic nanoparticles or ferrofluid The total amount ofprotein absorbed by the P(3HB)FF and P(3HB)MNP com-posite scaffolds increased by 83 and 91 respectively withrespect to neat P(3HB) A statistically significant difference(119901 lt 005) was found between total proteins absorbed byeither P(3HB)MNP or P(3HB)FF and the neat P(3HB)However among the composite specimens (P(3HB)MNPand P(3HB)FF) the extent of protein adsorption did notdiffer significantly though both composites followed the

8 Journal of NanomaterialsPr

otei

n co

ncen

trat

ion

(120583g

cm2)

0

50

100

150

200

P(3HB)MNP P(3HB)FFP(3HB)

Figure 9 Total protein adsorption study on P(3HB)MNP(54mgmL) and P(3HB)FF (54mgmL) films using fetal bovineserum (FBS) (119899 = 3 error bars = plusmnSD)

P(3HB)MNPP(3HB)FF

P(3HB)

25 3020155 100Time (days)

7

75

8

pH

Figure 10 Change in pH of SBF with time of immersion of P(3HB)P(3HB)MNP (54mgmL) and P(3HB)FF (54mgmL) compositefilms

same trend of increase in total protein adsorption comparedto neat P(3HB)

38 pH Changes of the SBF Solution Immersed with NeatP(3HB) and the Composites The pH of the immersed SBFfor neat P(3HB) and composite materials exhibited initialincrease in value and later decreased with increase in theincubation time However the pH of the SBF solution withneat P(3HB) film increased mostly after 7 days of incuba-tion followed by P(3HB)FF and P(3HB)MNP respectively(Figure 10)

39 Cytocompatibility Study of Neat P(3HB) P(3HB)MNPand P(3HB)FF Composites Themagnetic composites devel-oped in this work were further assessed for cytocompatibil-ity an essential property for a tissue engineering scaffoldThe increased hydrophilicity of the composites and themicronanosurfaces was expected to allow cell adhesion pro-liferation and functioning of human osteoblasts The humanosteoblast-like cell line MG-63 was used as the prototypefor osteoblastic cells This human osteosarcoma MG-63 cell

Time (days)1 3 6

0

40

80

120

Relat

ive c

ell p

rolif

erat

ion

()

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast

lowastlowastlowast

TCPNeat P(3HB)

P(3HB)MNP (54mgmL)P(3HB)FF (54mgmL)

Figure 11 Cell proliferation study using Alamar blue assay forTCP neat P(3HB) P(3HB)MNP (54mgmL) and P(3HB)FF(54mgmL) composite film performed on days 1 3 and 6 Allsamples are tested relative to the control set at 100 (119899 = 3 error =plusmnSD significant at lowast119901 = 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 005)

line has been extensively characterized and validated as amodel to test biocompatibility of various materials intendedfor bone tissue engineering [16] Despite being a tumourcell line MG-63 exhibits many osteoblastic traits includinghigh levels of 125-dihydroxyvitamin D3 (125-(OH)2D3)responsive alkaline phosphatase activity and the inhibition ofcell proliferation after 125-(OH)2D3 treatment Studies havealso shown its ability to synthesize osteocalcin and collagentype 1 which are characteristic of bone-forming cells Tissueculture plastic (TCP) was used as the positive control onwhich all cell types are known to attach and proliferate

391 Cell Proliferation Study The in vitro biocompatibilityof the composite materials was investigated using the MG-63 cell line described above A histogram representing cellproliferation and growth on the composite films produced byincorporation of either 14mg MNP or ferrofluid to the poly-mer matrix is shown in Figure 11 The result shows that thecells proliferated and grew well on all the tested samples Thecell proliferation on neat P(3HB) was found to be lower thanthat on TCP and the composite materials In contrast with thecontrol theMG-63 cells proliferated better on the P(3HB)FFcomposite material Thus a significant difference (119901 lt 001)was found in cell proliferation between the P(3HB)MNPandP(3HB)FF composite films At day 6 of the cell proliferationstudy the cell proliferation on P(3HB)MNP and P(3HB)FFcomposite specimens wasmuch better as compared to that onneat P(3HB) films

392 Total Protein Production by Human Osteosarcoma MG-63 Cell Lines on the P(3HB)MNPand P(3HB)FF CompositesThe total protein production by the MG-63 cell lines on thecomposite specimens was investigated from the supernatantof the cells grown in both osteogenic media and normalgrowth media at different time points Since total proteinproduction by the cells correlates with mineralisation by thegrowing cells the protein produced by the growing MG-63


lowast

lowastlowast

lowastlowast lowast

0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

TCPP(3HB)


Figure 12 Protein production by MG-63 cells on TCP P(3HB)P(3HB)MNP (54mgmL) and P(3HB)FF (54mgmL) grown inosteogenic media at different time points Samples were measuredrelative to the control set at 100 (TCP) (119899 = 3 error = plusmnSD andlowast119901 lt 001)

cells on days 1 7 14 and 21 was quantified using commercialQubit Protein Assay Kits purchased from Invitrogen

Figures 12 and 13 show the histogram of the total proteinproduced by the growing cells on the testmaterials at differenttime points Total protein produced was measured for MG-63 cells grown on the TCP (control) neat P(3HB) and thecomposite specimens grown in both osteogenic and normalgrowth media A statistical difference (119901 lt 001) was foundbetween total protein produced by the MG-63 cells culturedin osteogenic media and those cultured in normal growthmedia Furthermore statistical difference (119901 lt 001) wasfound between total protein produced by the MG-63 cellsgrown on P(3HB)FF and P(3HB)MNP substrates and TCPafter 14 days of culture

310 Cell Morphology Figure 14 shows MG-63 cells grownon TCP neat P(3HB) P(3HB)MNP and P(3HB)FF filmson day 1 and day 7 Both P(3HB)MNP and P(3HB)FFcomposite films contained 14mg of either magnetic particlesor magnetic fluid The figure highlights the attachment ofcells on the surface of the materials Also MG-63 cells werefound to be flattened on the magnetic composite films asobserved on either the tissue culture plastic or the P(3HB)films indicating that the composite materials were conducivefor cell adhesion and proliferation At day 7 the MG-63 cellsspread very well throughout the surface of both TCP and thetest materials forming a monolayer on the surfaces

