Carbon Nanotube Functionalization Decreases Osteogenic

Article pubs.acs.org/journal/abseba

Carbon Nanotube Functionalization Decreases Osteogenic Differentiation in Aluminum Oxide Reinforced Ultrahigh Molecular Weight Polyethylene Anup Kumar Patel, Pramanshu Trivedi, and Kantesh Balani* Biomaterials Processing and Characterization Laboratory, Department of Materials Science and Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India S Supporting Information *

ABSTRACT: Ultrahigh molecular weight polyethylene (UHMWPE) is one of the most preferred materials as an acetabular cup-liner for bone implant applications. The current work develops a correlation between wettability, protein adsorption with osteogenic differentiation upon reinforcement of functionalized carbon nanotube (f-CNT) and 10 wt % aluminum oxide (Al2O3) in compression molded UHMWPE composites. Phase characterization has confirmed the retention of CNTs after compression molding. The loading of 2 wt % f-CNT in UHMWPE has shown to increase the contact angle (CA, from 88.9° to ∼97.3°), decrease the surface free energy (SFE, 23.20 to ∼20.85 mJ/m2) and elicit enhanced adsorbed protein density (PD, from ∼0.26 to ∼0.32 mg/cm2) in comparison to that of virgin polymer. Similar trend also has observed with 5 and 10 wt % f-CNT reinforcement. Initially, a high density of L929 mouse fibroblast cells is observed for 10 wt % unfunctionalized CNT (u-CNT) loading (48 h of incubation) with high values of dispersion fraction of surface free energy (σd), i.e., 0.967, whereas a decrease in cell density after 48 h is attributed to significant apatite mineralization and low dispersion fraction (σd) of CNT−Al2O3−UHMWPE biocomposites. Interestingly, gene expression studies have corroborated low osteogenic differentiation (i.e., weaker intensity osteopontin and β-actin) in 2−10 wt % f-CNT reinforced Al2O3−UHMWPE biocomposites in comparison to that of similar wt % reinforcement of u-CNT. Thus, implant material can be engineered, (bulk or surface-modified), possessing osteoanalogous and cytocompatible properties based on f-CNT−Al2O3-reinforced UHMWPE nanocomposites. KEYWORDS: ultrahigh molecular weight polyethylene (UHMWPE), carbon nanotube (CNTs), wettability, cytocompatibility, osteogenic differentiation inorganic fillers, natural (nacre), and metallic fillers have been incorporated in polymer matrices.13,19−24 Aluminum oxide (Al2O3) is inert in nature, exhibits high hardness (389.5 GPa), and possesses high compressive strength (416 MPa), and thus is a potential candidate for reinforcing UHMWPE. Osman Asi et al.25 have reported that 10 wt % Al2O3 loading increases the bearing strength by 20% (∼290 MPa to ∼350 MPa) in comparison to that of virgin epoxy resin. A. Chanda et al.26 have reported a reduced wear rate (3.1 × 10−7 mm3/N.m) and lowering of coefficient of friction by a factor of 2 in water medium than that in dry cases (5.5 × 10−7 mm3/N.m). S. Omar et al.23 have shown that loading of silane treated Al2O3 particles in dental material (bisphenol A-glycidyl methacrylate base monomer) enhances the hardness and modulus when compared to that of Al2O3 from 14.4 to 23.5 kg/mm2 and from 1.5 to 5.7 GPa, respectively, whereas flexural

1. INTRODUCTION Ultrahigh molecular weight polyethylene (UHMWPE) is a widely used material for biomedical applications due to its excellent mechanical properties (elastic modulus ∼1.2 GPa), wear endurance (COF-0.08-0.12 against alumina counter body), corrosion resistance, and good cytocompatibility with human tissues.1−5 Formation of wear debris (1< μm) and metal ions on the mating surfaces causes failure and aseptic loosening of arthroplasty only after 10−12 years of implantation.6−8 It has been reported that ∼25% of implant failure occurred only because of debris generated at the interfaces.9−11 The debris may also be the cause of a number of diverse biological effects, such as inflammatory cell influx, osteolysis, bone resorption, and loss of biological activity of the implant.9−11 Kurtz et al. and other researchers have reported that cross-linked polyethylene shows a lower wear rate and relatively longer life span in comparison to that of virgin UHMWPE, used in orthopedic implant applications.12−18 To enhance the life span of implants, organic (graphene, carbon fiber, and carbon nanotubes), © XXXX American Chemical Society

Received: March 19, 2016 Accepted: July 5, 2016

A

DOI: 10.1021/acsbiomaterials.6b00154 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

Figure 1. SEM images and particle size distribution of as received (a, b) UHMWPE, (c, d) Al2O3, and (e, f) silane-functionalized CNT powders, respectively.

modulus slightly decreased from 84.5 to 74.2 MPa. Carbon nanotubes (CNTs) are extremely strong with tensile strength (∼200 GPa), Young’s modulus (∼1 TPa) and flexible (with break strain ∼10−30%).27,28 The reinforcement of UHMWPE with 1 wt % multiwalled CNTs enhanced toughness from ∼60 MPa to ∼150 MPa (∼150%) and modulus ∼ from 977.4 MPa to ∼1352.3 MPa (∼38%).29 It has been reported that reinforcement of silane-modified CNTs in UHMWPE has lowered the specific wear rate by ∼59%, compared to that of virgin UHMWPE and formed sheek-kebab crystalline structure, which enhances the modulus, fracture toughness and crystallinity.30−33 It has been observed that the functionalized CNT reinforcement in UHMWPE reduces wear debris.33 For a good articulating surface, the leaching-out of reinforcements like CNT and alumina as well as matrix must be highly restricted under biotribomotion. Also, it has been reported that the hydroxides of alumina may induce neurological disorders, specially alzheimer disease.34−36

Importantly, a successful orthopedic implant must exhibit an effective biocompatibility and osteogenic affinity toward hosts. Therefore, a thorough understanding is required of correlation among biocompatibility, surface wetting, cell−material interaction, cell adhesion, proliferation, differentiation, durability, as well as physicochemical aspects of implant materials.37−40 Attachment of the protein layer on biomaterials surface occurs immediately after implantation. As host cells approaches (interrogate) the biomaterial implant, giant cells (cytokines) form and initiate protein signaling. Further, protein signals help the arrival of fibroblasts. The synthesis of collagen triggers the encapsulation of the implant within a cellular collagens bag.41−45 The receptors present on the cells interact with the hundreds of proteins and regulate a plethora of responses, such as organogenesis, adipogenesis,wound healing around material surface, etc.38,46 The surface perturbations (receptors) are related to wettability and surface energy.17,44,47 Very few studies have been reported on the effect of alumina and CNT reinforcement in polymer matrix in terms of wettability B


Article


ratio of (3:1 by volume) at ∼50 °C for about 20 h with constant stirring according to previous work.61 Hybrid UHMWPE-Al2O3− CNTs nanocomposites were blended of varying wt % of pristine and silane functionalized CNTs (2, 5 and 10), fixed wt % of Al2O3 (10%) and UHMWPE powder for ∼4 h. The composite (sheet of dimensions of 90 mm × 90 mm × 3 mm, and pellets of the dimensions of diameter 12 mm and thickness ∼3 mm) were fabricated using compression molding (SCM-30, M/s Santec Automation Pvt. Ltd., Delhi) at processing temperature of 200 °C, pressure of 7.5 MPa and holding time of 60 min. The schematic of processing is shown in Figure 2, and

(changes from ∼88° ± 2° to ∼118° ± 4°) and surface energy (∼23.20 to ∼17.75 m N/m).17,47−53 However, no work has yet been reported on the combined effect of Al2O3 and CNTs reinforcement on correlating the surface energy, wettability, protein-adhesion with osteogenic differentiation of mesenchymal stem cells (MSCs), and cell viability of UHMWPE. The effect of CNTs and Al2O3 reinforcement in UHMWPE is evaluated in terms of change in surface energy and density of adsorbed protein at the surface, which regulate cell adhesion and osteogenic differentiation.54 Osteogenic differentiation is a prime biological function of MSCs to form bone-creating osteocytes on the periphery of implant biomaterials. Few carbon materials like graphene based biomaterials and metallic substrate have been studied for osteogenic differentiation of human adipose-derived stem cells (hADSCs) and bone marrow multipotent stromal cells (BMSCs) coupled with characteristic gene expression using gene markers.55,56 There are list of gene biomarkers that have been used to analyze the gene expression.57,58 CD10 and CD92, have been used as surface markers to demonstrate expression of hBMSCs differentiation for osteogenic and adipogenic lineages as well as immune response.59 Though immune response, osteolytic reactions and gene expressions are available for UHMWPE, however, the correlation of gene expression with wettability, surface energy and cytocompatibility are still not well studied, which is indeed a prime factor in appropriately designing an implant material.59,60 Therefore, current work aims at establishing the role of wettability on the protein adhesion (using bovine serum albumin (BSA) protein). Further, the leaching out of alumina, if any, during in vitro tests was estimated using energy dispersive spectroscopy. The aspect of protein adsorption is connected to the apatite precipitation and cytocompatibility (using mouse fibroblast cell line (L929)) on the UHMWPE-based surfaces. In addition, correlation of osteogenic differentiation and gene expression mechanism of stem cells is established with dispersion fraction (of surface energy) and apatite mineralization on CNT−Al2O3-reinforced UHMWPE.

