Materials Science and Engineering C 33 (2013) 1380–1388

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Materials Science and Engineering C

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Novel polypropylene biocomposites reinforced with carbon nanotubes andhydroxyapatite nanorods for bone replacements

Cheng Zhu Liao a, Kai Li a, Hoi Man Wong b, Wing Yin Tong b, Kelvin Wai Kwok Yeung b, Sie Chin Tjong a,⁎a Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kongb Department of Orthopedics and Traumatology, The University of Hong Kong, Hong Kong

⁎ Corresponding author. Tel.: +852 3442 7702; fax: +E-mail address: aptjong@cityu.edu.hk (S.C. Tjong).

0928-4931/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.msec.2012.12.039

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 August 2012Received in revised form 8 October 2012Accepted 4 December 2012Available online 13 December 2012

Keywords:Carbon nanotubeHydroxyapatiteNanocompositePolypropyleneOsteoblastBiocompatibility

Multi-walled carbon nanotubes (MWNTs) of 0.1 and 0.3 wt.% and hydoxyapatite nanorods (nHAs) of 8–20 wt.% were incorporated into polypropylene (PP) to form biocomposites using melt-compounding andinjection molding techniques. The structural, mechanical, thermal and in vitro cell responses of the PP/MWNT–nHA hybrids were investigated. Tensile and impact tests demonstrated that the MWNT additions arebeneficial in enhancing the stiffness, tensile strength and impact toughness of the PP/nHA nanocomposites.According to thermal analysis, the nHA andMWNTfillerswere found to be very effective to improve dimensionaland thermal stability of PP. The results of osteoblast cell cultivation and dimethyl thiazolyl diphenyl tetrazolium(MTT) tests showed that the PP/MWNT–nHA nanocomposites are biocompatible. Such novel PP/MWNT–nHAhybrids are considered to be potential biomaterials for making orthopedic bone implants.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Recently, significant interest has emerged in the development of bio-materials capable of repairing bone defects owing to a large increase inaging populations and patients suffering from trauma and bone cancer[1–5]. Metallic materials such as stainless steel, Co–Cr and Ti-6Al-4Valloys are typically used for the bone replacements in order to restoretheir lost functions. However, these materials often suffer from thewear and stress-shielding effect upon incorporation into the humanbody. The stress-shielding effect results from a large difference in theYoung'smodulus between themetals and human bones. Thus bone is in-sufficiently loaded compared to metallic implants, thereby causing a re-duction in the bone density [1]. These shortcomings and concerns havedriven materials scientists to develop polymer composites with tailoredmechanical stiffness and strength for making orthopedic load-bearingimplants with excellent biocompatibility.

Bone tissue is a biocomposite consisting of a collagen matrixand reinforcing hydroxyapatite (HA) nanoplatelets. Synthetichydroxyaptite-collagen composites generally exhibit low mechanicalstrength, hence aremainly used as biodegradable scaffolds for bone tissueengineering applications [2,3]. Bonfield and coworkers made pioneeringdevelopment of the HAPEX™ polymer composite composing of the dis-persion of 40 vol.% hydroxyapatite microparticles in high-density poly-ethylene (HDPE) matrix [4–6]. The incorporation of large volume

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content of hydroxyapatite microparticles leads to a seven-fold increasein the elastic modulus of HDPE. However, this polymer composite doesnot have adequate mechanical strength for load-bearing purposes, andthus can be only used for the low-strength orbital floor prosthesis,middleear implant and maxillofacial surgery. Tang et al. demonstrated thatlarge hydroxyapatite microparticles debond easily from the polymermatrix, resulting in inefficient stress-transfer mechanism across thematrix-filler interface during the tensile tests [7]. Moreover, large HAmicroparticles act as stress concentrators for the polymer composites,and fracture readily during mechanical loading [8].

Recent advances in nanotechnology enable chemists and materialsscientists to synthesize nanohydroxyapatite of various morphologieswith good biocompatibility [9–12]. Synthetic nanohydroxyapatite hasbeen reported to be very effective for anchoring osteoblasts andpromot-ing their growth [13,14]. Thus polymer composites reinforced withnanohydroxyapatite particles are promising biomaterials for orthopedicapplications. In previous work, we fabricated polypropylene (PP) com-posites containing various loadings of HA nanorods (nHA). The struc-ture, bioactivity and mechanical properties of such nanocompositeswere studied [15]. Tensile tests showed that the nHA additions stiffenand reinforce PP but reduce its tensile ductility. According to the resultsof simulated body fluid immersion tests, it deems necessary to add nHAcontent ≥15 vol.% to PP for achieving good bioactivity because pure PPis bioinert. Generally, the mechanical performance, bioactivity and bio-compatibility of the nHA/polymer composites depend greatly on thenHA volume fractions and the type of polymers selected [15–20]. Fur-thermore, the successful development of biocomposite implantsrequires a detailed understanding of their biological cell responses.

Table 1The designation and compositions of the composite specimens.