4 Discussion

Recently much effort is being dedicated towards the devel-opment of sustainable technologies for the fabrication ofcustomized tissue engineering scaffolds with reproducibleinternal morphology that can ensure enhanced oxygen and

0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

lowast

ControlP(3HB)


Figure 13 Protein production by MG-63 cell lines on the controlP(3HB) P(3HB)MNP (54mgmL) and P(3HB)FF (54mgmL)with normal growth media at different time points Samples weremeasured relative to the control (TCP) set at 100 (119899 = 3 error bars= plusmnSD and lowast119901 lt 001)

nutrient transport throughout the scaffold Among the suc-cessfully developed scaffolds there are still limitations anddifficulties in controlling cell differentiation and angiogenesisas well as obtaining stable scaffold implantation in the patho-logical site Hence the conceptual provision of a scaffoldthat can not only provide architectural frame work andphysicomechanical support but also enhance cell growthproliferation and differentiation in vivo has been proposedin this study Two different forms of magnetite (MNPsand FF) were incorporated into polymeric P(3HB) in orderto achieve these objectives The SEM imaging confirmedthe 2D composite materials to have micronanosurfacesMisra et al have observed similar micronanosurfaces onthe addition of Bioglass in the P(3HB) matrix to produceP(3HB)Bioglass 2D and 3D composites [17] The crystalsfound on the surface of the P(3HB)MNP composite were themagnetic particles incorporated within the polymer matrixThe well dispersed nanoparticles and their aggregates areknown to be very useful in the provision of large surfacearea for protein adsorption and cellular adhesions Alsothe presence of the highly distributed magnetic particlesand aggregates of MNPs is expected to aid in reducingthe hydrophobic properties of the polymer matrix Electrondiffraction patterns of the composite materials confirmedthe presence of the magnetic nanoparticles and ferrofluid aswell as the homogenous distributions of aggregates of theincorporated materials throughout the composite specimensThe diffraction rings of magnetite and polymeric P(3HB)shown in Figure 2 indicated that aggregates of magnetitenanoparticles and ferrofluid were randomly distributed inthe composite films (P(3HB)MNP and P(3HB)FF) Thisobservation is in agreementwith the crystallographic featuresobserved in the XRD spectra of the composite samples(figure not shown) The homogenous distribution of the


times150

times200

times2000

Figure 14 SEM images of MG-63 cells growing on (A1) TCP (B1) neat P(3HB) (C1) P(3HB)MNP and (D1) P(3HB)FF film on day 1 SEMimages ofMG-63 cells growing on (A2) tissue culture plastic (B2) neat P(3HB) (C2) P(3HB)MNP and (D2) P(3HB)FF film at day 7 Highermagnification of MG-63 cells growing at day 7 revealing healthy cells that flattened out and attached on the surface of the materials and oneach other forming a monolayer on (A3) tissue culture plastic (B3) neat P(3HB) (C3) P(3HB)MNP and (D3) P(3HB)FF films Arrowshighlight cells attached on the materials

incorporated materials in the composites is necessary toobtain composites with high mechanical performance anduniform degradation kinetics throughout the materials

The hysteresis loops of the P(3HB)magnetic nanoparti-cles and P(3HB)magnetic fluid composites almost saturateat plusmn1 T without coercivity or remnant magnetization Thisbehaviour is typical for paramagnetic or superparamagneticmaterials As shown in Figures 3 and 4 all the magnetichysteresis loops passed through the grid origin In additionit was also shown that the residual magnetization (120590119903) of allthe particles was zero This shows that both the magnetitenanoparticles and ferrofluid composites have good super-paramagnetism (ie small superparamagnetic iron oxideparticles lt20 nm forming asymp100 nm large aggregates) Alsocomparing their saturation magnetization (120590119904) it is apparentthat 120590119904 increased as the amount of magnetite nanoparticlesin the scaffold increased The absence of a coercive fieldat temperatures close to 300K is characteristic of super-paramagnetic material and therefore confirmed that bothP(3HB)MNP and P(3HB)FF composites had superparam-agnetic properties [18] For a superparamagneticmaterial theresulting magnetic scaffold may reach appropriate magneti-zation values (ie up to 15 emu gminus1 at 10 kOe) upon applica-tion of an externalmagnetic fieldThesemagnetization values

can in future be used to attract cells or other bioagents boundto the MNPs [19] Also the superparamagnetic propertiesconfirm the ability of the scaffold to be magnetized byapplying amagnetic fieldwithout any remnantmagnetizationonce the field is removed [1 20 21]The hysteresis loop of neatP(3HB) was found to be composed of two magnetic phasesfirst corresponding to the paramagnetic or superparamag-netic phase and second belonging to the diamagnetic phaseas is evident from the profile of the hysteresis curve at lowerand higher applied fields respectively The appearance ofweak paramagnetic or superparamagnetic phase could haveresulted from contaminations during sample preparation Asexpected neat P(3HB) has a very low maximum magneti-zation (119872max+) at 1 T (0004 emug) when compared to thecompositematerialsHence neat P(3HB) can be confirmed tohave negligible magnetic properties and would not produceany effects when an external magnetic field is applied

The result of the DMA analysis shown in Figure 5demonstrated that the mechanical properties of P(3HB)increased with the addition of either magnetic nanoparticlesor ferrofluid The increase in the mechanical properties ofthe composite materials was possibly due to enhanced crys-tallinity which in turn could be due to spherulite formationStronger intermolecular interactionwithin the lamellae of the


spherulite could account for the increased Youngrsquos modulusachieved in the compositematerials Similar trends have beenobserved for tensile strength yield stress and toughness byEhrenstein and Theriault on isotactic polypropylene [22]Hence the addition of MNP and ferrofluid had a positiveeffect on enhancing Youngrsquos modulus of the compositematerials Misra et al Chen and Wang and Wang et al havereported similar increase in Youngrsquos modulus on the additionof bioceramics to a polymer matrix [12 23 24] Enhancedmechanical performances of the composite materials arehighly desirable in the context of bone tissue engineering ahigh load bearing application as compared to soft tissues [25]