Figure 2. Schematic of processing UHMWPE composites reinforced with CNTs and Al2O3.

the nomenclature of processed compositions is provided in Table 1. The microstructural, phase, mechanical, and biological characterization of CNT−Al2O3−UHMWPE composites are presented in the following sections. 2.3. Physical and Mechanical Characterization. The density of the composites was measured by Archimedes’ principle by observing change in the weight of samples (CITIZEN CX 220 microbalance) using ethanol as immersion medium. Bulk hardness of the materials was determined using Vickers indentation (BAREISS-V-TEST) Bareiss Prüfgerätebau GmbH, Germany. An average hardness from ten indentations is reported herewith (taken at room temperature with 10 g load on each sample with a dwell time of 10 s). The indent diagonals were measured with the help of an optical microscope. 2.4. Phase and Microstructural Characterization. Presence of various phases in compression molded Al2O3 and CNTs reinforced UHMWPE nanocomposites were identified by analyzing X-ray diffraction pattern (Rich-Seifert, 2000D diffractometer, operated at 25 kV and 15 mA with Cu−Kα (λ= 1.541 Å) radiation scanned at step size of 0.5°/min). Fourier Transform Infrared Analysis (FT-IR, vortex 70, BRUKER) was carried out in the range of 400−4000 cm−1 in order to obtain the information about oxidation or/and degradation of materials. Micro-Raman spectroscopy of powder feedstock and compression molded composite pellets was performed to validate the retention of carbon nanotubes by WITec GmbH, Germany, Alpha 300, using 514 nm wavelength green laser. 2.5. Surface Energy and Protein Adsorption Estimation. The contact angle of distilled water on the processed composite samples was measured using contact angle goniometer (Dataphysics Contact Angle System OCA,) via sessile drop method at room temperature. A water droplet of ∼3−4 mm diameter was dropped on the sample using a syringe, and a charged coupled device (CCD) camera was used to capture the images. The contact angle for each sample was obtained by averaging 10 separate measurements. The surface energy was calculated by using the Owen−Wendt−Rabel−Kaelble (OWRK) geometric mean equations (see Supplementary 2).

2. MATERIALS AND METHODS 2.1. Materials. Medical grade UHMWPE powder (GUR1020) with a density of 0.93 g/cm3, molecular weight of 2.7 × 106 g/mol, and particle size of ∼10 to 300 μm was supplied in kind by Ticona GmbH (Werk Ruhrchemie) Germany. Particle size was measured by using a laser particle size analyzer (Analysette 22; Fritsch GmbH, Germany) and scanning electron microscope (SEM, ZEISS, EVOR50) as shown in Figures 1a and b. The α-Al2O3 (∼99.9% purity and density of ∼3.953 g/cm3) was procured from Allied Hi-tech Products, USA. SEM micrograph and its corresponding particle size are given in Figures 1c, d, respectively. The average particle size of acicular Al2O3 is 10−30 μm. Multiwalled CNTs of >95% purity (outer diameter 30−50 nm, inner diameter 5−15 nm, and length up to 10−20 μm with true density of ∼2.1 g/cm3)17 were procured from Nanostructured and Amorphous Materials Inc., NM, USA. 3-Aminopropyltriethoxysilane (APTES), trade name Dynsaylan AMEO, was procured from Evonik Degussa GmbH, Germany. To make it chemically reactive and enhance interfacial bonding with UHMWPE, we functionalized pristine CNT with APTES as per the protocol of previous work61 (also see Supplementary 1), briefly discussed in section 2.2. The morphology and particle size distribution of functionalized CNTs (fCNTs) are provided as Figure 1e, f, respectively. 2.2. Functionalization of CNTs and Processing of Nanocomposites. The modification of nanotubes was carried out by adding the reactive −COOH groups onto the surface by refluxing ∼3 g of pristine CNTs in 300 mL of acid mixture, (H2SO4−HNO3) in the C


Article


Table 1. Sample Composition, Densification, and Hardness of Fabricated Composites (where u-CNT and f-CNT represent pristine and silane-functionalized CNT, respectively) sample ID U U-10A U-10A-2uC U-10A-5uC U-10A10uC U-10A-2fC U-10A-5fC U-10A-10fC

theoretical density (g/cm3)

experimental density (g/cm3)

densification (%ρth)

average porosity (%)

hardness (MPa)

wt %Al2O3 wt %Al2O3 −2 wt % u-

0.934 1.007 1.017

0.932 1.001 1.004

99.8 ± 0.1 99.4 ± 0.1 98.7 ± 0.2

0.2 ± 0.1 0.6 ± 0.1 1.3 ± 0.0

36.1 ± 3.4 39.6 ± 3.8 63.6 ± 3.3

wt %Al2O3 −5 wt % u-

1.034

1.033

98.5 ± 0.1

1.5 ± 0.1

63.4 ± 5.7

wt %Al2O3 −10 wt % u-

1.063

1.025

96.6 ± 0.2

3.4 ± 0.1

63.9 ± 4.9

wt %Al2O3 −2 wt % f-

1.018

1.012

99.4 ± 0.1

0.6 ± 0.2

55.9 ± 2.1

wt %Al2O3 −5 wt % f-

1.034

1.018

98.5 ± 0.2

1.5 ± 0.1

56.9 ± 2.8

wt %Al2O3 −10 wt % f-

1.062

1.035

97.4 ± 0.1

2.6 ± 0.1

59.6 ± 7.9

composition UHMWPE UHMWPE-10 UHMWPE-10 CNT UHMWPE-10 CNT UHMWPE-10 CNT UHMWPE-10 CNT UHMWPE-10 CNT UHMWPE-10 CNT

using a standard protocol reported in a previous publication.17 The samples were sputter-coated with gold for observing cell adhesion, proliferation, and morphology under scanning electron microscope (ZEISS, EVOR50). 2.7.2. MTT Assay (ISO 10993−5). The MTT assay was performed for the compression molded composites pellets while taking gelatin coated glass coverslip as the negative control. The viability tests were performed for 2, 4, and 6 days using L929 mouse fibroblast cell line (ATCC). L929 cells were seeded on the samples and negative-control disc in 4 well plates at a density of ∼50 × 103/well, incubated for 2−6 days and then washed with 1X PBS. A 50 μL of MTT (5 mg/mL PBS)/100 μL of DMEM was added to each well and cells were reincubated for 4 h at 37 °C. The reaction of mitochondria (in viable cells) with tetrazolium component (present in MTT) forms an insoluble dark-blue formazan crystal, wich is dissolved by adding 200 μL of DMSO (Dimethyl sulfoxide) on each sample producing a blue colored solution. The solution was then transferred to a 96-well plate and cell viability was quantified in terms of optical density absorbance measured at 540 and 630 nm using an Automated Microplate Reader, Bio-Tek, model ELx800) against DMSO as blank solvent. 2.8. Osteogenic Differentiation. One of the most noteworthy characteristics of mesenchymal stem cells (MSCs) is their ability to differentiate into osteoblasts. Under in vitro conditions, such a behavior is easily captured by culturing MSCs in an appropriate induction medium. The C3H10t1/2 cells were seeded on the samples until they reached confluence. The medium was, then, removed and replaced with osteoblast differentiation medium; enriched with 20 mM β-glycerophosphate, 20 mM ascorbic acid, 1 × 10−7 M dexamethasone, 10% FBS, and 1% antibiotic solution. The cells were then incubated at 37 °C with 5% CO2 and the differentiation medium was replaced every 2 days. Once differentiated, the total cellular RNA was isolated using Guanidine thiocyanate as per the standard isolation protocol. The total RNA was reverse transcribed according to the manufacturer’s instructions (Bangalore genie) and analyzed for the expression of osteoblast related osteopontin gene marker by standard amplification of their respective mRNAs by PCR (polymer chain reaction). PCR conditions include an initial denaturation at 95 °C for 5 min, followed by a 30 cycle amplification consisting of denaturation at 95 °C for 15 s, annealing at 55−60 °C for 30 s, and an extension step at 72 °C for 30 s followed by a final extension step at 72 °C for 10 min. The relative expression of these genes was determined by normalizing gene expression to the housekeeping gene, β-actin. 2.9. Statistical Analysis. All experiments were triplicated and an average value is reported with standard deviation. Obtained data is statistically analyzed using Student’s t test with >95 percentage confidence level (p < 0.05).