Specimen PP (wt.%) MWNT (wt.%) nHA (wt.%)

PP/8%nHA 92 0 8PP/15%nHA 85 0 15PP/20%nHA 80 0 20PP/0.1%MWNT–8%nHA 91.9 0.1 8PP/0.1% MWNT–15%nHA 84.9 0.1 15PP/0.1% MWNT–20%nHA 91.9 0.1 20PP/0.3% MWNT–8%nHA 91.7 0.3 8PP/0.3% MWNT–15%nHA 84.9 0.3 15PP/0.3% MWNT–20%nHA 79.7 0.3 20

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Carbon nanotubes with large aspect ratio, extraordinary highYoung's modulus, superior flexibility and excellent electrical conduc-tivity have been widely used as reinforcing fillers for polymers toform nanocomposites with functional properties [21–23]. Carbon isthe main constituent of biomolecules, thus carbon nanotubes are con-sidered to be biocompatible with human tissues. Carbon nanotubesoffer a wide range of opportunities for orthopedic applications sincethey promote adhesion and growth of osteoblasts, myoblasts and neu-rons [24–28]. Accordingly, bone regeneration on the nanotube-basedcomposites is increasingly favored. Binary carbon nanotube/polymercomposites designed for biomedical applications have been studied byseveral researchers recently [29–31]. Carbon nanotubes generally canbe classified into single-wall, and multi-wall nanotubes based on theirdiameter dimensions. Chemical vapor deposited multiwalled carbonnanotubes (MWNTs) with diameters of about 3–20 nm are availablecommercially in large quantities at lower cost compared with single-walled nanotubes (SWNTs). By combining advantageous properties ofthe MWNT and nHA fillers, novel MWNT–nHA/polymer hybrid com-posites with excellent biocompatibility, good mechanical property andsuperior dimensional stability can be developed and realized. Recently,Singh et al. prepared MWNT–nHA/polymethyl methacrylate (PMMA)using freeze granulation process for a new generation of bone cementand implant coating [32]. Im et al. also used single-wall carbonnanotubesas reinforcement for the nHA/chitosan nanocomposite scaffolds [33].Such MWNT–nHA/PMMA and SWNT–nHA/chitosan nanocompositesare not designed for load bearing implants in orthopedics. To the bestof our knowledge, there has been no attempt at putting MWNT, nHAand PP together to form PP/MWNT–nHA hybrid nanocomposites for bio-medical applications. Polypropylene displays excellent chemical resis-tance, good stress crack resistance, and flexibility. It has been shownthat when implanted in tissue, polypropylene retains its tensile strengthover a long period of time [34]. This work aims to investigate the hybrid-izing additions of MWNT and nHA fillers on the biocompatibility, me-chanical and thermal properties of PP.

2. Experimental

2.1. Materials

Polypropylene pellets (Mophlen HP 500N) with a melt flow indexof 12 g/10 min at 230 °C were obtained commercially from Basell(Saudi Arabia). Hydroxyapatite nanorods with an average width of20 nm and length of 120 nm (aspect ratio of 6) were purchasedfrom Nanjing Emperor Nano Materials Co. (China). Multiwalled car-bon nanotubes with diameters ranging from 20 to 40 nm were sup-plied by Nanostructured & Amorphous Materials Inc. (USA).

2.2. Preparation of nanocomposites

The respective constituent materials of PP/nHA and PP/MWNT/nHAnanocomposites were melt-compounded in a Brabender twin-screwextruder (model TSE 20/40D) at 40 rpm. The length-to-diameter (L/D)ratio of the screw is 40, being long enough for processing thermoplastics.The barrel of the extruder is divided into six zones from the hopper tothe die due to the large L/D ratio of the screw. The compositions of bina-ry PP/nHA and ternary PP/MWNT–nHA nanocomposites were listed inTable 1. The raw materials were fed into the hopper by gravity, and thescrew extracted them continuously from the hopper. The blending tem-peratures of Brabender from the hopper to the die were maintainedat 215–230–230–220–190–180 °C, respectively. Different temperatureswere adopted in six barrel zones of the extruder in order to achieve com-plete melting (215 °C) and mixing (230–230–220 °C) of PP pellets withnHA andMWNTs, and smooth delivering (190–180 °C) of the extrudatesfrom the barrel. The melt-blending process required around 600 s foreach composite. The extrudates were cut into pellets and loaded intothe extruder for blending under the same conditions to achieve

homogeneous dispersion of nanofillers. The products were pellet-ized again, dried in an oven at 70 °C for 24 h and finally injectionmolded into rectangular plaques. The molding temperature wasmaintained at 40 °C, whereas the barrel zone temperatures werekept at 230–225–220 °C. For the purpose of comparison, pure PPwas fabricated under the same processing conditions.

2.3. Materials characterization techniques

2.3.1. Structure and morphologyX-ray diffraction (XRD) patterns of the specimens were obtained

using a Siemens D500 diffractometer equipped with Cu-Kα radiation(λ=0.154 nm). The diffractometer was operated at 40 kV and 30 mA.The data were collected from 10 to 40° at a scanning rate of 0.02°/s. Fou-rier transform infrared (FTIR) spectra were determined with a PerkinElmer spectrometer (16PC) under the transmission mode and scannedin the range of 4000–400 cm−1 with a resolution of 2 cm−1. The mor-phology of impact fracture surfaces of the composites was examinedin a JEOL field-emission scanning electron microscope (FE-SEM;JSM-7100F). The surfaces of the specimens were coated with a thincarbon layer prior to SEM examination. The chemical compositionof nHA was analyzed with the energy dispersive X-ray (EDX) system(Inca X-sight, Oxford Instrument) attached to field-emission SEM.