The important observations made from the dynamicmodulus (storage and loss modulus) analysis on thematerialsare firstly the addition of either MNPs or FF effectivelyincreased the storage modulus of the polymer as can be seenin Table 1 It is possible that the storage modulus increasewas due to partial immobilisation of the polymer chainsas a result of adsorption onto the filler surface Secondlythe addition of the magnetic materials also resulted in anincrease in tan 120575 hence the damping capacity of the materialincreased Such behaviour can occur due to the introductionof new damping mechanisms that are not present in the neatpolymer Some of the possible explanations for this increaseare (a) friction within the MNPs where particles touchone another as in weak agglomerates (b) friction betweenP(3HB) and MNPs where there is essentially no adhesionat the interface and (c) excess damping (tan 120575 ratio of theloss to storage modulus) in the polymer near the interfacebecause of induced thermal stresses or changes in polymerconformation due to incorporation of filler Finally thedynamic modulus studies revealed that the storage moduluswas found to increase with the addition of either MNPsor FF On increasing the temperature the storage moduluswas found to decrease more in the composite materials thanin the neat P(3HB) sample This further confirmed a weakpolymerMNP interfacial interaction

The FTIR spectra of the composite materials actuallyshowed modified adsorption peaks in shape and intensitywhen compared to the characteristic peaks of neat P(3HB)This could be due to hydrogen and ionic bond interactionsbetween the polymeric matrix and the magnetite Millanet al have observed similar weak hydrogen and strongionic bonds during the preparation of maghemite polymernanocomposite [26] The result of the analysis indicated thatthe crystallisation of the polymer matrix was affected by theadditionintroduction of the fillers This has therefore furtherstrengthened the possibility that changes in the crystallineproperties of the polymer microstructure occurred as a resultof the crystallisation process of the nanocomposite materialon introduction of the fillers

The surface property of biomaterials plays an impor-tant role in their performance in a biological environmentMuch research has been conducted to analyse the effects ofhydrophobicity and hydrophilicity on biological responses(ie protein adsorption and cell adhesion) Improved surfacewettability generally improves the interactions between thecompositematerials and the cells and results in controlled cel-lular adhesion and maintenance of differentiated phenotypic

expression [27] Many researchers including Li et al haveinvestigated the effect of addition of inorganic materials to abiodegradable polymer matrix Li et al in their investigationswith wollastonite composite scaffold observed that the incor-poration of wollastonite to the P(3HB-co-3HV) polymermatrix improved the hydrophilicity of the composite [28] Inthis study the introduction of either magnetic nanoparticlesor ferrofluid to the P(3HB) polymer matrix significantlyimproved the hydrophilicity of the composite material TheMNPs were washed with water and acetone after preparationand later dispersed in chloroform and used for compositefabrication This explains why the MNPs are hydrophilic andsubsequently reduced the hydrophobicity of the compositesupon their incorporation In the case of the FF thoughafter washing with water and methanol after preparation theFF was coated with oleic acid before heating under refluxThe coating with oleic acid reduced the hydrophilicity ofthe FF hence the slight reduction in hydrophilicity afterincorporation in the composite when compared to the neatP(3HB) and composite containing MNPs

Detailed knowledge of the relationship between thesurface properties of a biomaterial and its ability to absorbprotein when exposed to a protein-containing medium isvery important in the application of a specific biomate-rial in tissue regeneration Apart from the very importantphysicomechanical properties a biomaterial should be ableto support cell adhesion proliferation and differentiationand these can be achieved with the help of protein layerswhich provide support for the anchorage of cells onto abiomaterial When a biomaterial is exposed to cells sus-pended in a culture medium supplemented with FBS proteinin the serum is rapidly adsorbed onto the surface of thebiomaterial prior to cell adhesion The adsorption of celladhesive serum proteins such as fibronectin and vitronectinplays a critical role in cell adhesion onto a biomaterial surfaceand subsequently determines cell adhesion behaviour [29]The adsorption of serum protein onto the surface of the 2Dmagnetic nanocomposite was possibly due to the synergeticcontribution of increased hydrophilicity surface chemistryand surface charge provided by the incorporated magneticmaterials Also the larger surface area provided by themagnetic nanoparticles possibly played an important role inthe increased protein adsorption observed on the compositematerial

Previous research has shown that very hydrophobicmaterials such as polytetrafluoroethyene (PTFE) with awater contact angle between 105∘ and 116∘ inhibit cellularadsorption In contrast hydrophilic materials such as tissueculture plastic are known to support cellular adhesion [30]The observations made in this study using the Alamar blueassay showed that the cells were able to attach to the surfaceof the materials (ie TCP neat P(3HB) P(3HB)MNP andP(3HB)FF) on the first day of the assay This was most likelydue to the absorbed protein layer which in turn enabledanchorage for the cells However as the incubation timeincreased it was found that cell proliferation on the neatP(3HB) film reduced as compared to the cell proliferationon the control and the composite materials This couldbe explained based on the possibility that the absorbed


protein on the surface of the neat P(3HB) interacted withthe hydrophobic surface of the neat P(3HB) in a manner thatled to changes in the protein conformation and thus resultedin reduced access of the cells to the ligand moieties neededfor integrin binding and cell attachment Cell proliferationwas found to increase more with time on the magneticnanocomposites than on TCP and the neat P(3HB) This isprobably due to the ability of themagnetic compositematerialto provide large surface area and good surface chemistrywhich enhanced protein adsorption onto the surface ofthe materials This result was in agreement with the watercontact angles and protein adsorption measured thereforeconfirming that the magnetic nanocomposites are excellentbiomaterials for cellular attachment and proliferation Thefact that the magnetic materials supported cell attachmentand proliferation showed that they are highly cytocompatibleand can be safely used to improve mechanical properties aswell as provide nanostructured surfaces for cell adhesion andproliferation in tissue regeneration

It has been suggested that increased bone formation leadsto increased total protein production by the bone cells [31]The result of the investigation depicted in Figure 14 showsthat neither MNPs nor ferrofluid had any adverse effecttowards proteins secreted by the cells