Protein adsorption behavior was estimated by using the standard protein adsorption protocol as given in Bicinchoninic Acid (BCA) protein quantification assay kit (Sigma-Aldrich, Bangalore, India). An equal amount of initial protein concentration of 2 mg/mL was poured into 24 well plate containing composite samples (with a gelatin coated coverslip taken as a negative control). These well plates were place in a specific incubator at 37 °C temperature, 5% CO2 and 95% humidity for 48 h. After completion of incubation period, each sample was gently rinsed with PBS in order to remove nonadhered proteins. Surface adsorption of protein is estimated according to standard protein adsorption protocol (Bicinchoninic Acid Protein Kit (96 well plate assay) Sigma-Aldrich), which is based on the formation of a “Cu2+- protein complex” under alkaline conditions and reduction of Cu2+ to Cu1+. Formation of protein- BCA complex results a change in color from blue to violet, which is quantified using UV−vis (Merk, Germany) at wavelength between 540 to 570 nm. The quantification of the protein adsorption is performed by dividing the absorbance value by absorbance coefficient of BSA (i.e., value of 63). Five tests were conducted for each composition and their average values are reported herewith. 2.6. Bioactivity, Quantification of Mineralized Apatite, and Leaching of Alumina. The mineralization of apatite on the material’s surface was carried out using DMEM (Dulbecco’s modified Eagle’s medium), containing 10% serum and 1% antibiotic and simulated body fluid (SBF) under specified conditions such as 5% CO2, 95% humidity and 37 °C temperature for 48 h incubation period, respectively. Mineralized apatite was quatified by evaluating the ratio of integrated intensity peaks of apatite to all other phases in the XRD pattern. Further, in vitro experiments were performed for all the composites in order to check the presence of leached aluminum, which may result via hydroxide formation under similar culture conditions in DMEM and SBF solution. Thus, after 48 h of incubation of UHMWPE-based samples, the solutions (DMEM and SBF) were taken out onto a fresh coverslip (in another well plate), dried in an oven at 37 °C, and an elemental analysis was performed (using energy dispersive spextroscopy (SEM, ZEISS, EVOR50) in order to observe the presence of any leached aluminum in the dried culture medium. 2.7. Cellular Response. 2.7.1. Cell Adhesion and Proliferation Study. L929 Mouse fibroblast cell line (ATCC) was cultured on the control and fabricated composite pellets. The gelatin (0.2%) coated glass coverslip was used as a negative control with varying concentration of CNTs (2, 5, and 10 wt %) in 10 wt % Al2O3 reinforced UHMWPE compression molded pellets. The samples were ultrasonicated with ethanol to remove impurities, if any, dried at room temperature and further sterilized by 70% ethanol in the UV chamber along with negative control disc (gelatin-coated coverslip). Subconfluent monolayer of L929 cells were used for the seeding. L929 cells were cultured in DMEM containing 10% serum and 1% antibiotic under 5% CO2, 95% relative humidity, and 37 °C temperature by D


Article


Figure 3. XRD pattern of: (a) starting raw materials, and (b) CNTs and Al2O3 reinforced UHMWPE composite pellets. FT-IR spectra of (c) starting powders CNTs, UHMWPE and Al2O3, and (d) polymer-based composites. (e) Raman spectra during CNT functionalization.

3.2. Phase and Microstructural Characterization. Figure 3 shows the XRD patterns of the initial powders (Figure 3a) where the characteristics peaks of UHMWPE are observed at 2θ values of 21.9 and 24.2° and Al2O3 at 35.2, 37.9, and 43.6°, respectively, whereas CNT showed peaks at 26.1 and 43.1°. The XRD pattern of CNTs-Al2O3 reinforced compression molded UHMWPE biocomposites is presented in Figure 3b. Characteristic peaks of CNTs in the composites are not detected in the XRD pattern due to its poor X-ray reflection and low fraction in comparison to that of UHMWPE. First, in order to confirm the silanized nature of CNTs, FTIR of powders is carried out (Figure 3c). The result shows the presence of Si−O and Si−OH bonds with the stretching frequencies at 1031 and at 753 cm−1, respectively. Phase retention and chemical degradation (if any) of compression molded composites as well as functionalized CNT is confirmed by FTIR with presence of carbonyl (CO) stretching peak at ∼1724 cm−1, but did not give any information about the damage of functionalized CNTs in the compression molded composites. Therefore, to observe the structural and morphological change (i.e., CNT damage) after functionalization, both Raman spectroscopy and electron microscopy examination (using TEM) have been performed (Figures 3 and 4, respectively). The characteristic defect D-peak (1340 cm−1) and graphitic G- peak (1572−1582 cm−1) is obtained to be

3. RESULTS AND DISCUSSION 3.1. Densification and Hardness. Table 1 presents the nomenclature, densification, and hardness of the compressionmolded UHMWPE-Al2O3−CNT composites. Table 1 shows that 2 wt % CNT with 10 wt % Al2O3 reinforcement (U-10A2uC) has substantially higher hardness when compared to that without CNT reinforcement (U-10A). Further, higher amount of CNT loading (up to 10 wt %) does not show any significant enhancement in the hardness. This may be due to an increase in porosity and probable enhancement in the agglomeration for high amount of CNT loading. All unfunctionalized-CNT (uCNTs) reinforced composites exhibited nearly the same densification within the error bars (>98.5% dense). It has been observed that Al2O3 addition only marginally increased the hardness by ∼11%, whereas synergistic reinforcement of CNTs with Al2O3, yielded comparatively higher values of hardness (∼78% with u-CNT and ∼66% with f-CNTs) in comparison to that of UHMWPE. The hardness value of UHMWPE is obtained to be ∼36.1 MPa, whereas the highest hardness has been observed for 10 wt % u-CNTs reinforced composite, i.e., ∼ 64 MPa. Lower hardness of f-CNT reinforced UHMWPE-Al2O3 (compared to that of u-CNT reinforcement) may be attributed to to surface damage of outer CNT layers during chemical functionalization. E


