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Graphene-reinforced calcium silicate coatings for load-bearing implants
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Biomed. Mater. 9 025009 (http://iopscience.iop.org/1748-605X/9/2/025009) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 86.129.102.113 This content was downloaded on 23/04/2014 at 15:19
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Biomedical Materials Biomed. Mater. 9 (2014) 025009 (7pp)
doi:10.1088/1748-6041/9/2/025009
Graphene-reinforced calcium silicate coatings for load-bearing implants Youtao Xie 1 , Hongqing Li 1 , Chi Zhang 2 , Xin Gu 3 , Xuebin Zheng 1 and Liping Huang 1 1
Shanghai Institute of Ceramics, Key Laboratory of Inorganic Coating Materials, Chinese Academy of Sciences, Shanghai 200050, People’s Republic of China 2 Department of Orthopaedics, Zhongshan Hospital, Fudan University, Shanghai 200032, People’s Republic of China 3 Department of Orthopaedics, Tenth People’s Hospital, Shanghai 200011, People’s Republic of China E-mail: [email protected] and [email protected] Received 9 August 2013, revised 9 October 2013 Accepted for publication 21 November 2013 Published 11 February 2014 Abstract
Owing to the superior mechanical properties and low coefficient of thermal expansion, graphene has been widely used in the reinforcement of ceramics. In the present study, various ratios of graphene (0.5 wt%, 1.5 wt% and 4 wt%) were reinforced into calcium silicate (CS) coatings for load-bearing implant surface modification. Surface characteristics of the graphene/calcium silicate (GC) composite coatings were characterized by scanning electron microscopy. Results show that the graphene plates (less than 4 wt% in the coatings) were embedded in the CS matrix homogeneously. The surfaces of the coatings showed a hierarchical hybrid nano-/microstructure, which is believed to be beneficial to the behaviors of the cell and early bone fixation of the implants. Wear resistance measured by a pin-on-disc model exhibited an obvious enhancement with the adoption of graphene plates. The weight losses of the GC coatings decreased with the increase of graphene content. However, too high graphene content (4 wt% or more) made the composite coatings porous and the wear resistance decreased dramatically. The weight loss was only 1.3 ± 0.2 mg for the GC coating containing 1.5 wt% graphene (denoted as GC1.5) with a load of 10 N and sliding distance of 500 m, while that of the pure CS coating reached up to 28.6 ± 0.5 mg. In vitro cytocompatibility of the GC1.5 coating was evaluated using a human marrow stem cell (hMSC) culture system. The proliferation and alkaline phosphatase, osteopontin and osteocalcin (OC) osteogenesis-related gene expression of the cells on the GC1.5 coating did not deteriorate with the adoption of graphene. Conversely, even better adhesion of the hMSCs was observed on the GC1.5 coating than on the pure CS coating. All of the results indicate that the GC1.5 coating is a good candidate for load-bearing implants. Keywords: graphene, coating, reinforcement, cytocompatibility, implant (Some figures may appear in colour only in the online journal)
performance of the implants, especially in a load-bearing environment [5, 6]. Particulate debris from orthopedic implant articulation has been an important issue for a long time [7, 8]. Shearing micro-movements often appear at the interface between the implant and bone tissue due to the mismatch of mechanical properties of the two materials in contact, movements of the limb, or insufficient initial fixing [9, 10]. The oscillatory micro-movements at the interface induce fretting
1. Introduction Hydroxylapatite (HA) and calcium silicate (CS) ceramics possess excellent bioactivity and biocompatibility. They are good coating candidates on metallic biomedical implants for improving biocompatibility and accelerating early osseointegration [1–4]. However, the poor fracture toughness and wear resistance of these materials restrict the long-term 1748-6041/14/025009+07$33.00
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Biomed. Mater. 9 (2014) 025009
wear and sometimes fatigue cracks, causing the early failure of joint prosthesis. The formation of composite coatings through the addition of a second phase is a good solution for enhancing the mechanical properties of the coatings and thus the longterm stability of the implants in a biological environment. Biocompatible ceramics [1, 11–13], metals [14] and other materials like bio-glasses [15] have been widely used for reinforcement in these coatings, and obvious improvements have been demonstrated. For example, the Young’s modulus and Vicker’s hardness of the HA composite coatings increased by 17.6% and 16.3% respectively with zirconia and Ti–6Al– 4V doping [16]. Carbon nanotubes (CNTs) are one of the best reinforcements to plasma-sprayed HA coatings for improving the fracture toughness [17–20], hardness [17] and wear resistance [17, 20]. The oxidation of CNTs can be avoided by the formation of a ceramic protective layer during plasmaspraying processes and the inert shroud of the powder carrier gas (argon) [21, 22]. Furthermore, a slight decrease in the defect density of CNTs was found after plasma spraying due to the increase of graphitization on CNT walls with rapid high temperature exposure [17]. CNTs were found to enhance the adhesion of ceramic splats on metallic substrate by means of CNT bridging [23]. In this context, HA/CNT composite coatings retain greater integrity than HA coatings due to CNT bridging at the splat–splat and splat–substrate interfaces. The fracture toughness increased by 56% and the crystallinity increased from 53.7% to 80.4% through reinforcement of 4 wt% CNT in HA coatings [18]. Graphene is a kind of two-dimensional carbon nanofiller with a one-atom-thick planar sheet of sp2 bonded carbon atoms that are densely packed in a honeycomb crystal lattice. It is the basic structural unit of certain carbon allotropes, including graphite, CNTs and fullerenes. Owing to its high surface area, superior mechanical properties, thermal conductivity, flexibility and low coefficient of thermal expansion, graphene has been widely used as a reinforcement to improve the toughness and wear resistance of the ceramic matrix [20, 24–28]. For example, the elastic modulus and fracture toughness of spark plasma sintered HA doped graphene could be enhanced by 25% and 92%, respectively [27], and the wear resistance increased by 66% as compared to that of pure HA [20, 27, 28]. The cytotoxicity and biocompatibility of these carbon materials, like graphene and CNT, have also been widely studied. Several reports have demonstrated that apatite was formed on the CNT surface after immersion in simulated body fluids [29, 30]. Due to the selective absorption and attachment of proteins from cell culture medium by means of non-covalent C–C bonds, increased proliferation and adhesion of osteoblast cells on CNT or graphene-based surfaces have been reported [31–33]. Enhanced adhesion of osteoblast cells was found on the CNT-coated collagen surface in comparison with pure collagen, which is the main organic component of natural bone and widely used in orthopedic scaffolds [32]. Usui et al further demonstrated good in vivo bone tissue compatibility of CNT. Accelerated bone growth was found for CNT-coated collagen in a mouse model [34]. It has also been illustrated that graphene