2.3.2. Thermal analysisDifferential scanning calorimetry (DSC) tests were determined with

a TA Instruments model 2910 under a protective nitrogen atmosphere.The previous thermal history of the specimens was removed by an ini-tial heating cycle of 100 °C/min from ambient to 200 °C, followed byholding for 3 min at this temperature. The specimens were then cooledat a rate of 10 °C/min and the cooling traceswere recorded at this stage.They were subjected to a second heating cycle at the same rate to200 °C and the heating curves recorded.

Thermogravimetric (TGA) measurements were performed with aTA Instrument (TGA Q50) in nitrogen atmosphere at a heating rateof 20 °C/min. The weight percentage change of the specimens from30 to 700 °C was recorded. The degradation temperature at 5% weightloss (T5%) can be determined accordingly.

The heat deflection temperature (HDT) of the specimens was deter-mined with a TA Instrument dynamic mechanical analyzer (DMA,model 2980) under a three-point bending mode according to theASTM D648 from 25 °C to 150 °C at a heating rate of 2 °C/min. Duringthe measurement, a force (F) was applied to the specimen, accordingto the relation: F ¼ 2B2Wσ

�3L, where σ is the maximum tensile stress

(0.455 MPa), B the thickness (3 mm), W the width of specimen(12.7 mm), and L the span length (50 mm). The HDT was taken as thetemperature when the specimen deflected 0.566 mm from its initialroom temperature deflection.

2.3.3. Mechanical testsInjection-molded plaques were cut into dumb-bell shaped tensile

bars and notched impact specimens along the melt flow direction.Tensile tests were carried out using an Instron tester (model 5567)

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at ambient temperature and 50% relative humidity at a crossheadspeed of 10 mm min−1. The Young's modulus of the specimens wasdetermined with an extensometer. The tensile tests were conductedaccording to the ASTM D638-08. The impact tests were performedwith an Izod impact tester (Ceast model 6545) according to theASTM D256-05. At least six specimens for each composition weretested for both measurements and the average values reported.

2.4. In vitro cytocompatibility

2.4.1. Cell cultivationHuman osteoblast cell line (Saos-2) was cultured in Dulbecco's

Modified Eagle Medium (DMEM) (Thermo Scientific) supplementedwith 10% (v/v) fetal bovine serum, and antibiotics (100 U/ml of pen-icillin and 100 mg/ml of streptomycin). The specimens with a dimen-sion of 4×4×0.5 mmwere cut from the injection plaques and groundwith 600 grade SiC paper. Prior to the cell cultivation, the specimenswere sterilized with 70% ethanol and rinsed thoroughly with sterilephosphate-buffered saline (PBS). The specimens were cultured witha 100 μl cell suspension containing 104 cells at 37 °C in a humidifiedatmosphere of 5% CO2/95% air. After seeding for 24 and 72 h respec-tively, the specimens were washed with PBS, and treated with 10%formaldehyde solution to fix the attached cells. Subsequently, thespecimens were dehydrated in a series of ethanol solutions (30, 50,70, 90, 100% v/v) followed by critical point drying (CPD). They werefinally coated with a thin gold film, and examined in a scanning elec-tron microscope (JEOL, JSM-820).

2.4.2. Cell proliferation assayDimethyl thiazolyl diphenyl tetrazolium (MTT) colorimetric assay

was performed to determine the cell viability of the PP/MWNT andPP/MWNT/nHA nanocomposites. The cell suspension of 100 μl with104 cells was seeded on each specimen with a dimension of4×4×0.5 mm placed in a 96-well plate and incubated at 37 °C in ahumidified atmosphere of 5%CO2/95% air for 2, 4, 7 and 10 days, respec-tively. The DMEMmediumwas changed every 3 days. After incubation,sterilized MTT solution of 10 μl (5 mg MTT in 1 ml PBS) was added toeach well and left for another 4 h for the cell response to occur. Atthis stage, the cells metabolized the MTT, yielding a formazan productinside them. This was followed by adding 100 μl of 10% sodium dodecylsulfate (SDS) in 0.01 M hydrochloric acid (HCl) to dissolve formazancrystal. The light absorbance of the resulting solution was measured bya multimode detector (Beckman Coulter DTX 880) at 570 nm wave-length, with a reference wavelength of 640 nm. Positive control (osteo-blasts in the 96-well plate having the same seeding density as thespecimens) and negative control (DMEM only) were also performed forcomparison purposes. Mean standard deviation (±SD) of five identicalspecimenswas determined. The statistical significancewas set at pb0.05.