Article


to decreased surface energy (18.54 mJ/m2) and high dispersion fraction (0.967) when compared to that of U-10A composite (CA of 96.5°, SFE of 22.86 mJ/m2 (σsd of 0.780), and adsorbed protein ∼0.32 mg/cm2). Further, a synergistic ∼30% increase in the contact angle has been observed in comparison to that of virgin UHMWPE, showing higher hydrophobicity due to its higher dispersion fraction17 of free energy (Figure 5). Further, a slight decrease in the contact angle has been observed for fCNT-reinforced composites in comparison to that of u-CNTs (115.7 to 108.5°, see Table 2). The prevailing reason is the presence of reactive moieties and polar hydrophilic groups (−OH, −COOH, hydrogen bonds, etc.) on the surface due to silane functionalization, which ultimately changes the surface chemistry (see Supplementary 2) and wettability.64 Thus, higher surface energy, wettability and surface chemistry account for the change in the contact angle on UHMWPE-Al2O3−CNT composites. Both Al2O3 and CNTs reinforcement have shown to retain the roughness of compression molded UHMWPE (Table 2). Thus, the effect of surface roughness is not incorporated in the current analysis. The wetting nature of virgin UHMWPE, Al2O3, and CNTreinforced UHMWPE composites were further analyzed using three most significant biological liquid mediums (i.e., DI water, DMEM, and SBF) by sessile drop method. The SBF was prepared by Kokubo’s method,65 which has been used for the apatite growth studies. Surface energy, wettability, and amount of adsorbed protein on an implant surface are the significant parameters to affect the cell adhesion, spreading, and differentiation behaviors. Saturated hydrocarbons containing −CH3 group promotes the protein adsorption, and favors enhanced cellular interactions.17,48 Figure 5b shows the density of adsorbed protein on virgin UHMWPE and Al2O3 (10 wt %) and CNT-reinforced (2, 5, and 10 wt %) UHMWPE compression molded composites. It is evinced from the Figure 5b that the density of adsorbed protein increases significantly to ∼0.35 mg/cm2 for U-10A-10uC when compared to that of UHMWPE (from ∼0.26 mg/cm2). The 10 wt % alumina enhances the adsorbed protein density by 24%, whereas CNT reinforcements (with fixed 10 wt % alumina) enhances the same by 38% in comparison to that of UHMWPE. The 5 and 10 wt % of CNT-reinforced Al2O3−UHMWPE composite exhibited a nearly similar amount of adsorbed protein density (i.e., 0.34 and 0.35 mg/cm2, respectively). This might be due to their very similar value of dispersion fraction of surface free energy (σd), wettability (±10°) and surface free energy, SFE (see Table 2). It is well-established that the σd plays an important role in the preliminary cell adhesion and proliferation.17 Al2O3 (10 wt %) and CNTs (2, 5, and 10 wt %) reinforced UHMWPE composite system is hydrophobic in

Figure 4. Bright-field image of silane-treated CNTs with corresponding SAED pattern. The diffused ring appears because of the presence of amorphous carbon, and sharp rings correspond to the presence of CNTs.

similar to that of starting CNT, confirming its retention even after silane functionalization. The decreased G/D ratio (from 1.18 to 1.04) does indicate a slight damage to CNT during acid treatment and functionalization. The overall morphology of functionalized CNT seems to have remained intact and relatively less agglomerated than the pure CNTs.17 Figure 4 shows the selected area diffraction pattern (SAED) of silane CNTs (see inset) and gives the sharp rings (crystalline) and diffuse ring (amorphous carbon) corresponding to characteristic CNT planes. 3.3. Wettability, Surface Energy, and Protein Adsorption. The surface free energy (SFE) was calculated by the measurement of contact angle (θ) via sessile drop method and has been reported in Table 2 with their polar and dispersion fractions. About 10 readings were taken with a polar (water) and nonpolar solvent (n-hexane) and their average values are presented. Surface energy has an important role on dictating the adsorption of protein, and, consequently, proliferation of cellular response on any material surface. The total SFE is contributed by dispersion (nonpolar) and polar interactions.62 The dispersion interactions are always dominant, which are present in hydrophobic surfaces and saturated hydrocarbons. The SFE and it is polar and dispersion fractions of the components were calculated by utilizing Owen−Wendt− Rabe−-Kaelble (OWRK) geometric mean equations (see Supplementary 2).17,63 Figure 5a, b reveal that the contact angle and the amount of adsorbed BSA protein increases with increasing amounts of reinforcement (of CNTs and Al2O3) compared to that of virgin polymer. The U-10A-10C composite has shown higher values of contact angle (∼115°, Figure 5), roughness (0.40 μm) and amount of adsorbed protein (0.35 mg/cm2), which is attributed

Table 2. Surface Free Energy of Compression-Molded Composites sample ID U U-10A U-10A-2uC U-10A-5uC U-10A-10uC U-10A-2fC U-10A-5fC U-10A-10fC a

Ra (μm) 0.29 0.30 0.35 0.31 0.40 0.34 0.35 0.32

± ± ± ± ± ± ± ±

0.03 0.01 0.03 0.02 0.04 0.03 0.02 0.04

CA with water (deg) 88.9 96.5 109.2 111.6 115.7 97.3 104.6 108.5

± ± ± ± ± ± ± ±

2 3 5 1 4 4 3 5

CA with hexane (deg) 12.8 14.8 14.4 14.6 13.8 14.9 15.1 15.3

± ± ± ± ± ± ± ±

2 1 2 2 3 1 1 1

SFE (mJ/m2)

polar component

dispersion component

σs P

σsd

23.20 22.86 18.64 18.49 18.54 20.85 19.06 18.42

5.16 5.03 0.71 0.56 0.63 3.04 1.26 0.64

18.04 17.83 17.94 17.94 17.93 17.81 17.80 17.78

0.223 0.220 0.038 0.030 0.033 0.146 0.066 0.035

0.777 0.780 0.962 0.970 0.967 0.854 0.934 0.965

CA, contact angle; SFE = surface free energy; Ra = average roughness; σsp and σsd are polar and dispersion fraction of SFE, respectively. F


Article


Figure 5. (a) Contact angle of the compression-molded UHMWPE composites with distilled water, culture medium (DMEM), and simulated body fluid (SBF); and (b) adsorbed BSA protein concentration on UHMWPE composites. (* means that the mean values are significantly different than that on a control sample (U), with p < 0.002.)

Figure 6. XRD patterns of apatite mineralization, grown on surfaces of gelatin-coated cover clip, negative control, virgin UHMWPE, and composite samples (a) DMEM and (b) SBF.

nature (CA ≈ 115°).48 These hydrophobic interactions entropically favor the adsorption of protein on the surface. It has been seen that the wetting behavior of surface increases after the silane functionalization of CNTs (in 10 wt % reinforced Al2O3-UHMWPE composites). A slight decrease in the dispersion fraction of SFE, increased wettability, and reduction in the concentration of adsorbed protein has been observed in f-CNTs in comparison to those of u-CNT reinforced UHMWPE composites. The behavior may be attributed to enhanced hydrophilic interactions with bovine serum albumin protein. After 48 h of incubation period, the higher density of metabolically active L929 mouse fibroblast cells (3794 cell/mm2) was observed for 5 and 10 wt % CNT loading in UHMWPE matrix. This supports the formations of intense osteogenic differentiation bands shown later. Thus, the amount of adsorbed protein, dispersion fraction of surface free energy, wettability, bone remodeling, and osteogenic differentiation show a direct correlation with the cell adhesion, migration, and proliferation. 3.4. Bioactivity, Quantification of Mineralized Apatite, and Leached Alumina. Figure 6a, b show the XRD pattern of

mineralized apatite on composites after immersion in DMEM and SBF, respectively. The SEM images of grown apatite crystals on the surfaces (provided in Figures 7a−h, with elemental distribution of samples shown in Figure 8) after immersion in DMEM has confirmed the presence of Ca and P. The apatite mineralization upon immersed in SBF is not reported here, but elicited a similar response as that of DMEM. The apatite morphology appears as dendrimers, plate like and spherulite, nucleated on the surface under same culture conditions (Figure 7). The mineralization of apatite crystals is a very complex phenomenon and depends on several factors like mineral content (Ca/P ratio), pH of medium, adsorption and rate of release of ions at the interface, interactions with the immersion solutions, and nucleation and growth kinetics.17,66−70 The apatite content was quantified by taking the ratio of integrated area of the apatite XRD peaks divided by the integrated area of all the other peaks (Figure 6, see Table 3). It is clear from the Table 3, that the amount of % apatite is increasing with Al2O3 and CNTs reinforcement in comparison to that of UHMWPE. U-10A-10uC and U-10A-10fC possess G


Article


Figure 7. SEM micrographs of apatite mineralization on surface of materials in the presence of Dulbecco’s modified Eagles medium (DMEM) for (a) gelatin-coated coverslip (negative control), (b) U, (c) U-10A, (d) U-10A-5uC, (e) U-10A-10uC, (f) U-10A-2fC, (g) U-10A-5fC, and (h) U-10A10fC composites (in 48 h incubation period).