showed good compatibility to human osteoblasts and human marrow stem cells (hMSCs). The adhesion, proliferation and differentiation were notably improved on graphene surfaces compared to those on the SiO2 substrates [35]. The CS coating possesses not only good biocompatibility but also high bonding strength with titanium alloy substrates [4, 36]. To improve the mechanical properties and long-term stability of CS coating, graphene doping was selected in this study. Vacuum plasma spraying was applied for fabrication of the graphene/calcium silicate (GC) composite coatings. Mechanical properties such as wear resistance and in vitro cytocompatibility of the coatings have been studied. 2. Experimental processes 2.1. Material preparation and coating characterization
CS powder (synthesized in this laboratory, with a size of 20– 100 μm) and graphene plates (with a size of 0.5–20 μm and thickness of 5–25 nm, and mass ratios of 0.5 wt%, 1.5 wt% and 4 wt%) were dispersed in a suspension with water-soluble organic binder. The suspension was sprayed in an atomized chamber and dried subsequently to obtain micron-size spherical agglomerates. A vacuum plasma-spraying system (VPS, Sulzer Metco, Switzerland) was used for preparation of the GC composite coatings (denoted as GC0.5, GC1.5 and GC4 according the graphene content in the coatings) on Ti–6Al–4V substrates. Pure CS and medically widely used titanium (Ti) coatings were deposited for comparison purposes. The surface morphologies of the GC and CS coatings were observed using scanning electron microscopy (SEM; JEOL JSM-6700F, Japan). 2.2. Wear resistance of the GC coatings
A universal micro-tribometer tester (UMT-3, CETR, USA) was used to evaluate the wear resistance and coefficient of friction (COF) of the coatings with a ball-on-disc model. A stainless steel ball was used as the counter surface. The wear load and sliding distance were chosen by comprehensively considering the condition of an implant inside the living body and the experimental requirement, and were assigned at a 10 N load, a sliding rate of 0.33 m s−1, and a sliding distance of 500 m. All of the wear tests were performed in an ambient condition without any lubricant. 2.3. Cytocompatibility evaluation of the coatings
The hMSCs were isolated and expanded as described in the reference [37]. Cells were cultured in α-MEM culture medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and antibiotics at 37 ◦ C in a humidified incubator of 5% CO2 and 95% air. hMSCs passaged up to the fourth generation were used for the experiments described below. Growth medium described as above was used for cell adhesion and proliferation evaluation. Osteogenic medium obtained by growth medium supplemented with 50 μM L-ascorbic acid, 10 mM glycerophosphate and 100 nM dexamethasone was used for cell differentiation measurements. 2
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(A)
(B )
(C )
(D )
(E )
Figure 1. Surface morphologies of the GC0.5 (A), GC1.5 (B) and CS (C) coatings. (D) is the high magnification of (B). (E) shows the graphene plates homogeneously embedded in the CS matrix (as indicated by the arrows) in the GC1.5 coating.
Cells cultured for 12 h were used for morphological observation by SEM. The sample preparation process is as follows: cells were fixed overnight in 2% glutaraldehyde, then rinsed twice in PBS, then dehydrated in a graded series of ethanol (30%, 50%, 70%, 80%, 95% and 100%) and ethanol/hexamethyl disilazane (HMDS) at various proportions (2:1, 1:1 and 1:2 of ethanol to HMDS and 100% HMDS). Before SEM measurement, the samples were dried in an oven at 37 ◦ C overnight. Cell proliferation was monitored using the 3(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Sigma, St. Louis, MO, USA) assay. One milliliter of cell suspension (about 1 × 104 cells) was cultured on a 10 × 10 mm2 sample for two, four and six days. At each pre-determined time point, 0.1 ml of MTT solution was added, and the optical density (OD) was measured at 570 nm using an automated plate reader (Synergy HT multi-detection microplate). In the proliferation assay, the OD value of day 1 was measured as a standard. The proliferation of hMSCs was expressed as the ratio of the OD value with that of day 1 of the same specimen [38]. The osteogenic-associated gene expression of hMSCs was quantified by real-time PCR with an ABI 7500 Real-Time PCR
System (Applied Biosystems, USA) and a PCR kit (SYBR Premix EX Taq, TaKaRa, Japan). Cells with a density of 3 × 105 cells/well were cultured in osteogenic medium for 4, 14 and 21 days. The comparative Ct-value method was used to calculate the relative quantity of alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OC). 2.4. Statistical analysis
Statistical analysis was performed using Origin (OriginLab, Northampton, MA, USA). Measurements were performed in triplicate, and results are expressed as the mean ± standard deviation. Statistical differences were determined by an analysis of variance. Values of p < 0.05 were considered to be statistically significant. 3. Results and discussions 3.1. Characterization of the coatings
The surface morphologies of the GC0.5 and GC1.5 coatings are shown in figures 1(A) and (B). SEM views and the following examinations of the GC4.0 coating are not shown here 3
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because of the poor quality. The high porosity of the GC4.0 coating may have resulted from the high graphene content and the partial destruction in the plasma spraying process. Compared with that of the pure CS coating (figure 1(C)), the surfaces of the GC0.5 and GC1.5 coatings demonstrated a lot of small particles adhering on the relatively large particle surfaces. The greater the graphene content in the composite coatings, the more nano- or microparticles were found. A high magnification view (figure 1(D)) shows that these small particles were formed by a cluster of nano- or microparticles. However, in the pure CS coating, the particle surfaces were relatively smooth and nanoparticles were rarely found. This kind of hierarchical hybrid nano-/microstructure is considered to be favorable to the cell adhesion, proliferation and differentiation behaviors. In our previous works [39], not only strong in vitro cytocompatibility, but also excellent in vivo biocompatibility and early new bone formation were found in the hierarchical hybrid nano-/microstructured surface of chemically treated Ti coating implants. The similar structure of the GC coating is expected to be beneficial to the behaviors of the hMSCs. It could also be seen from figure 1(E) that the graphene plates were homogeneously embedded in the CS matrix (as indicated by arrows). There were minor parts of the graphene plates destroyed in the plasma-spraying process. However, most of them survived the plasma-spraying process and remained complete. The homogeneous embedding of the graphene (as indicated by the arrows in figure 1(E)) indicated good adhering behaviors of graphene with the CS matrix. Balani et al [21] studied the wetting behaviors of the CNTs by Al2O3 and found that a CNT bridge formed an anchor between two plasma-sprayed splats. An alumina layer (about 20–25 nm thick) was formed on the CNT surface. The neck formation at the two splats and uniform thickness indicate excellent Al2O3 wetting on the CNT surface.
Figure 2. COFs of the GC0.5, GC1.5 and CS coatings.