3. Results and discussion

3.1. Structure and morphology of hybrids

Fig. 1a–d shows the SEMmicrographs of the PP/0.1%MWNT–8%nHA,PP/0.1%MWNT–20%nHA, PP/0.3%MWNT–8%nHA and PP/0.3%MWNT–20%nHA specimens, respectively. The inset of thesemicrographs displaysEDX spectrum of the nHA fillers. Fig. 1e reveals high magnification viewof the PP/0.3%MWNT–20%nHA composite. It can be seen that the dimen-sions of the fillers of this composite fall in the nanometer scale. Themor-phology of nHA is also shown for the comparison purposes (Fig. 1f) [19].MWNTs generally showgooddispersion in the polymericmatrix of thesenanocomposites as indicated by black arrows in Fig. 1a–d. However, anagglomeration of nHA into fine clusters occurs in the PP/0.1%MWNT–20%nHA and PP/0.3%MWNT–20%nHA nanocomposites containinghigher nHA contents. It is difficult to prevent nHA clustering at high fillerloadings due to their large surface areas. This is a typical characteristic of

the polymer nanocomposites reinforced with large filler contents. Themorphology of binary PP/nHA nanocomposites has been reported previ-ously. A similar clustering behavior of nHA is found in the polymermatrix of binary PP/nHA nanocomposites with high filler loadings [15].As recognized, functional polymer nanocomposites designed for indus-trial applications generally contain very low filler loadings (ca≤5%) toavoid agglomeration of nanomaterials [35–38]. On the contrary, polymernanocomposites used for the bone replacements must contain highernHA contents in order to facilitate adhesion and growth of osteoblastson their surfaces [15,19,20]. Furthermore, the EDX analysis reveals thatthe Ca/P ratio of nHA is 1.65±0.04, being close to the stoichiometricvalue of 1.67. This indicates the presence of nHA in the PP matrix.

Fig. 2 shows the typical XRD patterns for PP, PP/0.3%MWNT, PP/20%nHA, PP/0.3%MWNT–20%nHA and nHA specimens. For pure PP,the patterns display characteristic peaks at 2θ=14.1, 16.9, 18.5, 21.2and 21.8°, corresponding to the (110), (040), (130), (111) and (041)planes of the α-PP crystals. The characteristic diffraction peaks of nHAlocated at 26.0°, 28.3°, 29.0° 31.9°, 33.0° and 34.2° can be assigned tothe (002), (102), (210), (211) (112) and (300) reflections accordingto the standard XRD pattern of hydroxyapatite (JCPDS No. 09-0432).Obviously, no structural change is induced in the PP composites byadding MWNT and/or nHA nanofillers.

Fig. 3a shows FTIR spectrum of nHA revealing the presence of PO43−

absorption bands [39]. The peaks at 963 cm−1 and 470 cm−1 areassigned to the γ1 and γ2 modes. Peaks at 1034 and 1092 cm−1 aredue to the γ3 mode of P\O symmetric stretching vibration, and 565and 601 cm−1 correspond to the γ4 P\O bending vibration. The bandsat 3569 and 631 cm−1 are associatedwith the OH− stretching vibration.Furthermore, the 870 and 1420 cm−1 can be attributed to the CO3

2−,resulting from the partial substitution of carbonate group into PO4

3−.This is due to the CO2 in the atmosphere being dissolved in the solutionduring the nHA synthesis [40]. The FTIR spectra of pure PP and its repre-sentative composites are shown in Fig. 3b. By incorporating nHA into PPand PP/MWNT, the spectra of the PP/20%nHA and PP/0.3%MWNT–20%nHA nanocomposites show the presence of nHA having PO4

3−

group at ~1050, 565 and 601 cm−1, and the OH− peak at 3569 cm−1,as well as the characteristic bands of PP.

3.2. Thermal behavior

Fig. 4 shows typical DSC cooling curves of pure PP and PP/MWNT–nHA nanocomposites. The onset crystallization temperature (To),peak crystallization (Tc) temperature and the crystallization enthalpy(ΔHc) can be determined from these curves, and listed in Table 2. Thedegree of crystallinity (Χc) of PP and its nanocomposites is evaluatedfrom the following expression:

Xc %ð Þ ¼ 100�ΔHc.

ΔHm 1−ϕð Þð1Þ

whereΔHm is themelting enthalpy of the 100% crystalline PP, i.e. 209 J/g[41,42], and ϕ is the weight fraction of the filler of the nanocomposites.The Χc values of the specimens studied are also listed in Table 2.

From Fig. 4 and Table 2, the incorporation of nHA rods into pure PP af-fects its crystallization behaviormarkedly. Both the To and Tc values of thePP/nHA nanocomposites increase considerably as the nHA contentincreases. The Tc value of pure PP is 115.7 °C, and increases to 119.1,121.4 and 123.9 °C by adding 8%, 15% and 20% nHA, respectively. Forthe PP/8%nHA composites, the Tc values can be increased from 119.1 °Cto 124.9 °C and 123.5 °C by adding 0.1% and 0.3% MWNT, respectively.By increasing nHA content of the composites to 15%, the Tc values increaseto 123.0 and123.1 °C by adding0.1% and0.3%MWNT, respectively.More-over, there is no increment in the Tc value of the PP/20% nHA compositeby incorporating MWNTs. The Tc value remains nearly unchanged.These results reveal that both nHA and MWNTs act as nucleating agentsfor PP crystallites. However, the effectiveness of MWNTs for nucleating

Fig. 1. SEM morphologies for (a) PP/0.1%MWNT–8%nHA, (b) PP/0.1%MWNT–20%nHA, (c) PP/0.3%MWNT–8%nHA, (d,e) PP/0.3%MWNT–20%nHA nanocomposites. MWNTs in thesemicrographs are denoted with black arrows. Dash square shows nHA fillers together with their EDX spectrum. (f) TEM micrograph of hydroxyapatite nanorods [19].