the highest amount of mineralized apatite content, i.e., ∼67.7% and ∼62.10%, respectively, on the surface after 48 h of incubation period. This suppresses the cell proliferation at higher times because of lower values of the dispersion fraction (i.e., weak hydrophobic interactions upon apatite mineralization) of SFE. Further, the in vitro experiments were performed for all composites in order to check the presence of leached aluminum, which may result via hydroxide formation under similar culture conditions in DMEM and SBF. Thus, after 48 h of incubation, the medium solutions (DMEM and SBF) were taken out onto a fresh coverslip (in another well plate) and were dried in an oven at 37 °C. The dried powders were characterized via energy-dispersive spectroscopy for elemental analysis (Figure 8). The EDS spectrum of dried DMEM (Figure 8) does not give any peak corresponding to aluminum, confirming that a short

period in vitro test (48 h, incubation period) is not sufficient to leach out the alumina from compression molded U−Al2O3− CNT. A similar observations is made for all the samples. In addition, Figure 8 also shows the presence of elements like Na, Mg, K, C, O, and Cl, which exist in the basic compositions of SBF as well as DMEM. An additional peak of sulfur arises from MgSO4 upon immersion in the DMEM solution. The presence of Ca and P (observed in all the dried mediums) indicates the precipitation of apatite minerals on the surface of petridish during incubation. Figure 9a, b, represent the FTIR spectra of mineralized apatite (after 48 h of incubation period) on the surface of composites in the presence of DMEM and SBF, respectively. The mineralized apatite on the composite surface is confirmed from the corresponding characteristics peaks (i.e., PO4−3, CO3−2, OH−, and HPO4−2) in the FTIR spectrum,39 Figure 9a (under DMEM) and b (under SBF). CO32− group gives an H


Article


Figure 8. Presence of elements in dried DMEM after 48 h of incubation of: (a) U, (b) U-10A, (c) U-10A-10 cm3, and (d) U-10A-10fC, and dried SBF after 48 h incubation of: (e) U, (f) U-10A, (g) U-10A-10 cm3, and (h) U-10A-10fC.

intense band between 1460 and 1530 cm−1 and weak band in the range of 870 and 880 cm−1, whereas PO43− bands is observed at 560 and 600 cm−1 and at 1000−1100 cm−1, respectively. A strong band and a split weak peak have been observed between 3600 to 2600 cm−1 and at 630 cm−1 for O− H vibrational stretching frequencies in H2O molecule.

3.5. Cellular Adhesion, Proliferation, and Quantification. SEM images of L292 mouse fibroblast cells cultured for 48 h on the composite samples in DMEM medium (Figure 10a−h) confirm that the processed composites exhibit good cytocompatibility toward L929 cell line. The cells have proliferated without eliciting any cytotoxicity on the material I


Article


Table 3. Quantification of Mineralized Apatite Fraction with DMEM and Corresponding Change in Surface Free Energy and Wettability sample-ID U U-10A U-10A-2uC U-10A-5uC U-10A-10uC U-10A-2fC U-10A-5f C U-10A-10fC

CA with H2O (deg) 66.0 56.6 67.9 57.5 49.6 52.2 62.4 67.2

± ± ± ± ± ± ± ±

3.6 1.7 7.2 1.0 3.4 5.2 4.9 4.4

CA with n-hexane (deg) 13.5 13.6 11.8 13.7 11.5 12.3 12.5 15.1

± ± ± ± ± ± ± ±

1.0 1.2 2.2 1.5 2.0 1.4 2.1 0.5

SFE (mJ/m2) 40.26 44.20 36.02 43.47 49.45 47.49 39.97 36.49

± ± ± ± ± ± ± ±

dispersion component

polar component

σsd

σs p

apatite (%)

17.62 17.91 18.04 17.91 18.05 18.01 17.99 17.79

22.64 26.28 17.98 25.56 31.40 29.48 21.98 18.70

0.43 0.41 0.51 0.41 0.36 0.38 0.45 0.49

0.57 0.59 0.49 0.59 0.64 0.62 0.55 0.51

48.5 54.7 57.8 58.4 67.7 54.6 55.9 62.1

3.1 5.2 3.8 5.7 2.4 2.9 2.7 2.4

± ± ± ± ± ± ± ±

3.3 1.6 2.1 1.5 2.4 3.0 1.4 5.3

CA, contact angle, SFE = surface free energy, σsd and σsp = dispersion and polar fraction of SFE; apatite fraction = [apatite/(apatite + UHMWPE +Al2O3)] (%). a

Figure 9. FTIR spectra of grown apatite oncomposites in (a) DMEM and (b) SBF.

surfaces. The confluence of cells at higher amount of u-CNTs reinforced composites (5 and 10 wt %) in 10 wt % Al2O3− UHMWPE composites is attributed to higher protein adsorption and enhanced dispersion fractions of SFE (Table 3), which dominate the hydrophobic interactions, thereby appropriately orienting the protein on biomaterial surface. Although comparatively less amount of cells were present on fCNTs reinforced composites (5 and 10 wt %) in 10 wt % Al2O3−UHMWPE. But the protein adsorption and cell density in U-10A-uC composites is higher than that of U sample. Figure 11 reports the metabolically active L929 mouse fibroblast cells (using MTT-assay), cultured and proliferated on the fabricated composite surfaces after 2, 4, and 6 days of incubation period under culture conditions (95% humidity, 5% CO2, and at 37 °C). The metabolic activity (Figure 11a) and optical density of live L929 cells in CNT−Al2O3-reinforced UHMWPE composites (Figure 11b) is statistically higher (with p < 0.002) with respect to that of negative control and UHMWPE. Whereas, for higher incubation periods (after

fourth and sixth day), it has been observed that the cell density of metabolically active cells decreases (see Figure 11a) for CNT−Al2O3−UHMWPE composites (when compared to that of negative control and UHMWPE sample). Such a decrease in cell density may be attributed to the confluence of L929 cells on the surface of Al2O3 and CNTs reinforced composites as the initial adhesion and proliferation is predominantly dictated by the dispersion fraction of surface free energy and the amount of adsorbed protein on the material surfaces.17,48 The highest amount of adsorbed protein has been observed for CNT-Al2O3reinforced composites in comparison to that of virgin UHMWPE (see Figure 5b). Furthermore, during the incubation, apatite has been mineralized on the sample surfaces, which increase the polar fraction of SFE (see Table 3) and lowers the dispersion fraction as well as hydrophobic interactions (because of changes in surface chemistry and reactive moieties present at the surfaces).48 The highest cell viability was observed for U-10A-10uC composites (3794 cells/ mm2) because higher amount of adsorbed protein on the J


Article


Figure 10. SEM micrographs showing proliferation of L929 mouse fibroblast cells on (a) gelatin-coated coverslip (negative control), (b) U, (c) U10A, (d) U-10A-5uC, (e) U-10A-10uC, (f) U-10A-2fC, (g) U-10A-5fC, and (h) U-10A-10fC composites (48 h incubation period).