Gopferich has observed that pH is an important factorthat influences the rate of hydrolysis during the degradationof the polymers [32] The pH of SBF in which both types ofsamples were immersed increased a little above the initial pHof the buffer (735) Since the degradation product of P(3HB)is a 3-hydroxy butyric acid and the pKa of 3-hydroxybutyricacid is 470 at pH 735 the 3-hydroxybutyric acid will be inthe anionic form that is the basic form leading to a risein pH of the SBF However once the concentration of 3-hydroxybutyric acid reaches beyond a critical concentrationdue to the degradation of the polymer the SBF is no longerable to maintain the pH at 735 and the pH falls slightly dueto the acidic nature of 3-hydroxybutyric acid

In vivo biomaterial surfaces are usually coated bycomponents present in the extracellular fluid Howeverthe adsorption of extracellular fluid to the biomaterial islargely dependent on the surface chemistry charge wet-tability and free energy of the biomaterial which is reg-ulated by the microstructural features on the biomaterialsurface The spreading and proliferation observed after day1 is possibly due to material surface-protein interactionsmicronanosurfaces and chemistry which favoured adhe-sion proliferation and differentiation of cells

Cell responses to micronanosurfaces are expressed intheir ability to attach proliferate and differentiate [33]Ordinarily it is difficult to determine which structures areresponsible for individual phenotypic traits expressed bycells grown on different materials The cells were found tohave anchored to the surfaces of the materials tested withflattened morphology that were most often characterizedby extended filopodia reaching out to neighbouring cellsIt is possible that favourable nanostructural surfaces of thematerials provided by the fillers could have influenced thespreading of the filopodia by the cells Boyan et al havesuggested that architectural features present on biomaterial

surface could have influence on the morphology of thecells [34] Such architectural features include micro roughsurfaces and shorter peak-to-peak distances (Figure 1) ofpores comparable to the length of the cell body Boyan etal later concluded that cells growing on biomaterial surfaceswith these features are prone to exhibit cuboidal shape whileanchoring to the surface with long dendritic filopodia [34]Brunette et al on the other hand observed that cells spreadout and lay flat resulting in a fibroblastic appearance onsmooth surfaces [35 36] However the cells seeded onto bothTCP and the 2D test materials conform to the characteristicbehaviour of osteoblasts on smooth surfaces In addition thecells grown on the magnetic composite materials producedthread-like denticles which are crucial for cell to cell com-munications

5 Conclusions

This work presents an in-depth analysis of the potentialapplication of P(3HB)MNPandP(3HB)FF nanocompositesfor bone tissue repair The favourable thermomechanicalproperties in combinationwith the biocompatibility achievedin this work provided evidence of the future potential ofthe P(3HB)MNP and P(3HB)FF composites in bone tissueengineering Further studies using these promising materialswill involve 3D scaffold fabrication and detailed in vivo workwhich will allow the generation of preclinical data Hencein conclusion the results obtained in this work confirmthe huge potential of the P(3HB)MNP and P(3HB)FFcomposites in the development of bone tissue repair implantsto meet the current unmet needs in magneto-mechanicalstimulationactivation of cells magnetic cell-seeding andcontrolled cell proliferation and differentiation in bone repair

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

Thiswork was partly supported by theMinistry of EducationYouth and Sports of the Czech Republic (Project no LO1305and the Research Infrastructure NanoEnviCz Project noLM2015073) and by the Czech Science Foundation (Projectno 14-11516S) The authors also thank Jirı Tucek for theSQUID measurements and Ondrej Malina for technicalassistance (both are from Regional Centre of AdvancedTechnologies and Materials Palacky University OlomoucCzech Republic) Dr Everest Akaraonye acknowledges theUniversity of Westminster Scholarship Committee for theaward of the Cavendish Scholarship which financially sup-ported him during this work

References

[1] N Bock A Riminucci C Dionigi et al ldquoA novel route inbone tissue engineering magnetic biomimetic scaffoldsrdquo ActaBiomaterialia vol 6 no 3 pp 786ndash796 2010


[2] J L Corchero J Seras E Garcıa-Fruitos E Vazquez andAVil-laverde ldquoNanoparticle-assisted tissue engineeringrdquo Nanobio-tech pp 13ndash15 2010

[3] D K Kim M Toprak M Mikhailova et al ldquoSurface mod-ification of superparamagnetic nanoparticles for in-vivo bio-medical applicationsrdquo Materials Research Society SymposiumProceedings vol 704 pp 369ndash374 2002

[4] K Shimizu A Ito J-K Lee et al ldquoConstruction of multi-layered cardiomyocyte sheets using magnetite nanoparticlesand magnetic forcerdquo Biotechnology and Bioengineering vol 96no 4 pp 803ndash809 2007

[5] H Akiyama A Ito M Sato Y Kawabe and M KamihiraldquoConstruction of cardiac tissue rings using a magnetic tissuefabrication techniquerdquo International Journal of Molecular Sci-ences vol 11 no 8 pp 2910ndash2920 2010

[6] A Ito E Hibino C Kobayashi et al ldquoConstruction anddelivery of tissue-engineered human retinal pigment epithelialcell sheets using magnetite nanoparticles and magnetic forcerdquoTissue Engineering vol 11 no 3-4 pp 489ndash496 2005

[7] A Ito M Hayashida H Honda et al ldquoConstruction andharvest of multilayered keratinocyte sheets using magnetitenanoparticles and magnetic forcerdquo Tissue Engineering vol 10no 5-6 pp 873ndash880 2004

[8] E Akaraonye C Moreno J C Knowles T Keshavarz andI Roy ldquoPoly(3-hydroxybutyrate) production by Bacillus cereusSPV using sugarcane molasses as the main carbon sourcerdquoBiotechnology Journal vol 7 no 2 pp 293ndash303 2012

[9] RMassart ldquoPreparation of aqueousmagnetic liquids in alkalineand acidic mediardquo IEEE Transactions on Magnetics vol 17 no2 pp 1247ndash1248 1981

[10] H Kiwada J Sato S Yamada and Y Kato ldquoFeasibility ofmagnetic liposomes as a targeting device for drugsrdquo Chemicaland Pharmaceutical Bulletin vol 34 no 10 pp 4253ndash42581986