(A )
(B )
3.2. Wear resistance of the coatings
Shearing micro-movements at the contact interface can induce fretting wear and sometimes fatigue cracks, and finally the early failure of joint prosthesis. Good mechanical fixation of the implants obtained by the rough surface can partly prevent the micro-movements. However, the relatively higher friction coefficient and fretting wear resistance are also necessary for the long-term performance of joint prosthesis. In this study, the COF of the GC coatings was measured by a pin-on-disc method. The COFs of the GC and CS coatings with respect to sliding distance at 10 N loads are shown in figure 2. The GC coatings showed a higher COF at almost all of the sliding distances and much lower weight losses than the CS coating. The weight loss of the CS coating reached up to 28.6 ± 0.5 mg with a sliding distance of 500 m (sliding rate of 0.33 m s−1), while it was only 1.3 ± 0.2 mg for GC1.5 and 7.8 ± 0.6 mg for GC0.5 coatings with a similar sliding distance. This improvement was attributed to the uniform dispersion of graphene plates and bridging effects between the splats and particles.
Figure 3. SEM images of the hMSCs cultured on the GC1.5 (A) and CS (B) coatings for 12 h.
3.3. In vitro cytocompatibility
To evaluate the in vitro cytocompatibility of the GC coatings, the GC1.5 coating was selected with pure CS and biomedically widely used Ti coating as comparisons. The morphologies of the hMSCs cultured on the GC1.5 and CS coatings were observed by SEM and are shown in figure 3. Cells on the GC1.5 coatings displayed a spindle-like morphology with relatively larger sizes, while those on the CS coating exhibited 4
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(A)
Figure 4. Proliferation of the hMSCs on the GC1.5 coatings, with CS and Ti as comparisons. (∗ ) p < 0.05.
elongated fusiform-shaped morphology. The pseudopodia of the cells on the CS coating were relatively larger and the amount was less than that on the GC1.5 coating. It is believed that the distribution and size of the pseudopodia depended on the substrate properties. Large pseudopodia indicate firm anchoring of the cells incapable of moving or proliferating, while the relatively smaller protruding ends of the cells always correspond to an active state [35]. Large size and lots of small protruding ends of the cells on the GC coatings indicate good proliferation as well as possible motility. The proliferation of the cells monitored by MTT assay shown in figure 4 further demonstrates the good cytocompatibility of the GC1.5 coating. During the six days of culture, cells on the GC1.5 coating exhibited a higher proliferation rate compared to those cultured on the Ti controls. At day 2 and 4, cell numbers on the GC1.5 and CS coatings were very close. With the culture time prolonged to six days, the cell number on the GC1.5 coating was notably higher than that on the CS coating. To further investigate the osteogenic differentiation behaviors of the cells on the GC1.5 coating, ALP, OPN and OC mRNA expressions were monitored. ALP is a well-defined early marker expressed by hMSCs during osteogenesis. It is mainly involved in the degradation of inorganic pyrophosphate to provide a sufficient local concentration of phosphate for mineralization [40]. OPN is an acidic glycoprotein excreted into the bone extracellular matrix, and is considered to be the mid-stage marker of osteogenic differentiation and mineralization [41]. As the late-stage marker, OC plays key biological roles in mineralization [42]. The expression of ALP (figure 5(A)), OPN (figure 5(B)) and OC (figure 5(C)) of the cells on the GC1.5 coating were monitored at 4, 14 and 21 days. Results show that these mRNA expressions arrived at a peak value after 14 days of culture except for the later marker of OC, which continued to increase for 21 days. Comparing with the Ti coating, cells on the GC1.5 and CS coatings exhibited much higher mRNA activity, and no apparent differences were found between these two kinds of coating. We attribute the good in vitro cytocompatibility of the GC1.5 coating to two factors. On the one hand, good
(B )
(C )
Figure 5. Comparison of osteogenesis-related gene expression of hMSCs cultured on GC1.5, CS and Ti coatings for 4, 14 and 21 days. (A) Relative expression level of ALP mRNA. (B) Relative expression level of h-OPN mRNA. (C) Relative expression level of OC mRNA. (∗ ) p < 0.05.
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cytocompatibility of the CS and graphene components in the GC1.5 coating has been demonstrated by early studies. Our in vivo study in a dog model has illustrated that new bone formation was notably more rapid on the CS coating than on the Ti controls [4]. Si, as the main component of the CS ceramic, has been reported to be mitogenic for human osteoblast-like cells [43] and acted through the insulin-like growth factor II, a known inducer of osteoblast proliferation [44]. Sun et al [45] have studied the stimulate efficiency of Si on the proliferation and differentiation of hMSCs. The good cytocompatibility of the carbon materials like graphene has also been illustrated [31–32] and explained by the selective absorption and attachment of proteins from cell culture medium by means of non-covalent C–C bonds. Apatite was found on the CNT surface after immersion in simulated body fluid [29, 30] and increased proliferation and adhesion of osteoblast cells on CNT or graphene-based surfaces were also reported [32, 43]. On the other hand, the hierarchical hybrid nano/microstructure formed on the GC1.5 coating is considered to be beneficial to the cell behaviors. The hierarchical structured titanium surface fabricated by acid etching followed by anodization has been reported to retain or promote nearly all the cell functions compared to the acid etching surface. Especially, the initial cell adhesion and osteogenesis-related gene expression was dramatically enhanced, and obvious synergistic effects in osteoblast functions were demonstrated [46, 47]. In our previous works [39], positive responses of hMSCs have also been illustrated in hierarchical hybrid micro/nanostructured Ti coating. The in vivo results which followed further demonstrated the early nova-bone formation on the hierarchical hybrid surface.