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PP depends on the nHA content. Thus both the nHA and MWNT fillerscompete with each other for the nucleation of PP macromolecules.MWNTs are beneficial for nucleating PP in the composites containing 8%and 15% nHA, particularly the former with low nHA content. AndMWNTs have no effect for the composites with high nHA content(i.e. 20%), possibly due to the severe agglomeration of nHA fillers.The Xc values are found to decrease slightly with increasing nHA con-tents. However, further additions of 0.1 and 0.3 wt.% MWNT to thePP/8% nHA, PP/15% nHA and PP/20% nHA nanocomposites improvetheir crystallinity. The melting temperature of PP increases by2–4.3 °C by incorporating 8–20% nHA and 0.1, 0.3% MWNT.

Non-resorbable polymer nanocomposites should exhibit good ther-mal and dimensional stability [15,19,20], thereby enabling the use in

long-lasting implants. This is because degradation components or prod-ucts of nonresorbable polymers may cause inflammatory response tothe human tissues. Thermal stability is the ability of a material to main-tain its property (mass) uponheating [43–45]. TGA is a useful techniquefor assessing thermal stability of pure polymers and their compositesupon heating. The raise of thermal decomposition temperature isoften regarded as an indicator for an improvement in the thermal stabil-ity. In addition, heat deflection temperature (HDT) is a key indicator forrevealing heat resistance and dimensional stability of the polymers andtheir composites under an applied load. Dimensional stability is relatedto the ability of a material to maintain accuracy when subjected to anexternal load. For orthopedic applications, the distortion of load-bearing polymer composite implants must be avoided.

Fig. 2. Typical XRD patterns for PP, PP/0.3%MWNT, PP/20%nHA, PP/0.3%MWNT–20%nHA and nHA.

Fig. 4. DSC cooling traces for pure PP, PP/0.1%MWNT–8%nHA, PP/0.1%MWNT–15%nHA,PP/0.1%MWNT–20%nHA, PP/0.3%MWNT–8%nHA, PP/0.3%MWNT–15%nHA and PP/0.3%MWNT–20%nHA nanocomposites.

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Fig. 5 displays representative TGA curves showing the changes inmass loss as a function of temperature for pure PP and PP/0.1%MWNT–nHA nanocomposites. The T5% temperatures of the specimens investigat-ed are summarized in Table 2. The results show that the T5% values ofPP-based nanocomposites increase markedly with increasing nHA con-tent. They can further increase to higher values by adding MWNTs. TheT5% value of the PP/0.3%MWNT–20%nHA hybrid is 451.7 °C, which is~86 °C higher than that of PP (365.3 °C). This implies that both nHAand MWNT additions are beneficial in improving the thermal stabilityof PP. From Table 2, the nHA additions lead to a significant incrementof the HDT value of PP. The HDT value increases from 105.1 °C for PP to

Fig. 3. Representative FTIR spectra for (a) nHA, and (b) PP, PP/0.3%MWNT, PP/20%nHA,PP/0.3%MWNT–20%nHA specimens.

127.5–130.5 °C by adding 8–20% nHA. The additions of small amountsof MWNTs to the PP/nHA composites further enhance their HDT values.This implies that both nHA and MWNTs are beneficial in improving thethermal resistance and dimensional stability of PP under an applied load.

3.3. Mechanical properties

Fig. 6a and b shows the Young's modulus and tensile strength as afunction of nHA content of the PP/nHA and PP/MWNT–nHA composites,respectively. For the PP/nHA nanocomposites, the stiffness increaseswith increasing nHA content. By adding 20 wt.% nHA to PP, themodulusenhancement is nearly 67%. The stiffness of the PP/nHA composites canbe further increased by incorporating low loading levels of MWNTsinto their matrices. Considering the tensile strength of PP/nHA compos-ites, it increases and saturates at 8–15 wt.% nHA content, then decreasesslightly with further nHA addition. The strength reduction is related tothe agglomeration of nHA fillers with the contents >15 wt.%. Similarly,the tensile strength of PP/nHA composites also improves considerablyby addingMWNTs of large aspect ratios and exceptional highmechanicalstrength. As aforementioned, largeHA contents are added to the polymerbiocomposites designed for bone implants in order to induce the adhe-sion and growth of osteoblasts. Liu andWang have prepared PP/HAcom-posites reinforced with 10, 20 and 25 vol.% HA microparticles [46]. Theyfound that the tensile strength of PP decreases from 29.55 MPa to 26.32,22.34 and 20.16 MPa respectively by adding 10, 20 and 25 vol.% HA. It isnoted that the nHA contents of the PP/nHA composites in this study are8, 15 and 20 wt.%, corresponding to 2.42, 4.80, 6.67 vol.%, respectively.Thus the filler volume contents of the PP/nHA nanocomposites aremuch lower than those of the PP/HA microcomposites prepared by Liuand Wang. From Fig. 6b, the tensile strength of PP is 29 MPa, andincreases to 31 MPa by adding 8 wt.% (2.42 vol.%) and 15 wt.%(4.80 vol.%) nHA, follows by a decrease to 30.2 MPa by adding 20 wt.%nHA (6.67 vol.%). Thus the additions of nHA contents of lowvolume frac-tions up to 4.60 vol.% to PP can enhance its tensile strength. The tensilestrength of PP can be further enhancedbyhybridizing nHAwithMWNTs.

