Improving osseointegration of Co-Cr by nanostructured titanium coatings
© Pham; licensee Springer. 2014
Received: 11 January 2014
Accepted: 8 April 2014
Published: 21 April 2014
This study reports the deposition of nanostructured Ti films on Co-Cr substrates to improve their surface characteristics and biocompatibility. The microstructure of the Ti films was controlled by application of negative substrate bias voltages. The surface roughness of Co-Cr implants was increased significantly after Ti coatings. The nanostructured Ti films are found to improve osteointergration of Co-Cr implants as indicated by enhancing cellular attachment, proliferation and differentiation, which was attributed mainly to the application of a biocompatible Ti coating, possessed a higher surface area for cell attachments and growth.
Co-Cr is the most extensively studied metallic biomedical implant due to its outstanding properties such as high strength, high corrosion resistance, flexibility and biocompatibility (Ohmori et al. 2006; Reclare et al. 2005). However, lack of osseointegration is limited its application (Okazaki and Gothoh 2005; Granchi et al. 1999; Ingham and Fisher 2005). Previous studies have shown that surface modification of Co-Cr by coating their surface with a bioinert material such as diamond-like carbon (DLC) (Choubey et al. 2004), titanium oxide (TiO2) (Han et al. 2009; Dicu et al. 2008) and titanium nitride (TiN) (Pham et al. 2011a, b) would offer improved the osseointegration, supporting bone growth on the Co-Cr implants. Nevertheless, there is a concern about instability of the coating-substrate interface because there are a lot of differences in physical and chemical properties between the coating and the substrates.
Ti and its alloys are some of the most biocompatible metals and they have been proven to be the effective materials for improving the osseointegration properties in vitro (Jayaraman et al. 2004; Citeau et al. 2005) and even in vivo (Li et al. 2010; Matsuno et al. 2001). In recent years, researchers have been shown that nanostructured Ti coating can enhance the biocompatibility of biomedical metals significantly (Vetrone et al. 2009; Khang et al. 2008) because of its high surface area (Liu et al. 2007), which provided higher binding sites and interlocking for osteoblast growth (Khang et al. 2012; Venkatsurya et al. 2012; Kim et al. 2012). More recent studies reported that nanoscale surface roughness, which directly correspond to the size of protein and cell membrane receptor, could also be sensitive to osteoblast proliferation and differentiation in vitro (Lipski et al. 2008; Webster et al. 2000), bioactive in vivo (Xue et al. 2005), protein adhesion (Dolatshahi-Pirouz et al. 2010) and gene expression (Mendonca et al. 2009). Nevertheless, there are no reports on the effect of nanostructured Ti coating on Co-Cr alloy for potential applications as the orthopaedic and dental implants.
Therefore, in this study, a Ti film was deposited on a Co-Cr substrate by DC sputtering. The microstructure and the surface roughness of the Ti films deposited Co-Cr substrates were evaluated (FE-SEM) and atomic force microscopy (AFM) testing, respectively. Pre-osteoblast (MC3T3-E1) were used for an osseointegration evaluation in terms of cell attachment, proliferation and differentiation and compared with those of the uncoated Co-Cr.
Materials and methods
Ti films were deposited onto Co-Cr substrates by DC sputtering (Ultech, Daegu, Korea). Prior to deposition, the Co-Cr substrates (Bukang Coalloy, Korea) with dimensions of 10 mm × 10 mm × 1 mm or 20 mm × 20 mm × 1 mm were ground with a 2000-grit SiC abrasive paper and cleaned ultrasonically. The deposition chamber was pumped to 5×10−4 Pa using rotary and diffusion pumps. The substrate was then subjected to ion bombardment in an argon flow discharge under a negative bias voltage of 600 V for 30 min to remove any residual surface contamination. Subsequently, the Ti films were deposited by DC sputtering of a Ti target (diameter 75 mm, thickness 5 mm, purity 99.99%, Kahee Metal, Korea) at a deposited power of 60 W in high purity argon (99.998% pure). The sputtering process of Ti film was carried out by varying the application of substrate bias voltages (Vb) up to 100 V to the Co-Cr substrate to control the structure of Ti films. The deposition of Ti films was carried out without the application of a negative substrate bias to the Co-Cr substrate. For the Ti films studied herein, the working pressure of 0.6 Pa were employed during reactive sputtering, while the substrate temperature of 100°C was maintained using a halogen heater with a programmable temperature controller.