Acknowledgments This work was supported by the National Natural Science Foundation of China (grant nos 81071455 and 51172264) Reference [1] Zhao Y et al 2006 Microstructure and bond strength of HA(+ZrO2+Y2O3)/Ti6Al4V composite coatings fabricated by RF magnetron sputtering Surf. Coat. Technol. 200 5354–63 [2] Yu L et al 2003 Effect of spark plasma sintering on the microstructure and in vitro behavior of plasma sprayed HA coatings Biomaterials 24 2695–705 [3] Yang Y et al 2011 Osteoblast interaction with laser cladded HA and SiO2-HA coatings on Ti–6Al–4V Mater. Sci. Eng. C 31 1643–52 [4] Xue W et al 2005 In vivo evaluation of plasma-sprayed wollastonite coating Biomaterials 26 3455–60 [5] Hoppner D and Chandrasekaran V 1994 Fretting in orthopedic implants: a review Wear 173 189–197 [6] Sivakumar M et al 1995 Failures in stainless steel orthopedic implant devices: a survey J. Mater. Sci. Lett. 14 351–4 [7] Dobbs H and Minski M 1980 Metal ion release after total hip replacement Biomaterials 1 193–8 [8] Cheng C and Gross A 1988 Loosening of the porous coating in total knee replacement J. Bone Joint Surg. Am. B 70 377–81 [9] Walker P et al 1987 Strains and micromotions of press-fit femoral stem prostheses J. Biomech. 20 693–702 [10] Riues J et al 1995 Fretting wear corrosion of surgical implants alloys: effects of ion implantation and ion nitriding on fretting behavior of metals/PMMA contact Surface Modification Technologies VIII ed T S Sudarshan and M Jeandin (London: The Institute of Materials) pp 43–52 [11] Li J et al 1996 Sintering of partially-stabilized zirconia and partially-stabilized zirconia-hydroxyapatite composites by hot isostatic pressing and pressureless sintering Biomaterials 17 1787–90 [12] Li J, Fartash B and Hermansson L 1995 Hydroxyapatite alumina composites and bone-bonding Biomaterials 16 417–22 [13] Gautier S et al 1997 Processing, microstructure and toughness of Al2O3 platelet-reinforced hydroxyapatite J. Eur. Ceram. Soc. 17 1361–9 [14] Zheng X et al 2000 Bond strength of plasma-sprayed hydroxyapatite/Ti composite coatings Biomaterials 21 841–9 [15] Goller G et al 2003 Processing and characterization of bioglass reinforced hydroxyapatite composites Ceram. Int. 29 721–4 [16] Khor K et al 2004 Microstructure and mechanical properties of plasma sprayed HA/YSZ/Ti–6Al–4V composite coatings Biomaterials 25 4009–17 [17] Lahiri D et al 2011 Wear behavior and in vitro cytotoxicity of wear debris generated from hydroxyapatite-carbon nanotube composite coating J. Biomed. Mater. Res. A 96 1–12 [18] Balani K et al 2007 Plasma-sprayed carbon nanotube reinforced hydroxyapatite coatings and their interaction with human osteoblasts in vitro Biomaterials 28 618–24 [19] Tercero J et al 2009 Effect of carbon nanotube and aluminum oxide addition on plasma-sprayed hydroxyapatite coating’s mechanical properties and biocompatibility Mater. Sci. Eng. C 29 2195–202 [20] Balani K et al 2007 Tribological behavior of plasma-sprayed carbon nanotube-reinforced hydroxyapatite coating in physiological solution Acta Biomater. 3 944–51 [21] Balani K and Agarwal A 2008 Wetting of carbon nanotubes by aluminum oxide Nanotechnology 19 165701
4. Conclusions In this paper, GC composite coatings have been prepared using vacuum plasma-spraying technology. Results show that the composite coatings with lower than 4 wt% graphene possessed a hierarchical hybrid nano-/microstructured surface and good wear resistance. The graphene exhibited good wetting behaviors with the CS matrix and could be embedded in the CS matrix homogeneously. The weight loss from wear testing was only 1.3 ± 0.2 mg for the GC1.5 coating, while that of the CS coating was 28.6 ± 0.5 mg with the load at 10 N and sliding distance of 500 m. In vitro cytocompatibility evaluation experiments show that the proliferation and ALP, OC and OPN osteogenesis-related gene expression of the hMSCs on the GC1.5 coating were notably higher than on the Ti coating control, and similar to the CS coating. The results demonstrated that the addition of graphene did not deteriorate the cytocompatibility of the composite coatings. Conversely, as a result of the adoption of graphene, and therefore the formation of a hierarchical nano-/microstructured surface, the cytocompatibility of the GC1.5 coating was even slightly better than the pure CS coating. 6
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[36] Liu X and Ding C 2001 Apatite formed on the surface of plasma sprayed wollastonite coating immersed in simulated body fluid Biomaterials 22 2007–12 [37] Peng Z et al 2010 Adjustment of the antibacterial activity and biocompatibility of hydroxypropyltrimethyl ammonium chloride chitosan by varying the degree of substitution of quaternary ammonium Carbohydr. Polym. 81 275–83 [38] Tan H et al 2012 Physical characterization and osteogenic activity of the quaternized chitosan-loaded PMMA bone cement Acta Biomater. 8 2166–74 [39] Xie Y et al 2012 Influence of hierarchical hybrid micro/nano-structured surface on biological performance of titanium coating J. Mater. Sci. 47 1411–7 [40] Jennifer L and Xu H 2009 Mesenchymal stem cell proliferation and differentiation on an injectable calcium phosphate–chitosan composite scaffold Biomaterials 30 2675–82 [41] Frank O et al 2002 Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro J. Cell. Biochem. 85 737 [42] Ramaswamy Y et al 2008 The responses of osteoblasts, osteoclasts and endothelial cells to zirconium modified calcium-silicate-based ceramic Biomaterials 29 4392–402 [43] Keeting P et al 1992 Zeolite A increases proliferation, differentiation, and transforming growth factor beta production in normal adult human osteoblast-like cells in vitro J. Bone Miner. Res. 7 1281–9 [44] Xynos I et al 2000 Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II mRNA expression and protein synthesis Biochem. Biophys. Res. Commun. 276 461–5 [45] Sun H et al 2006 Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics Biomaterials 27 5651–7 [46] Zhao L et al 2010 The influence of hierarchical hybrid micro/nano-textured titanium surface with titania nanotubes on osteoblast functions Biomaterials 31 5072–82 [47] Zhang W et al 2013 The synergistic effect of hierarchical micro/nano-topography and bioactive ions for enhanced osseointegration Biomaterials 34 3184–95
[22] Balani K et al 2007 Role of powder treatment and carbon nanotube dispersion in the fracture toughening of plasma-sprayed aluminum oxide-carbon nanotube nanocomposite J. Nanosci. Nanotechnol. 7 3553–62 [23] Keshri A et al 2011 Carbon nanotubes improve the adhesion strength of a ceramic splat to the steel substrate Carbon 49 4340–7 [24] Dreyer R et al 2010 The chemistry of graphene oxide Chem. Soc. Rev. 39 228–40 [25] Wang G et al 2008 Facile synthesis and characterization of graphene nanosheets J. Phys. Chem. C 112 8192–5 [26] Wang G et al 2009 Synthesis and characterization of hydrophilic and organophilic graphene nanosheets Carbon 47 1359–64 [27] Lahiri D et al 2010 Carbon nanotube toughened hydroxyapatite by spark plasma sintering: microstructural evolution and multi-scale tribological properties Carbon 48 3103–20 [28] Chen Y et al 2007 Wear studies of hydroxyapatite composite coating reinforced by carbon nanotubes Carbon 45 998–1004 [29] Beuvelot J et al 2010 In vitro calcification of chemically functionalized carbon nanotubes Acta Biomater. 6 4111–7 [30] Niu L et al 2010 Bonelike apatite formation utilizing carbon nanotubes as template Langmuir 26 4069–73 [31] Akasaka T et al 2010 Thin films of single-walled carbon nanotubes promote human osteoblastic cells (Saos-2) proliferation in low serum concentrations Mater. Sci. Eng. C 30 391–9 [32] Li X et al 2010 Current investigations into carbon nanotubes for biomedical application Biomed. Mater. 5 022001 [33] Lee W et al 2011 Origin of enhanced stem cell growth and differentiation on graphene and graphene oxide ACS Nano 5 7334–41 [34] Usui Y et al 2008 Carbon nanotubes with high bone-tissue compatibility and bone-formation acceleration effects Small 4 240–6 [35] Kalbacova M et al 2010 Graphene substrates promote adherence of human osteoblasts and mesenchymal stromal cells Carbon 48 4323–9
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Graphene-reinforced calcium silicate coatings for load-bearing implants
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Biomed. Mater. 9 025009 (http://iopscience.iop.org/1748-605X/9/2/025009) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 86.129.102.113 This content was downloaded on 23/04/2014 at 15:19
Please note that terms and conditions apply.