Singh et al. added 0.01, 0.1, 0.5 and 1 wt.% MWNT to reinforcePMMA/HA composites of 1:2 weight ratio using a freeze-granulationtechnique [32]. Such hybrids designed to be used as the bone cementand implant coating. They reported that the hardness and elastic mod-ulus of the hybrid nanocomposites increase with MWNT content up to0.1 wt.%. Above 0.1 wt.% MWNT, both the hardness and modulus ofthe hybrids decrease markedly. They attributed this to the formationof voids and internal cavities, which can negatively impact themechan-ical performance of the hybrids. The tensile strength of their hybridswas not reported. The tensile test results of this study also reveal thatthe Young's modulus and tensile strength of the PP/nHA composites

Table 2Crystallization and thermal parameters of the specimens investigated.

Specimen To (°C) Tc (°C) Tm (°C) ΔHc (J/g) Xc (%) HDT (°C) T5% (°C)

PP 112.1±0.3 115.7±0.1 161.9±0.2 98.9 47.3 105.1±1.0 365.3±1.5PP/8%nHA [15] 114.7±0.1 119.1±0.1 164.0±0.4 90.3 46.9 127.5±0.5 400.3±1.1PP/15%nHA [15] 118.8±0.2 121.4±0.2 164.6±0.2 83.2 46.8 128.6±0.6 432.9±0.5PP/20%nHA [15] 119.7±0.2 123.9±0.1 165.0±0.3 75.4 45.1 130.5±0.3 438.9±0.9PP/0.1%MWNT–8%nHA 127.9±0.2 124.9±0.3 164.8±0.1 95.0 49.5 132.6±0.4 405.7±1.6PP/0.1%MWNT–15%nHA 126.8±0.3 123.0±0.2 166.2±0.1 85.5 48.2 133.3±0.8 447.4±2.1PP/0.1%MWNT–20%nHA 126.9±0.1 123.4±0.1 165.0±0.3 84.6 50.7 134.9±0.5 448.3±1.3PP/0.3%MWNT–8%nHA 126.3±0.3 123.5±0.2 164.2±0.3 96.2 50.2 131.1±0.9 417.4±0.3PP/0.3%MWNT–15%nHA 126.2±0.1 123.1±0.1 164.7±0.3 87.3 49.3 133.5±0.3 448.8±1.4PP/0.3%MWNT–20%nHA 127.7±0.2 123.5±0.3 165.9±0.1 81.7 49.1 135.5±1.2 451.7±2.5

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can be increased considerably by adding 0.1 wt.%MWNT. Further addi-tion of MWNT to 0.3 wt.% only leads to a slight improvement of bothproperties (Fig. 6a and b). Im et al. also used single-wall carbonnanotubes as reinforcement for the nHA/chitosan nanocompositescaffolds [33]. They found that the tensile modulus of the chitosan/20%HA composite scaffold can be improved by adding 1 wt.% SWNT. Itis noted that the composite scaffolds possess numerous voids thatrequire high nanotube content. In contrast, our PP/MWNT–nHAnanocomposites are dense and compact, i.e., having no porosities at all.Furthermore, SWNTs are available commercially at high price comparedwith MWNTs due to their high production cost. Such MWNT–nHA/PMMA and SWNT–nHA/chitosan nanocomposites are not designed forload-bearing implants in orthopedics.

Fig. 7 shows the impact strength vs. nHA content for the PP/nHAand PP/MWNT–nHA composites. The impact strength of the PP/nHAcomposites decreases markedly with increasing nHA content. Howev-er, the impact strength of the PP/nHA composites can be restored andimproved by adding nanotubes. This can be ascribed to the high flex-ibility and large strain-to-failure of carbon nanotubes [22]. The incor-poration of MWNTs into polymers generally does not affect theirductility and toughness. This is because flexible MWNTs can bridgethe propagating cracks in these materials during mechanical defor-mation [47].

As aforementioned, MWNT additions can increase the crystallizationtemperature (Tc) of PP/nHA composites containing 8 and 15% nHA con-tents. From Table 2, the degree of crystallinity of PP/nHA composites in-creases by adding low loading levels of 0.1 and 0.3%MWNT. ThusMWNTadditions favor crystallization of the PP molecules. This leads to the in-crease of Young's modulus, tensile strength and impact strength of thePP/MWNT–nHA nanocomposites because of the MWNT additions(Fig. 6a and b, Fig. 7). Generally, polymer composites with higher degreeof crystallinity exhibit high tensile strength and elastic modulus [48,49].Very recently, Han et al. reported that the tensile strength and impact

Fig. 5. Representative TGA curves for pure PP, PP/0.1%MWNT–8%nHA, PP/0.1%MWNT–15%nHA and PP/0.1%MWNT–20%nHA nanocomposites.

strength of the poly(L-lactic acid) (PLLA)/carbon fiber composites in-crease markedly with increasing fiber content [50] This is because car-bon fibers act as a nucleation agent for the crystallization of PLLA,thereby enhancing crystallinity and Tc of the PLLA matrix. Comparedwith the PP/nHA composites, the high elastic modulus, tensile strengthand impact strength of the PP/MWNT–nHA hybrids can be attributed totheir higher crystallinity coupledwith the reinforcing effect andflexibil-ity of carbon nanotubes.