[11] T Kokubo N Takashi and M Fumiaki ldquoApatite formation onceramics metals and polymers induced by a CaO-SiO2 basedglass in a simulated body fluidrdquo in Bioceramics W Bonfield GW Hastings and K E Tanner Eds Oxford UK Butterworth-Heinemann 1991

[12] S K Misra P C P Watts S P Valappil S R P Silva I Roy andA R Boccaccini ldquoPoly(3-hydroxybutyrate)Bioglass compos-ite films containing carbon nanotubesrdquoNanotechnology vol 18no 7 Article ID 075701 2007

[13] J Rich J Tuominen J Kylma J Seppala S N Nazhat and K ETanner ldquoLactic acid based PEUHA and PEUBCP compositesdynamic mechanical characterization of hydrolysisrdquo Journal ofBiomedical Materials Research Part B Applied Biomaterials vol63 no 3 pp 346ndash353 2002

[14] I MWardMechanical Properties of Solid Polymers JohnWileyamp Sons New York NY USA 1985

[15] A Padermshoke Y Katsumoto H Sato S Ekgasit I Noda andY Ozaki ldquoMelting behavior of poly(3-hydroxybutyrate) inves-tigated by two-dimensional infrared correlation spectroscopyrdquoSpectrochimica ActamdashPart A Molecular and Biomolecular Spec-troscopy vol 61 no 4 pp 541ndash550 2005

[16] N Price S P Bendall C Frondoza R H Jinnah and D SHungerford ldquoHuman osteoblast-like cells (MG63) proliferateon a bioactive glass surfacerdquo Journal of Biomedical MaterialsResearch vol 37 no 3 pp 394ndash400 1997

[17] S K Misra D Mohn T J Brunner et al ldquoComparison ofnanoscale and microscale bioactive glass on the properties of

P(3HB)Bioglass compositesrdquo Biomaterials vol 29 no 12 pp1750ndash1761 2008

[18] AAkbarzadehM Samiei and S Davaran ldquoMagnetic nanopar-ticles preparation physical properties and applications inbiomedicinerdquoNanoscale Research Letters vol 7 article 144 2012

[19] A Gloria T Russo U DrsquoAmora et al ldquoMagnetic poly(120576-caprolactone)iron-doped hydroxyapatite nanocomposite sub-strates for advanced bone tissue engineeringrdquo Journal of theRoyal Society Interface vol 10 no 80 2013

[20] Q A Pankhurst J Connolly S K Jones and J Dobson ldquoAppli-cations of magnetic nanoparticles in biomedicinerdquo Journal ofPhysics D Applied Physics vol 36 no 13 pp R167ndashR181 2003

[21] A Tampieri T DrsquoAlessandro M Sandri et al ldquoIntrinsic mag-netism and hyperthermia in bioactive Fe-doped hydroxyap-atiterdquo Acta Biomaterialia vol 8 no 2 pp 843ndash851 2012

[22] G W Ehrenstein and R P Theriault Polymeric MaterialsStructure Properties Applications Hanser Munich Germany2001

[23] U Chen and M Wang ldquoProduction and evaluation ofbiodegradable composite based on PHB-PHV copolymerrdquoBiomat vol 23 no 13 pp 2631ndash2639 2002

[24] MWang L L Hench andW Bonfield ldquoBioglasshigh densitypolyethylene composite for soft tissue applications preparationand evaluationrdquo Journal of Biomedical Materials Research vol42 no 4 pp 577ndash586 1998

[25] T Russo A Gloria V DrsquoAnto et al ldquoPoly(120576-caprolactone)reinforced with sol-gel synthesized organic-inorganic hybridfillers as composite substrates for tissue engineeringrdquo Journalof Applied Biomaterials and Biomechanics vol 8 no 3 pp 146ndash152 2010

[26] A Millan F Palacio A Falqui et al ldquoMaghemite polymernanocomposites with modulated magnetic propertiesrdquo ActaMaterialia vol 55 no 6 pp 2201ndash2209 2007

[27] N Galego C Rozsa R Sanchez J Fung A Vazquez andJ S Tomas ldquoCharacterization and application of poly(120573-hydroxyalkanoates) family as composite biomaterialsrdquo PolymerTesting vol 19 no 5 pp 485ndash492 2000

[28] J Li H Yun Y Gong N Zhao and X Zhang ldquoEffectsof surface modification of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) on physicochemical propertiesand on interactionswithMC3T3-E1 cellsrdquo Journal of BiomedicalMaterials Research Part A vol 75 no 4 pp 985ndash989 2005

[29] F Grinnell and M K Feld ldquoFibronectin adsorption onhydrophilic and hydrophobic surfaces detected by antibodybinding and analyzed during cell adhesion in serum-containingmediumrdquo Journal of Biological Chemistry vol 257 no 9 pp4888ndash4893 1982

[30] M Chen P O Zamora P Som L A Pena and S Osaki ldquoCellattachment and biocompatibility of polytetrafluoroethylene(PTFE) treated with glow-discharge plasma of mixed ammoniaand oxygenrdquo Journal of Biomaterials Science Polymer Editionvol 14 no 9 pp 917ndash935 2003

[31] T Paz K Wade T Kiyoshima et al ldquoTissue and bone cell-specific expression of bone sialprotein is directed by a 90 kbpromoter intranspegenic micerdquo Matrix Biology vol 24 no 5pp 341ndash352 2005

[32] A Gopferich ldquoMechanisms of polymer degradation and ero-sionrdquo Biomat vol 17 no 2 pp 103ndash114 1996

[33] DM Brunette ldquoSpreading and orientation of epithelial cells ongrooved substratardquo Experimental Cell Research vol 167 no 1pp 203ndash217 1986


[34] B D Boyan T W Hummert K Kieswetter D SchraubD D Dean and Z Schwartz ldquoEffect of titanium surfacecharacteristics on chondrocytes and osteoblasts in vitrordquo Cellsand Materials vol 5 no 4 pp 323ndash335 1995

[35] D M Brunette ldquoThe effects of implant surface topographyon the behavior of cellsrdquo The International Journal of Oral ampMaxillofacial Implants vol 3 no 4 pp 231ndash246 1988