Biomedical Materials Biomed. Mater. 9 (2014) 025009 (7pp)
doi:10.1088/1748-6041/9/2/025009
Graphene-reinforced calcium silicate coatings for load-bearing implants Youtao Xie 1 , Hongqing Li 1 , Chi Zhang 2 , Xin Gu 3 , Xuebin Zheng 1 and Liping Huang 1 1
Shanghai Institute of Ceramics, Key Laboratory of Inorganic Coating Materials, Chinese Academy of Sciences, Shanghai 200050, People’s Republic of China 2 Department of Orthopaedics, Zhongshan Hospital, Fudan University, Shanghai 200032, People’s Republic of China 3 Department of Orthopaedics, Tenth People’s Hospital, Shanghai 200011, People’s Republic of China E-mail: [email protected] and [email protected] Received 9 August 2013, revised 9 October 2013 Accepted for publication 21 November 2013 Published 11 February 2014 Abstract
Owing to the superior mechanical properties and low coefficient of thermal expansion, graphene has been widely used in the reinforcement of ceramics. In the present study, various ratios of graphene (0.5 wt%, 1.5 wt% and 4 wt%) were reinforced into calcium silicate (CS) coatings for load-bearing implant surface modification. Surface characteristics of the graphene/calcium silicate (GC) composite coatings were characterized by scanning electron microscopy. Results show that the graphene plates (less than 4 wt% in the coatings) were embedded in the CS matrix homogeneously. The surfaces of the coatings showed a hierarchical hybrid nano-/microstructure, which is believed to be beneficial to the behaviors of the cell and early bone fixation of the implants. Wear resistance measured by a pin-on-disc model exhibited an obvious enhancement with the adoption of graphene plates. The weight losses of the GC coatings decreased with the increase of graphene content. However, too high graphene content (4 wt% or more) made the composite coatings porous and the wear resistance decreased dramatically. The weight loss was only 1.3 ± 0.2 mg for the GC coating containing 1.5 wt% graphene (denoted as GC1.5) with a load of 10 N and sliding distance of 500 m, while that of the pure CS coating reached up to 28.6 ± 0.5 mg. In vitro cytocompatibility of the GC1.5 coating was evaluated using a human marrow stem cell (hMSC) culture system. The proliferation and alkaline phosphatase, osteopontin and osteocalcin (OC) osteogenesis-related gene expression of the cells on the GC1.5 coating did not deteriorate with the adoption of graphene. Conversely, even better adhesion of the hMSCs was observed on the GC1.5 coating than on the pure CS coating. All of the results indicate that the GC1.5 coating is a good candidate for load-bearing implants. Keywords: graphene, coating, reinforcement, cytocompatibility, implant (Some figures may appear in colour only in the online journal)
performance of the implants, especially in a load-bearing environment [5, 6]. Particulate debris from orthopedic implant articulation has been an important issue for a long time [7, 8]. Shearing micro-movements often appear at the interface between the implant and bone tissue due to the mismatch of mechanical properties of the two materials in contact, movements of the limb, or insufficient initial fixing [9, 10]. The oscillatory micro-movements at the interface induce fretting
1. Introduction Hydroxylapatite (HA) and calcium silicate (CS) ceramics possess excellent bioactivity and biocompatibility. They are good coating candidates on metallic biomedical implants for improving biocompatibility and accelerating early osseointegration [1–4]. However, the poor fracture toughness and wear resistance of these materials restrict the long-term 1748-6041/14/025009+07$33.00
1
© 2014 IOP Publishing Ltd
Printed in the UK
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wear and sometimes fatigue cracks, causing the early failure of joint prosthesis. The formation of composite coatings through the addition of a second phase is a good solution for enhancing the mechanical properties of the coatings and thus the longterm stability of the implants in a biological environment. Biocompatible ceramics [1, 11–13], metals [14] and other materials like bio-glasses [15] have been widely used for reinforcement in these coatings, and obvious improvements have been demonstrated. For example, the Young’s modulus and Vicker’s hardness of the HA composite coatings increased by 17.6% and 16.3% respectively with zirconia and Ti–6Al– 4V doping [16]. Carbon nanotubes (CNTs) are one of the best reinforcements to plasma-sprayed HA coatings for improving the fracture toughness [17–20], hardness [17] and wear resistance [17, 20]. The oxidation of CNTs can be avoided by the formation of a ceramic protective layer during plasmaspraying processes and the inert shroud of the powder carrier gas (argon) [21, 22]. Furthermore, a slight decrease in the defect density of CNTs was found after plasma spraying due to the increase of graphitization on CNT walls with rapid high temperature exposure [17]. CNTs were found to enhance the adhesion of ceramic splats on metallic substrate by means of CNT bridging [23]. In this context, HA/CNT composite coatings retain greater integrity than HA coatings due to CNT bridging at the splat–splat and splat–substrate interfaces. The fracture toughness increased by 56% and the crystallinity increased from 53.7% to 80.4% through reinforcement of 4 wt% CNT in HA coatings [18]. Graphene is a kind of two-dimensional carbon nanofiller with a one-atom-thick planar sheet of sp2 bonded carbon atoms that are densely packed in a honeycomb crystal lattice. It is the basic structural unit of certain carbon allotropes, including graphite, CNTs and fullerenes. Owing to its high surface area, superior mechanical properties, thermal conductivity, flexibility and low coefficient of thermal expansion, graphene has been widely used as a reinforcement to improve the toughness and wear resistance of the ceramic matrix [20, 24–28]. For example, the elastic modulus and fracture toughness of spark plasma sintered HA doped graphene could be enhanced by 25% and 92%, respectively [27], and the wear resistance increased by 66% as compared to that of pure HA [20, 27, 28]. The cytotoxicity and biocompatibility of these carbon materials, like graphene and CNT, have also been widely studied. Several reports have demonstrated that apatite was formed on the CNT surface after immersion in simulated body fluids [29, 30]. Due to the selective absorption and attachment of proteins from cell culture medium by means of non-covalent C–C bonds, increased proliferation and adhesion of osteoblast cells on CNT or graphene-based surfaces have been reported [31–33]. Enhanced adhesion of osteoblast cells was found on the CNT-coated collagen surface in comparison with pure collagen, which is the main organic component of natural bone and widely used in orthopedic scaffolds [32]. Usui et al further demonstrated good in vivo bone tissue compatibility of CNT. Accelerated bone growth was found for CNT-coated collagen in a mouse model [34]. It has also been illustrated that graphene