3.4. In vitro cellular behavior

3.4.1. Cell morphologySurface chemistry and topography of the implanting materials are

the most influential factors affecting their bioactivity and biocompati-bility. Fig. 8a and b shows the SEM micrographs of pure PP cultivatedwith osteoblasts for 1 and 3 days, respectively. Only few cells are ob-served on the surface of bioinert PP. However, numerous osteoblasts

Fig. 6. Variations of (a) Young's modulus and (b) tensile strength with nHA content forPP/nHA and PP/MWNT–nHA nanocomposites.

Fig. 7. Variation of Izod impact strength with nHA content for PP/nHA and PP/MWNT–nHA nanocomposites.

Fig. 8. SEM micrographs showing the morphologies of human osteoblasts cultured onpure PP for (a) 1 day and (b) 3 days.

Fig. 9. SEM micrographs showing the morphologies of human osteoblasts cultured onPP/0.3%MWNT composite for (a) 1 day and (b) 3 days.

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are seen to adhere firmly on the surface of the PP/0.3%MWNTcomposite(Fig. 9a). This composite was prepared in our previous study under thesame processing conditions [51]. The number of anchored cells in-creases markedly after culturing for 3 days. The cells almost cover theentire surface of the composite (Fig. 9b). This implies that carbonaceousfillers of nanometric dimensions serve as effective seeding sites for theosteoblasts. Considering the PP/0.1%MWNT–20%nHA composite, thecells are well anchored and spread over the surface as expected(Fig. 10a and b). Apparently, nHA fillers with chemical compositionclose to the inorganic phase of human bones also facilitate adhesion ofthe osteoblasts. Nanohydroxyapatite provides large surface areas forinteracting with the osteoblasts [14]. A high magnification view of thecell anchored on this composite surface is shown in Fig. 10c. The cell de-velops flattened and elongated feature, displaying a large and thin cyto-plasmic feature. Numerous filopodias stretch out from the cell bodyacross the specimen surface for anchoring. The attachment of osteo-blasts is more pronounced for the PP/0.3%MWNT–20%nHA compositehaving higher nanotube loading. The cells colonize the whole materialand completely cover the surface after culturing for 3 days (Fig. 11).

As recognized, bone matrix composes of nanocrystalline HA of about70–100 nm long and 2–5 nm thick (aspect ratio of ~4), and collagen fi-bers [52,53]. Thus synthetic nHAmimic thenanostructure of the inorganicphase of bone tissue can serve as effective seeding site for the osteoblasts.Accordingly, the nanoscale topography of HA determines the cellular per-formance of osteoblasts. In this study, HA nanorods with the Ca/P ratio of1.65 (Fig. 1f) and MWNTs are used as fillers to reinforce PP and improveits biocompatibility. Upon adhesion to a substrate the cell probes its envi-ronment and moves using nanometer scale processes such as filopodia(Fig. 10(c)), followed by the establishment of focal adhesion [54]. Gener-ally, cell–substrate interactions are mediated by the presence of proteins(e.g. actin, vinculin) adsorbed on the substrate specimen. An importantissue in cellular adhesion is the formation of focal adhesions. Without ad-hesion, osteoblast cells cannot spread on the substrate. Vinculin is neededto form a strong actin filament network at the focal contact binding sites.In a previous study,we reported that the actinfilament can develop into awell-organized cytoskeleton for forming focal contact sites by culturingosteoblasts on the hydroxyapatite nanorods of PEEK/nHA nanocom-posites [20]. Very recently, Castros et al. [55], Liu et al. [56] and Liang etal. [57] reported that synthetic HA nanorods are biocompatible and veryeffective to promote the growth of human osteoblasts. Furthermore,MTT assay showed that the rod-like nanohydroxyapatite exhibits highercell viability than those with needle-like features. From the results invivo experiments with adult mice on calvarial bone, Castros et al. furtherdemonstrated that HA nanorods with a Ca/P ratio of 1.67 possessosteoconductive properties. After one month of implantation, osteoid tis-sue was present in proximity to nHA. Newly formed bone was observedin test sites after 3 months.

Pure carbon and carbonaceous nanomaterials such as carbonnanotubes and graphene are generally accepted as biocompatible

Fig. 10. SEMmicrographs showing the morphologies of human osteoblasts cultured onPP/0.1%MWNT–20%nHA composite for (a) 1 day, (b) 3 days, and (c) high magnifica-tion view of osteoblastic cell cultured on this specimen.

Fig. 11. SEM micrograph showing the morphology of human osteoblasts cultured onPP/0.3%MWNT–20%nHA nanocomposite for 3 days.