[36] B D Boyan S Lossdorfer L Wang et al ldquoOsteoblasts generatean osteogenic microenvironment when grown on surfaces withroughmicrotopographiesrdquo European Cells andMaterials vol 6pp 22ndash27 2003

Submit your manuscripts athttpwwwhindawicom

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TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


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MaterialsJournal of


Nano

materials


Journal ofNanomaterials


problem growth factors and cell ligands are incorporated asbiologically active molecules to facilitate cell attachment andexpansion

Current and future applications of magnetic nanoparti-cles in biology and medicine have been largely dependent ontheir nanometer-size particles (3ndash10 nm in diameter) whichexhibit novel magnetic chemical and biomedical properties[3] Maghemite (120574-Fe2O3) magnetite (Fe3O4) and FeIIFeIIIoxides are technologically important compounds widely usedfor the production of magnetic materials and catalysts Asa confirmation of cytocompatibility of magnetic material intissue engineering an emerging tissue engineering strategymagnetic force-based tissue engineering (Mag-TE) employscells that have been magnetically labelled with magnetitecationic liposomes (MCLs) Such MCL-labelled cells can bemanipulated and organised by magnetic force and theirfunctionality is maintained The use of magnetic material tomanipulate cells has been tested in many cell lines includingmesenchymal stem cells cardiomyocytes [4] human umbil-ical vein endothelial cells [5] retinal pigment epithelial cells[6] and keratinocytes [7]

In this study polymeric P(3HB) based magnetic ironoxide (mainly magnetite) composite scaffolds were suc-cessfully developed using a simple and inexpensive com-pression mouldingparticulate leaching technique The twodifferent types of magnetic composite scaffolds producedincluded P(3HB)magnetic nanoparticles P(3HB)MNP andP(3HB)ferrofluid P(3HB)FFThe developed magnetic scaf-folds were characterized for their magnetic properties as wellas the effects of addition of magnetic materials (particles andfluid) on the thermomechanical properties of the compositescaffolds FTIR was employed to study the crystallographicproperties of the scaffolds In vitro degradation study of thecomposite scaffolds was performed in simulated body fluid(SBF) to understand the effect of magnetic materials on thebiodegradation of the composite scaffoldMagnetite was usedfor the manipulation of the nano- and microenvironmentson P(3HB) scaffolds in order to promote protein adsorptionand subsequently stimulate cell proliferation Cytocompat-ibility studies on the magnetic scaffolds were carried outusing the human osteosarcoma MG-63 cell line and totalprotein production and cell proliferation were assessed Thisstudy confirmed the promising potential application of thedeveloped composites in bone tissue engineering and boneregeneration therapy

2 Experimental Procedures

21 Bacterial Strain Cells and Culture Medium Poly(3-hydroxybutyrate) was produced following previously devel-oped biotechnological methods [8] The proliferation assayswere performed using the HOS cell lineMG-63 grown in lowglucose Dulbeccorsquos Modified Eagle Medium (DMEM) sup-plemented with 10 fetal calf serum and 1 (wv) penicillinand 1 (wv) streptomycin solution

22 Magnetic Nanoparticle Preparation Magnetic nanocrys-tals of Fe3O4 were prepared by the classical coprecipitation

method where a solution containing ferric and ferrouschlorides was introduced in an alkaline solution [9] Tothe mixture of 20 g FeCl2sdot4H2O in 5mL 2M HCl and54 g FeCl3 sdot6H2O in 20mL 2M HCl 50mL 07M NH3 wasdropped under mixing The magnetic particles were washedwith water and acetoneThese particles were used in the formof aggregates particle size ca 100 nm (1mL of centrifugedsample corresponded to 67mg of dry weight)

23 Preparation of Magnetic Fluid (Ferrofluid FF) Stabilizedin Chloroform To the mixture of 20 g FeCl2 sdot4H2O in 5mL2M HCl and 54 g FeCl3 sdot6 H2O in 20mL 2M HCl 50mL07M NH3 was dropped under mixing Then concentrated(25) NH3 was added to the total volume 66mL and pHca 10 After that 13mL oleic acid was added and thoroughlymixed for 1 h Then the mixture was heated in water bath to95∘C with temperature increasing 2∘Cs (45ndash60min) Aftercooling down 15HNO3 was added to pH 5The suspensionwas then washed with water and subsequently with acetone(both four times) Magnetite in acetone was heated for 15 hunder mixing in water bath at 35ndash50∘C until all acetone wasevaporated Then chloroform was added in small portionsuntil ferrofluid was formed To remove solid particles theferrofluid was repeatedly centrifuged (90min 9500 rpm)under the same conditions up to 4ndash6 days The relativemagnetic fluid concentration (25mgmL) was determined bya colorimetric method (Fe3O4 used as standard) pH was ca70 [10]

24 Nanocomposite Film Preparation 1 g of P(3HB) wasdissolved in 10mL of CHCl3 at room temperature andstirred for 24 h The desired amount of magnetic nanoparti-clesferrofluid in CHCl3 was added to the P(3HB) solutionto give a final concentration of 14 27 and 54mgmL of eitherMNPor FF and themixturewas homogenized for 2minusinga homogenizer (Ultra-Turrax T25 basic Ika-Werke) Thesuspension was then degassed three times and poured intoa glass Petri-dish where the films were obtained by solventevaporation at room temperature Hence nanocompositefilms containing 14 27 and 54mgg of P(3HB)were prepared

25 Scanning Electron Microscopy (SEM) Scanning electronmicroscopy JEOL 5610LV instrument (JEOL USA) and anESEMFEIQuanta 200F was used to examine themicrostruc-ture of the test materials The samples were placed on 8mmdiameter aluminium stubs using a sticky tag to hold them Agold sputtering device (EMITECH-K550) was used to coatthe samples operating at a pressure of 7 times 10minus2 bar anddeposition current of 20mA for 2min Images were taken atvarious acceleration voltages (maximum of 20 kV) to avoidbeam damage on the polymer

26 SQUID Analysis A superconducting quantum inter-ference device magnetometer (SQUID MPMS XL-7 typeQuantum Design USA) was used for measurement of mag-netization of the solid samples The hysteresis loops of allstudied samples were collected at a temperature of 300Kunder an external magnetic field ranging from minus1 T to +1 T