showed good compatibility to human osteoblasts and human marrow stem cells (hMSCs). The adhesion, proliferation and differentiation were notably improved on graphene surfaces compared to those on the SiO2 substrates [35]. The CS coating possesses not only good biocompatibility but also high bonding strength with titanium alloy substrates [4, 36]. To improve the mechanical properties and long-term stability of CS coating, graphene doping was selected in this study. Vacuum plasma spraying was applied for fabrication of the graphene/calcium silicate (GC) composite coatings. Mechanical properties such as wear resistance and in vitro cytocompatibility of the coatings have been studied. 2. Experimental processes 2.1. Material preparation and coating characterization
CS powder (synthesized in this laboratory, with a size of 20– 100 μm) and graphene plates (with a size of 0.5–20 μm and thickness of 5–25 nm, and mass ratios of 0.5 wt%, 1.5 wt% and 4 wt%) were dispersed in a suspension with water-soluble organic binder. The suspension was sprayed in an atomized chamber and dried subsequently to obtain micron-size spherical agglomerates. A vacuum plasma-spraying system (VPS, Sulzer Metco, Switzerland) was used for preparation of the GC composite coatings (denoted as GC0.5, GC1.5 and GC4 according the graphene content in the coatings) on Ti–6Al–4V substrates. Pure CS and medically widely used titanium (Ti) coatings were deposited for comparison purposes. The surface morphologies of the GC and CS coatings were observed using scanning electron microscopy (SEM; JEOL JSM-6700F, Japan). 2.2. Wear resistance of the GC coatings
A universal micro-tribometer tester (UMT-3, CETR, USA) was used to evaluate the wear resistance and coefficient of friction (COF) of the coatings with a ball-on-disc model. A stainless steel ball was used as the counter surface. The wear load and sliding distance were chosen by comprehensively considering the condition of an implant inside the living body and the experimental requirement, and were assigned at a 10 N load, a sliding rate of 0.33 m s−1, and a sliding distance of 500 m. All of the wear tests were performed in an ambient condition without any lubricant. 2.3. Cytocompatibility evaluation of the coatings
The hMSCs were isolated and expanded as described in the reference [37]. Cells were cultured in α-MEM culture medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and antibiotics at 37 ◦ C in a humidified incubator of 5% CO2 and 95% air. hMSCs passaged up to the fourth generation were used for the experiments described below. Growth medium described as above was used for cell adhesion and proliferation evaluation. Osteogenic medium obtained by growth medium supplemented with 50 μM L-ascorbic acid, 10 mM glycerophosphate and 100 nM dexamethasone was used for cell differentiation measurements. 2
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(A)
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Figure 1. Surface morphologies of the GC0.5 (A), GC1.5 (B) and CS (C) coatings. (D) is the high magnification of (B). (E) shows the graphene plates homogeneously embedded in the CS matrix (as indicated by the arrows) in the GC1.5 coating.
Cells cultured for 12 h were used for morphological observation by SEM. The sample preparation process is as follows: cells were fixed overnight in 2% glutaraldehyde, then rinsed twice in PBS, then dehydrated in a graded series of ethanol (30%, 50%, 70%, 80%, 95% and 100%) and ethanol/hexamethyl disilazane (HMDS) at various proportions (2:1, 1:1 and 1:2 of ethanol to HMDS and 100% HMDS). Before SEM measurement, the samples were dried in an oven at 37 ◦ C overnight. Cell proliferation was monitored using the 3(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Sigma, St. Louis, MO, USA) assay. One milliliter of cell suspension (about 1 × 104 cells) was cultured on a 10 × 10 mm2 sample for two, four and six days. At each pre-determined time point, 0.1 ml of MTT solution was added, and the optical density (OD) was measured at 570 nm using an automated plate reader (Synergy HT multi-detection microplate). In the proliferation assay, the OD value of day 1 was measured as a standard. The proliferation of hMSCs was expressed as the ratio of the OD value with that of day 1 of the same specimen [38]. The osteogenic-associated gene expression of hMSCs was quantified by real-time PCR with an ABI 7500 Real-Time PCR
System (Applied Biosystems, USA) and a PCR kit (SYBR Premix EX Taq, TaKaRa, Japan). Cells with a density of 3 × 105 cells/well were cultured in osteogenic medium for 4, 14 and 21 days. The comparative Ct-value method was used to calculate the relative quantity of alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OC). 2.4. Statistical analysis
Statistical analysis was performed using Origin (OriginLab, Northampton, MA, USA). Measurements were performed in triplicate, and results are expressed as the mean ± standard deviation. Statistical differences were determined by an analysis of variance. Values of p < 0.05 were considered to be statistically significant. 3. Results and discussions 3.1. Characterization of the coatings
The surface morphologies of the GC0.5 and GC1.5 coatings are shown in figures 1(A) and (B). SEM views and the following examinations of the GC4.0 coating are not shown here 3
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because of the poor quality. The high porosity of the GC4.0 coating may have resulted from the high graphene content and the partial destruction in the plasma spraying process. Compared with that of the pure CS coating (figure 1(C)), the surfaces of the GC0.5 and GC1.5 coatings demonstrated a lot of small particles adhering on the relatively large particle surfaces. The greater the graphene content in the composite coatings, the more nano- or microparticles were found. A high magnification view (figure 1(D)) shows that these small particles were formed by a cluster of nano- or microparticles. However, in the pure CS coating, the particle surfaces were relatively smooth and nanoparticles were rarely found. This kind of hierarchical hybrid nano-/microstructure is considered to be favorable to the cell adhesion, proliferation and differentiation behaviors. In our previous works [39], not only strong in vitro cytocompatibility, but also excellent in vivo biocompatibility and early new bone formation were found in the hierarchical hybrid nano-/microstructured surface of chemically treated Ti coating implants. The similar structure of the GC coating is expected to be beneficial to the behaviors of the hMSCs. It could also be seen from figure 1(E) that the graphene plates were homogeneously embedded in the CS matrix (as indicated by arrows). There were minor parts of the graphene plates destroyed in the plasma-spraying process. However, most of them survived the plasma-spraying process and remained complete. The homogeneous embedding of the graphene (as indicated by the arrows in figure 1(E)) indicated good adhering behaviors of graphene with the CS matrix. Balani et al [21] studied the wetting behaviors of the CNTs by Al2O3 and found that a CNT bridge formed an anchor between two plasma-sprayed splats. An alumina layer (about 20–25 nm thick) was formed on the CNT surface. The neck formation at the two splats and uniform thickness indicate excellent Al2O3 wetting on the CNT surface.