Fig. 12. Cell viability of Saos-2 cells cultured on PP/8% nHA, PP/0.1%MWNT–8% nHA andPP/0.3%MWNT–8% nHA composites. The error bar represents the mean standard devi-ation (±SD) of five identical specimens. The statistical significance was set at pb0.05.

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materials [58]. Carbon nanotubes show promise for orthopedic implantapplications because they promote osteoblast adhesion and prolifera-tion aswell as sustain bone formation [24–28,59,60]. Thus cells culturedonMWNTs showed high viability and adhesiveness. The exceptional to-pography of the CNT surfaces provides enhanced osteoblastic adhesion,resulting in the selective absorption and attachment of proteins fromcell culture medium by means of C\C bonds [61,62]. With regard tosurface chemistry, proteins adsorbed and adhere onto hydrophobicCNT surfaces readily because of their graphitic structure and small di-ameter with a high surface energy and surface area [63]. By combiningadvantageous properties of MWNT and nHA, osteoblast cell populationand viability have been reported to be slightly higher on the nHA–CNTsurface than nHA [64,65]. The MTT results of this study also confirmthis finding as described in the next section.

3.4.2. Cell proliferation assayIn general, HA nanomaterials enhance the formation of new bone tis-

sue by increasing osteoblast adhesion and osteointegration on their sur-faces [13,14]. MTT assay was used to determine the proliferation rate ofosteoblasts on the binary PP/nHA nanocomposites and PP/MWNT–nHAhybrids (Figs. 12 and 13). Apparently, the osteoblastic proliferation onthese specimens increases with increasing culturing time up to 10 days.Comparing with the PP/20%nHA specimen, the PP/0.3%MWNT–20%nHAcomposite exhibits enhanced growth rate of the osteoblasts, especiallyon day 7. Therefore, the biocompatibility of PP is considerably improvedby reinforcing with appropriate concentrations of nHA and MWNT.From the literature, CNTs may induce cytotoxicity upon inhalation and

Fig. 13. Cell viability of Saos-2 cells cultured on PP/20% nHA, PP/0.1%MWNT–20% nHAand PP/0.3%MWNT–20% nHA composites. The error bar represents the mean standarddeviation (±SD) of five identical specimens. The statistical significance was set atpb0.05.

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exposure to human cells [66–68]. Some researchers have found reducedviability in human lung cells and epidermal keratinocytes [67,68].However, other workers have reported favorable cellular interactionswith nanotubes by seeding with osteoblasts and neurons [62,69].

The toxicity of CNTs is closely related to the type of biological cellsexamined, the nature of nanotubes including the type, length, catalyticimpurity, functionalization, etc., and the dose of nanotubes employedfor the cell interactions. The above mentioned studies on cytotoxicitywere conducted by exposing various doses of stand-alone CNTs to dif-ferent kinds of human cells. Those carbon nanotubes were loose andnot embedded in the polymer matrix of the composites. On the con-trary, favorable viability of osteoblasts was found by embedding carbonnanotubes in both the hydroxyapatite matrix [65,70] and the polymermatrix of the composites [20,33]. The present study also reveals thatthe incorporation of MWNTs into the polymer matrix of the PP/nHAcomposites has no negative effect on the viability and proliferation ofosteoblasts but rather improves these properties.

Finally,we concluded that differentweights of CNTwere added to thePP/nHA nanocomposites aiming to study the effects of nanotube addi-tions on their structural, mechanical and thermal properties as well asbiocompatibility. Since nanotubes with unique structural and mechani-cal behaviors as well as good biocompatibility show high tendency foragglomeration, thus only small MWNT loadings are incorporated intothe PP/nHA composites for enhancing their performance. Thus 0.1 and0.3 wt.%MWNT are loaded to the PP/nHA composites during the design-ing of ternary PP/MWNT–nHA composites for biomedical applications.Practically, only experimental measurements can verify the effects ofnanotube additions on the performance of ternary PP/MWNT–nHA com-posites. The experimental results show that the PP/nHA composites with0.1 wt.% MWNT have more or less same effect on the composites com-pared with those of 0.3 wt.%. This implies that the performance of thePP/MWNT–nHA hybrids can be greatly improved by adding very lowloading of 0.1 wt.% MWNT. The mechanical test results in this studyagree reasonably with those of Sing et al. [32] demonstrating that themechanical performance of ternary PMMA–MWNT–HA composites canbe improved by adding MWNT up to 0.1 wt.%.

4. Conclusions

This study deals with the design, preparation, mechanical andthermal assessments and biocompatibility evaluation of novel poly-mer hybrid composites reinforced with MWNT and nHA fillers. Itaims to introduce novel PP/MWNT–nHA hybrid composites as poten-tial implants for biomedical applications. Hybridizing MWNTs of lowloadings (0.1 and 0.3 wt.%) with nHA in the PP matrix have resultedin considerable improvements in mechanical strength, impact tough-ness, thermal and dimensional stability. The MTT calorimetric assayresults confirm the viability and proliferation activity of the osteo-blasts cultured on the PP/MWNT–nHA hybrids. The PP/0.3%MWNT–20%nHA composite is biocompatible with a better cell proliferationrate compared to the PP/20%nHA composite.

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