3 Results




100120583m 100120583m

100120583m

300120583m

60120583m 40120583m


MNP

P(3HB)

(a)

P(3HB)FF

(b)






119864 =120590

120576= 1198641015840 + 11986410158401015840

tan 120575 = 1198641015840

11986410158401015840

(1)



P(3HB)T = 300K

minus20

minus15

minus10

minus5

0

5

10

15

20M

agne

tizat

ionM

(10minus3

emu

g)



(a)

T = 300K

minus08

minus06

minus04

minus02

00

02

04

06

08

Mag

netiz

atio

nM

(em

ug)



14mg

minus 06

minus 04

minus 02

00

02

04

06

M(e

mu

g)


H (kOe)

(b)


T = 300K


H (kOe)

minus 08

minus 06

minus 04

minus 02

00

02

04

06

08

M(e

mu

g)



minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

Mag

netiz

atio

nM

(em

ug)

(a)

T = 300K



minus12

minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

12

Mag

netiz

atio

nM

(em

ug)

minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

M(e

mu

g)


H (kOe)

(b)












00

02

04

06

08

10

12

Youn

grsquos m

odul

us (G

Pa)











000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

175e + 9

200e + 9

225e + 9

250e + 9

275e + 9St

orag

e mod

ulus

(GPa

)

(a)



minus500e + 8

minus250e + 8

000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

Loss

mod

ulus

(GPa

)

(b)


2978

1720

1378

1275 1050

591

P(3HB)

P(3HB)FFP(3HB)MNP

minus04

minus02

00

02

04

06

08

Tran

smitt

ance

()






20

40

60

80M

ean

cont

act a

ngle

(deg

)





otei

n co

ncen

trat

ion

(120583g

cm2)

0

50

100

150

200



P(3HB)MNPP(3HB)FF

P(3HB)

25 3020155 100Time (days)

7

75

8

pH





Time (days)1 3 6

0

40

80

120

Relat

ive c

ell p

rolif

erat

ion

()


lowastlowastlowast


lowastlowastlowast

TCPNeat P(3HB)







lowast

lowastlowast

lowastlowast lowast

0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

TCPP(3HB)






4 Discussion


0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

lowast

ControlP(3HB)





times150

times200

times2000





















5 Conclusions


Competing Interests


Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials












3 Results




100120583m 100120583m

100120583m

300120583m

60120583m 40120583m


MNP

P(3HB)

(a)

P(3HB)FF

(b)






119864 =120590

120576= 1198641015840 + 11986410158401015840

tan 120575 = 1198641015840

11986410158401015840

(1)



P(3HB)T = 300K

minus20

minus15

minus10

minus5

0

5

10

15

20M

agne

tizat

ionM

(10minus3

emu

g)



(a)

T = 300K

minus08

minus06

minus04

minus02

00

02

04

06

08

Mag

netiz

atio

nM

(em

ug)



14mg

minus 06

minus 04

minus 02

00

02

04

06

M(e

mu

g)


H (kOe)

(b)


T = 300K


H (kOe)

minus 08

minus 06

minus 04

minus 02

00

02

04

06

08

M(e

mu

g)



minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

Mag

netiz

atio

nM

(em

ug)

(a)

T = 300K



minus12

minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

12

Mag

netiz

atio

nM

(em

ug)

minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

M(e

mu

g)


H (kOe)

(b)












00

02

04

06

08

10

12

Youn

grsquos m

odul

us (G

Pa)











000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

175e + 9

200e + 9

225e + 9

250e + 9

275e + 9St

orag

e mod

ulus

(GPa

)

(a)



minus500e + 8

minus250e + 8

000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

Loss

mod

ulus

(GPa

)

(b)


2978

1720

1378

1275 1050

591

P(3HB)

P(3HB)FFP(3HB)MNP

minus04

minus02

00

02

04

06

08

Tran

smitt

ance

()






20

40

60

80M

ean

cont

act a

ngle

(deg

)





otei

n co

ncen

trat

ion

(120583g

cm2)

0

50

100

150

200



P(3HB)MNPP(3HB)FF

P(3HB)

25 3020155 100Time (days)

7

75

8

pH





Time (days)1 3 6

0

40

80

120

Relat

ive c

ell p

rolif

erat

ion

()


lowastlowastlowast


lowastlowastlowast

TCPNeat P(3HB)







lowast

lowastlowast

lowastlowast lowast

0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

TCPP(3HB)






4 Discussion


0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

lowast

ControlP(3HB)





times150

times200

times2000





















5 Conclusions


Competing Interests


Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




100120583m 100120583m

100120583m

300120583m

60120583m 40120583m


MNP

P(3HB)

(a)

P(3HB)FF

(b)






119864 =120590

120576= 1198641015840 + 11986410158401015840

tan 120575 = 1198641015840

11986410158401015840

(1)



P(3HB)T = 300K

minus20

minus15

minus10

minus5

0

5

10

15

20M

agne

tizat

ionM

(10minus3

emu

g)



(a)

T = 300K

minus08

minus06

minus04

minus02

00

02

04

06

08

Mag

netiz

atio

nM

(em

ug)



14mg

minus 06

minus 04

minus 02

00

02

04

06

M(e

mu

g)


H (kOe)

(b)


T = 300K


H (kOe)

minus 08

minus 06

minus 04

minus 02

00

02

04

06

08

M(e

mu

g)



minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

Mag

netiz

atio

nM

(em

ug)

(a)

T = 300K



minus12

minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

12

Mag

netiz

atio

nM

(em

ug)

minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

M(e

mu

g)


H (kOe)

(b)












00

02

04

06

08

10

12

Youn

grsquos m

odul

us (G

Pa)











000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

175e + 9

200e + 9

225e + 9

250e + 9

275e + 9St

orag

e mod

ulus

(GPa

)

(a)



minus500e + 8

minus250e + 8

000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

Loss

mod

ulus

(GPa

)

(b)


2978

1720

1378

1275 1050

591

P(3HB)

P(3HB)FFP(3HB)MNP

minus04

minus02

00

02

04

06

08

Tran

smitt

ance

()






20

40

60

80M

ean

cont

act a

ngle

(deg

)





otei

n co

ncen

trat

ion

(120583g

cm2)