Figure 2. COFs of the GC0.5, GC1.5 and CS coatings.
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3.2. Wear resistance of the coatings
Shearing micro-movements at the contact interface can induce fretting wear and sometimes fatigue cracks, and finally the early failure of joint prosthesis. Good mechanical fixation of the implants obtained by the rough surface can partly prevent the micro-movements. However, the relatively higher friction coefficient and fretting wear resistance are also necessary for the long-term performance of joint prosthesis. In this study, the COF of the GC coatings was measured by a pin-on-disc method. The COFs of the GC and CS coatings with respect to sliding distance at 10 N loads are shown in figure 2. The GC coatings showed a higher COF at almost all of the sliding distances and much lower weight losses than the CS coating. The weight loss of the CS coating reached up to 28.6 ± 0.5 mg with a sliding distance of 500 m (sliding rate of 0.33 m s−1), while it was only 1.3 ± 0.2 mg for GC1.5 and 7.8 ± 0.6 mg for GC0.5 coatings with a similar sliding distance. This improvement was attributed to the uniform dispersion of graphene plates and bridging effects between the splats and particles.
Figure 3. SEM images of the hMSCs cultured on the GC1.5 (A) and CS (B) coatings for 12 h.
3.3. In vitro cytocompatibility
To evaluate the in vitro cytocompatibility of the GC coatings, the GC1.5 coating was selected with pure CS and biomedically widely used Ti coating as comparisons. The morphologies of the hMSCs cultured on the GC1.5 and CS coatings were observed by SEM and are shown in figure 3. Cells on the GC1.5 coatings displayed a spindle-like morphology with relatively larger sizes, while those on the CS coating exhibited 4
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Figure 4. Proliferation of the hMSCs on the GC1.5 coatings, with CS and Ti as comparisons. (∗ ) p < 0.05.
elongated fusiform-shaped morphology. The pseudopodia of the cells on the CS coating were relatively larger and the amount was less than that on the GC1.5 coating. It is believed that the distribution and size of the pseudopodia depended on the substrate properties. Large pseudopodia indicate firm anchoring of the cells incapable of moving or proliferating, while the relatively smaller protruding ends of the cells always correspond to an active state [35]. Large size and lots of small protruding ends of the cells on the GC coatings indicate good proliferation as well as possible motility. The proliferation of the cells monitored by MTT assay shown in figure 4 further demonstrates the good cytocompatibility of the GC1.5 coating. During the six days of culture, cells on the GC1.5 coating exhibited a higher proliferation rate compared to those cultured on the Ti controls. At day 2 and 4, cell numbers on the GC1.5 and CS coatings were very close. With the culture time prolonged to six days, the cell number on the GC1.5 coating was notably higher than that on the CS coating. To further investigate the osteogenic differentiation behaviors of the cells on the GC1.5 coating, ALP, OPN and OC mRNA expressions were monitored. ALP is a well-defined early marker expressed by hMSCs during osteogenesis. It is mainly involved in the degradation of inorganic pyrophosphate to provide a sufficient local concentration of phosphate for mineralization [40]. OPN is an acidic glycoprotein excreted into the bone extracellular matrix, and is considered to be the mid-stage marker of osteogenic differentiation and mineralization [41]. As the late-stage marker, OC plays key biological roles in mineralization [42]. The expression of ALP (figure 5(A)), OPN (figure 5(B)) and OC (figure 5(C)) of the cells on the GC1.5 coating were monitored at 4, 14 and 21 days. Results show that these mRNA expressions arrived at a peak value after 14 days of culture except for the later marker of OC, which continued to increase for 21 days. Comparing with the Ti coating, cells on the GC1.5 and CS coatings exhibited much higher mRNA activity, and no apparent differences were found between these two kinds of coating. We attribute the good in vitro cytocompatibility of the GC1.5 coating to two factors. On the one hand, good
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Figure 5. Comparison of osteogenesis-related gene expression of hMSCs cultured on GC1.5, CS and Ti coatings for 4, 14 and 21 days. (A) Relative expression level of ALP mRNA. (B) Relative expression level of h-OPN mRNA. (C) Relative expression level of OC mRNA. (∗ ) p < 0.05.
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cytocompatibility of the CS and graphene components in the GC1.5 coating has been demonstrated by early studies. Our in vivo study in a dog model has illustrated that new bone formation was notably more rapid on the CS coating than on the Ti controls [4]. Si, as the main component of the CS ceramic, has been reported to be mitogenic for human osteoblast-like cells [43] and acted through the insulin-like growth factor II, a known inducer of osteoblast proliferation [44]. Sun et al [45] have studied the stimulate efficiency of Si on the proliferation and differentiation of hMSCs. The good cytocompatibility of the carbon materials like graphene has also been illustrated [31–32] and explained by the selective absorption and attachment of proteins from cell culture medium by means of non-covalent C–C bonds. Apatite was found on the CNT surface after immersion in simulated body fluid [29, 30] and increased proliferation and adhesion of osteoblast cells on CNT or graphene-based surfaces were also reported [32, 43]. On the other hand, the hierarchical hybrid nano/microstructure formed on the GC1.5 coating is considered to be beneficial to the cell behaviors. The hierarchical structured titanium surface fabricated by acid etching followed by anodization has been reported to retain or promote nearly all the cell functions compared to the acid etching surface. Especially, the initial cell adhesion and osteogenesis-related gene expression was dramatically enhanced, and obvious synergistic effects in osteoblast functions were demonstrated [46, 47]. In our previous works [39], positive responses of hMSCs have also been illustrated in hierarchical hybrid micro/nanostructured Ti coating. The in vivo results which followed further demonstrated the early nova-bone formation on the hierarchical hybrid surface.