The microstructure and surface morphology of Ti films deposited on Co-Cr substrates were studied by field emission scanning electron microscopy (SUPRA 55 VP, CARL ZEISS, Germany) operated at 2 kV. In addition, the surface morphology and average surface roughness (RMS) of the samples were measured by atomic force microscopy (Nanostation II, Germany) in tapping mode with a 5-μm scan sizes for both x and y axis.
Pre-osteoblasts MC3T3-E1 (ATCC, CRL-2593) were used to examine the interaction between the cell and specimens (uncoated Co-Cr substrate and Ti-deposited Co-Cr). The cells were maintained in α-MEM containing 10% fetal bovine serum (FBS) and 1% antibiotic at 37°C in humidified air and 5% CO2. The cell cytoskeleton organization was visualized by confocal laser scanning microscopy (CLMS, Zeiss-LSM510, Carl Zeiss Inc., NY, USA). After culturing for 24 h and 72 h, the cells on the tested sample were fixed in 4% paraformaldehyde in PBS for 10 min, washed in PBS, permeabilized with 0.1% Triton X-100 in PBS in 7 min, washed in PBS and stained with fluorescent anti-tubulin for 30 min. The cell nuclei were counterstained with DAPI for 5 min. The stained samples were placed on a cover slide, and the cell morphology was observed.
The rate of proliferation was measured after culturing for up to 10 days using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(−4-sulfophenyl)-2H-tetrazolium (MTS, Promega, Madison, WI, USA) for mitochondrial reduction. The cells (2 × 104 cell/mL) were seeded on the specimens (uncoated Co-Cr substrate and Ti-deposited Co-Cr) and cultured for 10 days. They were then washed with PBS and placed in a culture medium containing the MTS solution and returned to the incubator at 37°C for 3 h. This assay is based on the ability of metabolically active cells to reduce a tetrazolium-based compound, MTS, to a purple formazan product. The quantity of formazan product, which is measured by the absorbance at 490 nm using a micro-reader (Biorad, Model 550, USA), is directly proportional to the number of living cells in the culture.
The extent of cell differentiation was assessed by measuring the alkaline phosphatase (ALP) activity of the cells cultured on the specimens (uncoated Co-Cr substrate and Ti-deposited Co-Cr). The cells (1 × 104 cell/mL) were seeded on the specimens and cultured for 21 days. They were then washed with PBS and detached using trypsin-ethylene diamine tetraacetic acid. The amount of protein in the cell lysates was quantified using a protein assay kit (biorad, Hercules, CA, USA) and the ALP activity was assayed calorimetrically using p-nitrophenyl phosphate (pNPP, Sigma-Aldrich, UK). This colorimetric assay is based on the conversion of pNPP to p-nitrophenol (pNP) in the presence of ALP, where the rate of pNP production is proportional to the ALP activity. The absorbance of the reaction product, p-nitrophenol, was measured at 405 nm using a microplate reader.
The data is presented as the mean ± standard deviation. Statistical analysis was performed using a t-test. A p value < 0.05 was considered significant.
Results and discussion
The effect of a nanostructured Ti film on the biocompatibility of a Co-Cr substrate was examined. DC sputtering allowed the successful deposition of a dense and uniform Ti films on Co-Cr. The surface roughness of the Co-Cr was increased remarkably by nanostructured Ti film. Furthermore, the Ti film enhanced the attachment, proliferation and differantiation of osteoblasts remarkably. This suggests the potential use of Ti-deposited Co-Cr as orthopaedic and dental implants.
This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number “103.99-2013.05”. Author thanks Prof. Hyoun-Ee Kim for contribution to this paper.
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