0

50

100

150

200



P(3HB)MNPP(3HB)FF

P(3HB)

25 3020155 100Time (days)

7

75

8

pH





Time (days)1 3 6

0

40

80

120

Relat

ive c

ell p

rolif

erat

ion

()


lowastlowastlowast


lowastlowastlowast

TCPNeat P(3HB)







lowast

lowastlowast

lowastlowast lowast

0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

TCPP(3HB)






4 Discussion


0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

lowast

ControlP(3HB)





times150

times200

times2000





















5 Conclusions


Competing Interests


Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




P(3HB)T = 300K

minus20

minus15

minus10

minus5

0

5

10

15

20M

agne

tizat

ionM

(10minus3

emu

g)



(a)

T = 300K

minus08

minus06

minus04

minus02

00

02

04

06

08

Mag

netiz

atio

nM

(em

ug)



14mg

minus 06

minus 04

minus 02

00

02

04

06

M(e

mu

g)


H (kOe)

(b)


T = 300K


H (kOe)

minus 08

minus 06

minus 04

minus 02

00

02

04

06

08

M(e

mu

g)



minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

Mag

netiz

atio

nM

(em

ug)

(a)

T = 300K



minus12

minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

12

Mag

netiz

atio

nM

(em

ug)

minus10

minus08

minus06

minus04

minus02

00

02

04

06

08

10

M(e

mu

g)


H (kOe)

(b)












00

02

04

06

08

10

12

Youn

grsquos m

odul

us (G

Pa)











000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

175e + 9

200e + 9

225e + 9

250e + 9

275e + 9St

orag

e mod

ulus

(GPa

)

(a)



minus500e + 8

minus250e + 8

000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

Loss

mod

ulus

(GPa

)

(b)


2978

1720

1378

1275 1050

591

P(3HB)

P(3HB)FFP(3HB)MNP

minus04

minus02

00

02

04

06

08

Tran

smitt

ance

()






20

40

60

80M

ean

cont

act a

ngle

(deg

)





otei

n co

ncen

trat

ion

(120583g

cm2)

0

50

100

150

200



P(3HB)MNPP(3HB)FF

P(3HB)

25 3020155 100Time (days)

7

75

8

pH





Time (days)1 3 6

0

40

80

120

Relat

ive c

ell p

rolif

erat

ion

()


lowastlowastlowast


lowastlowastlowast

TCPNeat P(3HB)







lowast

lowastlowast

lowastlowast lowast

0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

TCPP(3HB)






4 Discussion


0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

lowast

ControlP(3HB)





times150

times200

times2000





















5 Conclusions


Competing Interests


Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










00

02

04

06

08

10

12

Youn

grsquos m

odul

us (G

Pa)











000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

175e + 9

200e + 9

225e + 9

250e + 9

275e + 9St

orag

e mod

ulus

(GPa

)

(a)



minus500e + 8

minus250e + 8

000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

Loss

mod

ulus

(GPa

)

(b)


2978

1720

1378

1275 1050

591

P(3HB)

P(3HB)FFP(3HB)MNP

minus04

minus02

00

02

04

06

08

Tran

smitt

ance

()






20

40

60

80M

ean

cont

act a

ngle

(deg

)





otei

n co

ncen

trat

ion

(120583g

cm2)

0

50

100

150

200



P(3HB)MNPP(3HB)FF

P(3HB)

25 3020155 100Time (days)

7

75

8

pH





Time (days)1 3 6

0

40

80

120

Relat

ive c

ell p

rolif

erat

ion

()


lowastlowastlowast


lowastlowastlowast

TCPNeat P(3HB)







lowast

lowastlowast

lowastlowast lowast

0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

TCPP(3HB)






4 Discussion


0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

lowast

ControlP(3HB)





times150

times200

times2000





















5 Conclusions


Competing Interests


Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

175e + 9

200e + 9

225e + 9

250e + 9

275e + 9St

orag

e mod

ulus

(GPa

)

(a)



minus500e + 8

minus250e + 8

000

250e + 8

500e + 8

750e + 8

100e + 9

125e + 9

150e + 9

Loss

mod

ulus

(GPa

)

(b)


2978

1720

1378

1275 1050

591

P(3HB)

P(3HB)FFP(3HB)MNP

minus04

minus02

00

02

04

06

08

Tran

smitt

ance

()






20

40

60

80M

ean

cont

act a

ngle

(deg

)





otei

n co

ncen

trat

ion

(120583g

cm2)

0

50

100

150

200



P(3HB)MNPP(3HB)FF

P(3HB)

25 3020155 100Time (days)

7

75

8

pH





Time (days)1 3 6

0

40

80

120

Relat

ive c

ell p

rolif

erat

ion

()


lowastlowastlowast


lowastlowastlowast

TCPNeat P(3HB)







lowast

lowastlowast

lowastlowast lowast

0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

TCPP(3HB)






4 Discussion


0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

lowast

ControlP(3HB)





times150

times200

times2000





















5 Conclusions


Competing Interests


Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




otei

n co

ncen

trat

ion

(120583g

cm2)

0

50

100

150

200



P(3HB)MNPP(3HB)FF

P(3HB)

25 3020155 100Time (days)

7

75

8

pH





Time (days)1 3 6

0

40

80

120

Relat

ive c

ell p

rolif

erat

ion

()


lowastlowastlowast


lowastlowastlowast

TCPNeat P(3HB)







lowast

lowastlowast

lowastlowast lowast

0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

TCPP(3HB)






4 Discussion


0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

lowast

ControlP(3HB)





times150

times200

times2000





















5 Conclusions


Competing Interests


Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




lowast

lowastlowast

lowastlowast lowast

0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

TCPP(3HB)






4 Discussion


0

20

40

60

80

100

120

140

Relat

ive t

otal

who

le p

rote

in p

rodu

ctio

n (

)

7 14 211Time (days)

lowast

ControlP(3HB)





times150

times200

times2000





















5 Conclusions


Competing Interests


Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




times150

times200

times2000





















5 Conclusions


Competing Interests


Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials


















5 Conclusions


Competing Interests


Acknowledgments


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 p(3hb) based magnetic nanoc omposites...

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