Figure 11. (a) MTT assay results representing the relative number and (b) Density of metabolically active L929 cells on CNT−Al2O3-reinforced UHMWPE composites, respectively. (Club symbol means that the cell count and density is significantly different than that on neat UHMWPE and negative control sample (gelatin coated coverslip), with p < 0.002, whereas * means that the cell count is significantly different than that on a control sample, with p < 0.002.)

surface (Figure 11b, where the quantification of cells is performed with the help of ImageJ software). It is evident

that the cell density is always significantly higher for Al2O3 and CNTs reinforced UHMWPE compression-molded composites K


Article


Figure 12. (a) Gene expression analysis of osteogenic markers on gelatin coated coverslip (negative control), U, U-10A, U-10A-2uC, U-10A-5uC, U10A-10uC, U-10A-2fC, U-10A-5fC, and U-10A-10fC composites, and (b) schematic illustration showing effect of CNT functionalization on cell adhesion, proliferation and osteoblast differentiation (i.e., U, U-10A-5uC and U-10A-5fC composites).

total SFE and amount of adsorbed protein plays a significant role in the bone remodeling and osteogenic differentiation. Figure 12b also shows that the increasing amount of Al2O3 and CNTs, enhances σd of SFE. Higher dispersion-fraction of uCNT (compared to that of f-CNT) reinforcement results in higher protein adsorption, cell adhesion, and proliferation on the composite surfaces. It has observed that after 48 h of cell culture, a significant extent of apatite mineralization occurs on the biopolymer surfaces. Because of the presence of polar groups in the mineralized apatite crystals, the dispersion fraction (σd) gets reduced, which suppress the cell adhesion and proliferation on the biopolymer surfaces. Further, the dependency of wettability in terms of dispersion fraction of surface free energy (σd), density of adsorbed protein (BSA), and viable density of metabolically active cells (L929, mouse fibroblasts) is shown in Figure 13. From Figure 13, it is observed that the higher value of dispersion fraction of surface free energy (σd) is directly related to the density of adsorbed protein on the biopolymer surfaces. This is well correlated with data given in Table 2 and the plot of adsorbed protein (Figure 5). It is indicated that more hydrophobic surfaces adsorbed more amount of protein (∼0.35 mg/cm2) in U-10A-10uC when compared to that of less hydrophobic (∼0.26 mg/cm2) U. The relationship between cell viability, protein adsorption and the surface property (σd), as given in Figure 13, also concludes that the higher values of dispersion fraction of surface free energy (in u-CNT compared to that of f-CNT) favors high protein adsorption and higher metabolic activity (viable cell density) in primary stage of cell adhesion and proliferation.

in comparison to that of the negative control (gelatin-coated glass coverslip). 3.6. Osteogenic Differentiation Study. To estimate the osteogenicity of the developed implant materials, the osteogenic differentiation study was performed by incubating mesenchymal stem cells (MSCs). Mesenchymal cells condense to produce osteoblast, which deposit osteoid matrix, and array along the calcified region of the matrix. Osteoblast that are trapped within the bone matrix, later become osteocytes. By performing osteogenic differentiation, the presence of bone specific gene marker, i.e., osteopontin, confirmed the conversion of growing cells into bone developing cells. Figure 12 shows the PCR image of specific gene markers. It is clearly visible from Figure 12a that the band intensity of the osteopontin marker is high for higher weight percent of carbon nanotube reinforced composites when compared to that of alumina reinforced and UHMWPE matrix. It is well-known that cells never directly attach to the material surface,71−74 rather they get adhered with specific protein that serves as connecting ligand between implant surface and cells. Hence, proteinadsorption significantly affects the cell adherence and proliferation. The protein adsorption behavior (Figure 12b) confirms that more the protein adsorption; more is the band intensity in the PCR image (Figure 11a), which also reconfirms the results of enhanced cell viability of CNT−Al2O3-reinforced UHMWPE. It has been observed that the brightest bands correspond to u-CNTs in comparison to that of f-CNTs. The composites U-10A-5uC and U-10A-10uC possess the most intense bands compared to that of others, which is correlated with the amount of adsorbed protein. This result indicates that the surface energy and its dispersion fraction play significant role in the cellular growth, and that CNT functionalization necessitated for enhanced interfacial interaction with polymer matrix actually decreases the osteogenic differentiation. Figure 12b schematically shows the effect of Al2O3 and CNT reinforcements on UHMWPE matrix for osteoblast differentiation. Here, it is highlighted that the dispersion fraction of

4. CONCLUSIONS UHMWPE-based biopolymers reinforced with CNTs and Al2O3 (>98% densification) were processed by compression molding technique. The change in wettability of 10 wt % fCNT reinforced composite has been observed in terms of contact angle from ∼88° ± 2° to ∼108.5 ± 5° and surface L



Article

■

ACKNOWLEDGMENTS The authors acknowledge funding from the MHRD, Government of India. The authors also thank Celanese Pte. Ltd. Keppel Towers Singapore for providing UHMWPE medical grade polymer (GUR 1020) in kind. K.B. acknowledges the P.K. Kelkar Fellowship, IIT Kanpur, and funding from Department of Biotechnology (BT/PR13466/COE/34/26/ 2015).

■ Figure 13. Relationship between cell viability, protein adsorption, and the surface property (σd) for composites.

energy ∼23.20 to ∼18.42 mJ/m2 with respect to virgin UHMWPE. In addition, the change in density of adsorbed protein increased to ∼0.33 mg/cm2 in comparison to that of virgin UHMWPE (from ∼0.26 mg/cm2). But, higher dispersion fraction in u-CNT reinforcement has resulted enhanced protein adsorption (∼0.35 mg/cm2). It was observed that after 48 h of the incubation period, U-10A-5uC and U-10A-10uC composites exhibited the highest cell density of live cells on the surface, whereas lower density of cells with f-CNT reinforced composites is attributed to their higher dispersion fraction of surface free energy (0.970) and enhanced adsorbed protein density (∼0.34 mg/cm2). The absence of aluminum peak after 48 h of incubation of UHMWPE-samples in the SBF and DMEM concludes that the leaching out of alumina is completely absent for a short period in vitro test. Gene expression studies have indicated slightly weaker intensity of osteogenic differentiation bands for f-CNT in comparison to that of u-CNT-reinforced composites, which directly corroborates with the L929 cell density. Thus, high metabolic activity (with enhanced mineralization of apatite) in CNT−Al2O3reinforced UHMWPE nanocomposites makes it a potential candidate as osteoanalogous material for articulating surface.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00154. Supplementary 1, schematic of CNT functionalization with APTES and interactions; Supplementary 2, Owen− Wendt−Rabel−Kaelble (OWRK) geometric mean equations (PDF)

■

REFERENCES

(1) Pavoor, P. V.; Gearing, B. P.; Muratoglu, O.; Cohen, R. E.; Bellare, A. Wear reduction of orthopaedic bearing surfaces using polyelectrolyte multilayer nanocoatings. Biomaterials 2006, 27 (8), 1527−1533. (2) Vegetti, A.; Radnoczi, G.; Ossi, P. M. Role of embedded carbon particles on the morphology, microstructure and transport properties of sintered ultra-high molecular weight polyethylene. Carbon 2013, 65, 20−27. (3) Roualdes, O.; Duclos, M.-E.; Gutknecht, D.; Frappart, L.; Chevalier, J.; Hartmann, D. J. In vitro and in vivo evaluation of an alumina−zirconia composite for arthroplasty applications. Biomaterials 2010, 31 (8), 2043−2054. (4) An, J.-W.; You, D.-H.; Lim, D.-S. Tribological properties of hotpressed alumina−CNT composites. Wear 2003, 255 (1−6), 677−681. (5) Lewis, G. Properties of crosslinked ultra-high-molecular-weight polyethylene. Biomaterials 2001, 22 (4), 371−401. (6) Maloney, W. J.; S, R.; Schmalzried, T. P.; Chiba, J.; Huene, D.; Rubash, H. Isolation and characterization of wear particles generated in patients who have had failure of a hip arthroplasty without cement. J. Bone Jt. Surg. 1995, 77, 1301−1310. (7) Shanbhag, A.; Jacobs, J.; Glant, T.; Gilbert, J.; Black, J.; Galante, J. Composition and morphology of wear debris in failed uncemented total hip replacement. J. Bone Jt. Surg. Br. 1994, No. 76-B, 60−67. (8) Liao, Y.; Pourzal, R.; Wimmer, M. A.; Jacobs, J. J.; Fischer, A.; Marks, L. D. Graphitic tribological layers in metal-on-metal hip replacements. Science (Washington, DC, U. S.) 2011, 334 (6063), 1687−1690. (9) Wooley, P. H.; Schwarz, E. M. Aseptic loosening. Gene Ther. 2004, 11 (4), 402−407. (10) Boss, J. H.; Shajrawi, I.; Mendes, D. G. The nature of the boneimplant interface. The lessons learned from implant retrieval and analysis in man and experimental animal. Med. Prog. Technol. 1994, 20 (3−4), 119−142. (11) Langton, D. J.; Joyce, T.; Jameson, S. S.; Lord, J.; Van Orsouw, M.; Holland, J. P.; Nargol, A. V.; De Smet, K. A. Adverse reaction to metal debris following hip resurfacing: the influence of component type, orientation and volumetric wear. J. Bone Jt. Surg., Br. Vol. 2011, 93-B, 164−171. (12) Kostarelos, K.; Novoselov, K. S. Exploring the Interface of Graphene and Biology. Science 2014, 344 (6181), 261−263. (13) Puertolas, J. A.; Kurtz, S. M. Evaluation of carbon nanotubes and graphene as reinforcements for UHMWPE-based composites in arthroplastic applications: A review. J. Mech. Behavior Biomed. Mater. 2014, 39, 129−145. (14) Kurtz, S.; Gawel, H.; Patel, J. History and Systematic Review of Wear and Osteolysis Outcomes for First-generation Highly Crosslinked Polyethylene. Clin. Orthop. Relat. Res. 2011, 469 (8), 2262− 2277. (15) Kurtz, S. M.; Toth, J. M.; Siskey, R.; Ciccarelli, L.; MacDonald, D.; Isaza, J.; Lanman, T.; Punt, I.; Steinbeck, M.; Goffin, J.; van Ooij, A. The Latest Lessons Learned from Retrieval Analyses of Ultra-High Molecular Weight Polyethylene, Metal-on-Metal, and Alternative Bearing Total Disc Replacements. Seminars in Spine Surgery 2012, 24 (1), 57−70. (16) Kurtz, S. M.; Lau, E.; Ong, K. L.; Carreon, L.; Watson, H.; Albert, T.; Glassman, S. Infection risk for primary and revision instrumented lumbar spine fusion in the Medicare population. J. Neurosurg.: Spine 2012, 17 (4), 342−347.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +91-512-259-6194. Notes