Acknowledgments This work was supported by the National Natural Science Foundation of China (grant nos 81071455 and 51172264) Reference [1] Zhao Y et al 2006 Microstructure and bond strength of HA(+ZrO2+Y2O3)/Ti6Al4V composite coatings fabricated by RF magnetron sputtering Surf. Coat. Technol. 200 5354–63 [2] Yu L et al 2003 Effect of spark plasma sintering on the microstructure and in vitro behavior of plasma sprayed HA coatings Biomaterials 24 2695–705 [3] Yang Y et al 2011 Osteoblast interaction with laser cladded HA and SiO2-HA coatings on Ti–6Al–4V Mater. Sci. Eng. C 31 1643–52 [4] Xue W et al 2005 In vivo evaluation of plasma-sprayed wollastonite coating Biomaterials 26 3455–60 [5] Hoppner D and Chandrasekaran V 1994 Fretting in orthopedic implants: a review Wear 173 189–197 [6] Sivakumar M et al 1995 Failures in stainless steel orthopedic implant devices: a survey J. Mater. Sci. Lett. 14 351–4 [7] Dobbs H and Minski M 1980 Metal ion release after total hip replacement Biomaterials 1 193–8 [8] Cheng C and Gross A 1988 Loosening of the porous coating in total knee replacement J. Bone Joint Surg. Am. B 70 377–81 [9] Walker P et al 1987 Strains and micromotions of press-fit femoral stem prostheses J. Biomech. 20 693–702 [10] Riues J et al 1995 Fretting wear corrosion of surgical implants alloys: effects of ion implantation and ion nitriding on fretting behavior of metals/PMMA contact Surface Modification Technologies VIII ed T S Sudarshan and M Jeandin (London: The Institute of Materials) pp 43–52 [11] Li J et al 1996 Sintering of partially-stabilized zirconia and partially-stabilized zirconia-hydroxyapatite composites by hot isostatic pressing and pressureless sintering Biomaterials 17 1787–90 [12] Li J, Fartash B and Hermansson L 1995 Hydroxyapatite alumina composites and bone-bonding Biomaterials 16 417–22 [13] Gautier S et al 1997 Processing, microstructure and toughness of Al2O3 platelet-reinforced hydroxyapatite J. Eur. Ceram. Soc. 17 1361–9 [14] Zheng X et al 2000 Bond strength of plasma-sprayed hydroxyapatite/Ti composite coatings Biomaterials 21 841–9 [15] Goller G et al 2003 Processing and characterization of bioglass reinforced hydroxyapatite composites Ceram. Int. 29 721–4 [16] Khor K et al 2004 Microstructure and mechanical properties of plasma sprayed HA/YSZ/Ti–6Al–4V composite coatings Biomaterials 25 4009–17 [17] Lahiri D et al 2011 Wear behavior and in vitro cytotoxicity of wear debris generated from hydroxyapatite-carbon nanotube composite coating J. Biomed. Mater. Res. A 96 1–12 [18] Balani K et al 2007 Plasma-sprayed carbon nanotube reinforced hydroxyapatite coatings and their interaction with human osteoblasts in vitro Biomaterials 28 618–24 [19] Tercero J et al 2009 Effect of carbon nanotube and aluminum oxide addition on plasma-sprayed hydroxyapatite coating’s mechanical properties and biocompatibility Mater. Sci. Eng. C 29 2195–202 [20] Balani K et al 2007 Tribological behavior of plasma-sprayed carbon nanotube-reinforced hydroxyapatite coating in physiological solution Acta Biomater. 3 944–51 [21] Balani K and Agarwal A 2008 Wetting of carbon nanotubes by aluminum oxide Nanotechnology 19 165701
4. Conclusions In this paper, GC composite coatings have been prepared using vacuum plasma-spraying technology. Results show that the composite coatings with lower than 4 wt% graphene possessed a hierarchical hybrid nano-/microstructured surface and good wear resistance. The graphene exhibited good wetting behaviors with the CS matrix and could be embedded in the CS matrix homogeneously. The weight loss from wear testing was only 1.3 ± 0.2 mg for the GC1.5 coating, while that of the CS coating was 28.6 ± 0.5 mg with the load at 10 N and sliding distance of 500 m. In vitro cytocompatibility evaluation experiments show that the proliferation and ALP, OC and OPN osteogenesis-related gene expression of the hMSCs on the GC1.5 coating were notably higher than on the Ti coating control, and similar to the CS coating. The results demonstrated that the addition of graphene did not deteriorate the cytocompatibility of the composite coatings. Conversely, as a result of the adoption of graphene, and therefore the formation of a hierarchical nano-/microstructured surface, the cytocompatibility of the GC1.5 coating was even slightly better than the pure CS coating. 6
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[36] Liu X and Ding C 2001 Apatite formed on the surface of plasma sprayed wollastonite coating immersed in simulated body fluid Biomaterials 22 2007–12 [37] Peng Z et al 2010 Adjustment of the antibacterial activity and biocompatibility of hydroxypropyltrimethyl ammonium chloride chitosan by varying the degree of substitution of quaternary ammonium Carbohydr. Polym. 81 275–83 [38] Tan H et al 2012 Physical characterization and osteogenic activity of the quaternized chitosan-loaded PMMA bone cement Acta Biomater. 8 2166–74 [39] Xie Y et al 2012 Influence of hierarchical hybrid micro/nano-structured surface on biological performance of titanium coating J. Mater. Sci. 47 1411–7 [40] Jennifer L and Xu H 2009 Mesenchymal stem cell proliferation and differentiation on an injectable calcium phosphate–chitosan composite scaffold Biomaterials 30 2675–82 [41] Frank O et al 2002 Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro J. Cell. Biochem. 85 737 [42] Ramaswamy Y et al 2008 The responses of osteoblasts, osteoclasts and endothelial cells to zirconium modified calcium-silicate-based ceramic Biomaterials 29 4392–402 [43] Keeting P et al 1992 Zeolite A increases proliferation, differentiation, and transforming growth factor beta production in normal adult human osteoblast-like cells in vitro J. Bone Miner. Res. 7 1281–9 [44] Xynos I et al 2000 Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II mRNA expression and protein synthesis Biochem. Biophys. Res. Commun. 276 461–5 [45] Sun H et al 2006 Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics Biomaterials 27 5651–7 [46] Zhao L et al 2010 The influence of hierarchical hybrid micro/nano-textured titanium surface with titania nanotubes on osteoblast functions Biomaterials 31 5072–82 [47] Zhang W et al 2013 The synergistic effect of hierarchical micro/nano-topography and bioactive ions for enhanced osseointegration Biomaterials 34 3184–95
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