The authors declare no competing financial interest. M


Article

ACS Biomaterials Science & Engineering (17) Patel, A. K.; Balani, K. Dispersion fraction enhances cellular growth of carbon nanotube and aluminum oxide reinforced ultrahigh molecular weight polyethylene biocomposites. Mater. Sci. Eng., C 2015, 46, 504−513. (18) Reinitz, S. D.; Currier, B. H.; Levine, R. A.; Van Citters, D. W. Crosslink density, oxidation and chain scission in retrieved, highly cross-linked UHMWPE tibial bearings. Biomaterials 2014, 35 (15), 4436−4440. (19) Yamamoto, G.; Shirasu, K.; Hashida, T.; Takagi, T.; Suk, J. W.; An, J.; Piner, R. D.; Ruoff, R. S. Nanotube fracture during the failure of carbon nanotube/alumina composites. Carbon 2011, 49 (12), 3709− 3716. (20) Chen, Y.-F.; Bi, J.-Q.; Yin, C.-L.; You, G.-L. Microstructure and fracture toughness of graphene nanosheets/alumina composites. Ceram. Int. 2014, 40, 13883−13889. (21) Afzal, M. A. F.; Kesarwani, P.; Reddy, K. M.; Kalmodia, S.; Basu, B.; Balani, K. Functionally graded hydroxyapatite-alumina-zirconia biocomposite: Synergy of toughness and biocompatibility. Mater. Sci. Eng., C 2012, 32 (5), 1164−1173. (22) Patel, A. K.; Trivedi, P.; Balani, K. Processing and mechanical characterization of compression-molded ultrahigh molecular weight polyethylene biocomposite reinforced with aluminum oxide. J. Nanosci. Nanoeng. Appl. 2014, 4 (3), 1−11. (23) Alsharif, S. O.; Bin Md Akil, H.; Abbas Abd El-Aziz, N.; Arifin Bin Ahmad, Z. Effect of alumina particles loading on the mechanical properties of light-cured dental resin composites. Mater. Eng. 2014, 54, 430−435. (24) Herkendell, K.; Shukla, V. R.; Patel, A. K.; Balani, K. Domination of volumetric toughening by silver nanoparticles over interfacial strengthening of carbon nanotubes in bactericidal hydroxyapatite biocomposite. Mater. Sci. Eng., C 2014, 34, 455−467. (25) Asi, O. An experimental study on the bearing strength behavior of Al2O3 particle filled glass fiber reinforced epoxy composites pinned joints. Composite Structures 2010, 92 (2), 354−363. (26) Chanda, A.; Mukhopadhyay, A. K.; Basu, D.; Chatterjee, S. Wear and friction behaviour of UHMWPE-alumina combination for total hip replacement. Ceram. Int. 1997, 23 (5), 437−447. (27) Jin, L.; Bower, C.; Zhou, O. Alignment of carbon nanotubes in a polymer matrix by mechanical stretching. Appl. Phys. Lett. 1998, 73 (9), 1197−1199. (28) Bower, C.; Rosen, R.; Jin, L.; Han, J.; Zhou, O. Deformation of carbon nanotubes in nanotube−polymer composites. Appl. Phys. Lett. 1999, 74 (22), 3317−3319. (29) Ruan, S. L.; Gao, P.; Yang, X. G.; Yu, T. X. Toughening high performance ultrahigh molecular weight polyethylene using multiwalled carbon nanotubes. Polymer 2003, 44 (19), 5643−5654. (30) Yang, B. X.; Pramoda, K. P.; Xu, G. Q.; Goh, S. H. Mechanical Reinforcement of Polyethylene Using Polyethylene-Grafted Multiwalled Carbon Nanotubes. Adv. Funct. Mater. 2007, 17 (13), 2062− 2069. (31) Haggenmueller, R.; Fischer, J. E.; Winey, K. I. Single Wall Carbon Nanotube/Polyethylene Nanocomposites: Nucleating and Templating Polyethylene Crystallites. Macromolecules 2006, 39 (8), 2964−2971. (32) Sui, G.; Zhong, W. H.; Ren, X.; Wang, X. Q.; Yang, X. P. Structure, mechanical properties and friction behavior of UHMWPE/ HDPE/carbon nanofibers. Mater. Chem. Phys. 2009, 115 (1), 404− 412. (33) Lee, J.-H.; Kathi, J.; Rhee, K. Y.; Lee, J. H. Wear properties of 3aminopropyltriethoxysilane-functionalized carbon nanotubes reinforced ultra high molecular weight polyethylene nanocomposites. Polym. Eng. Sci. 2010, 50 (7), 1433−1439. (34) Krewski, D.; Yokel, R. A.; Nieboer, E.; Borchelt, D.; Cohen, J.; Harry, J.; Kacew, S.; Lindsay, J.; Mahfouz, A. M.; Rondeau, V. Human health risk assessment for aluminium, aluminium oxide, and aluminium hydroxide. J. Toxicol. Environ. Health, Part B 2007, 10 (Suppl 1), 1−269. (35) Fulgenzi, A.; Vietti, D.; Ferrero, M. E. Aluminium involvement in neurotoxicity. BioMed Res. Int. 2014, 2014, 1−5.

(36) Shaw, C. A.; Tomljenovic, L. Aluminum in the central nervous system (CNS): toxicity in humans and animals, vaccine adjuvants, and autoimmunity. Immunol. Res. 2013, 56 (2), 304−316. (37) Hanker, J. S.; Giammara, B. L. Biomaterials and biomedical devices. Science 1988, 242 (4880), 885−892. (38) Stevens, M. M.; George, J. H. Exploring and Engineering the Cell Surface Interface. Science 2005, 310 (5751), 1135−1138. (39) Ratner, B. D.; Hoffman, A. S.; Schoen, F. J.; Lemons, J. E. Biomaterials Science: An Introduction to Materials in Medicine, third ed.; Elsevier: Amsterdam, 2012; pp 406−407. (40) Haynes, C. A.; Norde, W. Globular proteins at solid/liquid interfaces. Colloids Surf., B 1994, 2 (6), 517−566. (41) Sinha, R. K.; Morris, F.; Shah, S. A.; Tuan, R. S. Surface composition of orthopaedic implant metals regulates cell attachment, spreading, and cytoskeletal organization of primary human osteoblasts in vitro. Clin. Orthop. Relat. Res. 1994, 305, 258−272. (42) Martin, J. Y.; Schwartz, Z.; Hummert, T. W.; Schraub, D. M.; Simpson, J.; Lankford, J., Jr; Dean, D. D.; Cochran, D. L.; Boyan, B. D. Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63). J. Biomed. Mater. Res. 1995, 29 (3), 389−401. (43) Ong, J. L.; Prince, C.; Raikar, G. N.; Lucas, L. C. Effect of surface topography of titanium on surface chemistry and cellular response. Implant Dent. 1996, 5 (2), 83−88. (44) Schwartz, Z.; Boyan, B. D. Underlying mechanisms at the bone−biomaterial interface. J. Cell. Biochem. 1994, 56 (3), 340−347. (45) Williams, D. F. On the mechanisms of biocompatibility. Biomaterials 2008, 29 (20), 2941−2953. (46) Wallner, E. I.; Yang, Q.; Peterson, D. R.; Wada, J.; Kanwar, Y. S. Relevance of extracellular matrix, its receptors, and cell adhesion molecules in mammalian nephrogenesis. Am. J. Physiol. 1998, 275, F467−F477. (47) Zenkiewicz, M. Methods for the calculation of surface free energy of solids. J. Achievements Mater. Manuf. Eng. 2007, 24 (1), 137− 145. (48) Gupta, A.; Tripathi, G.; Basu, B.; Balani, K. Dependence of Protein Adsorption on Wetting Behavior of UHMWPE−HA− Al2O3−CNT Hybrid Biocomposites. JOM 2012, 64 (4), 506−513. (49) Tripathi, G.; Basu, B. A porous hydroxyapatite scaffold for bone tissue engineering: Physico-mechanical and biological evaluations. Ceram. Int. 2012, 38 (1), 341−349. (50) Boonrungsiman, S.; Gentleman, E.; Carzaniga, R.; Evans, N. D.; McComb, D. W.; Porter, A. E.; Stevens, M. M. The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (35), 14170−14175. (51) Trivedi, P.; Patel, A. K.; Maurya, R.; Jayaganthan, R.; Balani, K. Nanomechanical Characterization and Protein Adsorption of ColdRolled Zirconium Alloy. JOM 2015, 67 (4), 726−732. (52) Bertazzo, S.; Gentleman, E.; Cloyd, K. L.; Chester, A. H.; Yacoub, M. H.; Stevens, M. M. Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification. Nat. Mater. 2013, 12 (6), 576−583. (53) Boyan, B. D.; Hummert, T. W.; Dean, D. D.; Schwartz, Z. Role of material surfaces in regulating bone and cartilage cell response. Biomaterials 1996, 17 (2), 137−146. (54) Lavenus, S.; Berreur, M.; Trichet, V.; Pilet, P.; Louarn, G.; Layrolle, P. Adhesion and osteogenic differentiation of human mesenchymal stem cells on titanium nanopores. Eur. Cell Mater. 2011, 22, 84−96. (55) Lyu, C.-Q.; Lu, J.-Y.; Cao, C.-H.; Luo, D.; Fu, Y.-X.; He, Y.-S.; Zou, D.-R. Induction of Osteogenic Differentiation of Human Adipose-Derived Stem Cells by a Novel Self-Supporting Graphene Hydrogel Film and the Possible Underlying Mechanism. ACS Appl. Mater. Interfaces 2015, 7 (36), 20245−20254. (56) Marycz, K.; Smieszek, A.; Grzesiak, J.; Siudzinska, A.; Maredziak, M.; Donesz-Sikorska, A.; Krzak, J. The Osteogenic Properties of Multipotent Mesenchymal Stromal Cells in Cultures on TiO2 Sol-GelDerived Biomaterial. BioMed Res. Int. 2015, 2015, 1−11. N



■

(57) Otto, W. R.; Wright, N. A. Mesenchymal stem cells: from experiment to clinic. Fibrog. Tissue Repair 2011, 4, 20. (58) Boxall, S. A.; Jones, E. Markers for Characterization of Bone Marrow Multipotential Stromal Cells. Stem Cells Int. 2012, 2012, 975871. (59) Granéli, C.; Thorfve, A.; Ruetschi, U.; Brisby, H.; Thomsen, P.; Lindahl, A.; Karlsson, C. Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach. Stem Cell Res. 2014, 12 (1), 153− 165. (60) Kauther, M. D.; Xu, J.; Wedemeyer, C. Alpha-Calcitonin GeneRelated Peptide Can Reverse The Catabolic Influence Of UHMWPE Particles On RANKL Expression In Primary Human Osteoblasts. Int. J. Biol. Sci. 2010, 6 (6), 525−536. (61) Lavorgna, M.; Romeo, V.; Martone, A.; Zarrelli, M.; Giordano, M.; Buonocore, G. G.; Qu, M. Z.; Fei, G. X.; Xia, H. S. Silanization and silica enrichment of multiwalled carbon nanotubes: Synergistic effects on the thermal-mechanical properties of epoxy nanocomposites. Eur. Polym. J. 2013, 49 (2), 428−438. (62) Fowkes, F. M. Surfaces and Interfaces; Burke, J. J., Ed.; Syracuse University Press: Syracuse, NY, 1967. (63) Alam, F.; Patel, A. K.; Sharma, R. K.; Balani, K. Processing, Characterization and Fretting Wear of Zinc Oxide and Silver Nanoparticles Reinforced Ultra High Molecular Weight Polyethylene Biopolymer Nanocomposite. JOM 2015, 67 (4), 688−701. (64) Preedy, E. C.; Brousseau, E.; Evans, S. L.; Perni, S.; Prokopovich, P. Adhesive forces and surface properties of cold gas plasma treated UHMWPE. Colloids Surf., A 2014, 460 (0), 83−89. (65) Kokubo, T.; Kim, H.-M.; Kawashita, M. Novel bioactive materials with different mechanical properties. Biomaterials 2003, 24 (13), 2161−2175. (66) Barrere, F.; Layrolle, P.; van Blitterswijk, C. A.; de Groot, K. Biomimetic calcium phosphate coatings on Ti6Al4V: a crystal growth study of octacalcium phosphate and inhibition by Mg2+ and HCO3−. Bone 1999, 25 (2), 107S−111S. (67) Graham, S.; Brown, P. W. Reactions of octacalcium phosphate to form hydroxyapatite. J. Cryst. Growth 1996, 165 (1−2), 106−115. (68) Dekker, R. J.; de Bruijn, J. D.; Stigter, M.; Barrere, F.; Layrolle, P.; van Blitterswijk, C. A. Bone tissue engineering on amorphous carbonated apatite and crystalline octacalcium phosphate-coated titanium discs. Biomaterials 2005, 26 (25), 5231−5239. (69) Drouet, C. Apatite Formation: Why It May Not Work as Planned, and How to Conclusively Identify Apatite Compounds. BioMed Res. Int. 2013, 2013, 490946. (70) Aparecida, A.; Fook, M. L.; Guastaldi, A. Biomimetic apatite formation on Ultra-High Molecular Weight Polyethylene (UHMWPE) using modified biomimetic solution. J. Mater. Sci.: Mater. Med. 2009, 20 (6), 1215−1222. (71) Dewez, J.-L.; Doren, A.; Schneider, Y.-J.; Rouxhet, P. G. Competitive adsorption of proteins: Key of the relationship between substratum surface properties and adhesion of epithelial cells. Biomaterials 1999, 20 (6), 547−559. (72) Yu, Q.; Zhang, Y.; Chen, H.; Zhou, F.; Wu, Z.; Huang, H.; Brash, J. L. Protein Adsorption and Cell Adhesion/Detachment Behavior on Dual-Responsive Silicon Surfaces Modified with Poly(Nisopropylacrylamide)-block-polystyrene Copolymer. Langmuir 2010, 26 (11), 8582−8588. (73) Xu, Y.; Takai, M.; Ishihara, K. Protein adsorption and cell adhesion on cationic, neutral, and anionic 2-methacryloyloxyethyl phosphorylcholine copolymer surfaces. Biomaterials 2009, 30 (28), 4930−4938. (74) Arima, Y.; Iwata, H. Effects of surface functional groups on protein adsorption and subsequent cell adhesion using self-assembled monolayers. J. Mater. Chem. 2007, 17 (38), 4079−4087.

Article

NOTE ADDED AFTER ASAP PUBLICATION This article was published ASAP on July 19, 2016, with an incorrect version of Figure 6. The corrected version was published ASAP on July 22, 2016.

O


Carbon Nanotube Functionalization Decreases Osteogenic

Recommend Documents