Bio-Medical Materials and Engineering 24 (2014) 173–179 DOI 10.3233/BME-130797 IOS Press

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The influence of protein concentration on the biotribological properties of the stem-cement interface1 Hong-Yu Zhang*, Ming Zhou

State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University, Beijing 100084, China

Abstract. The stem-cement interface in total hip replacement experiences fretting wear following debonding under cyclical physiological loading. However, the influence of protein concentration on the biotribological properties of this interface has not been well taken into consideration. In the present study, a series of fretting frictional tests were performed using polished Ti6Al4V and bone cement, lubricated by bovine serum albumin solutions of different concentrations (5%, 30%, and 75%). Surface characterizations of Ti6Al4V pins were conducted by optical interferometer, scanning electron microscope, and Raman spectroscopy. The results show that the friction coefficient decreases with the increase of protein concentration, although the difference is not significant. In addition, bovine serum albumin is adsorbed onto Ti6Al4V surface, forming a protective film to prevent the metal substrate from wear. The elemental and spectroscopic analyses of the film confirm the presence of protein molecules adsorbed on Ti6Al4V surface, with a thickness of 2.5 μm. It is indicated from this study that fretting wear at the stem-cement interface can be postponed by promotion of protein adsorption on the metal surface. Keywords: stem-cement interface, biotribology, surface characterization, protein adsorption, bovine serum albumin

1. Introduction Aseptic loosening has generally been accepted as the main reason for failure of cemented total hip replacement (THR), which can be further attributed to the generation of wear debris through wear of the femoral components [1]. Initially, wear primarily concentrated at the head-cup interface because this interface was designed for motion. Recently, great progress has been made to reduce the wear at the head-cup interface with introduction of cross-linked ultrahigh molecular weight polyethylene (UHMWPE) and renaissance of the hard-on-hard bearing systems, such as metal-on-metal and ceramic-on-ceramic configurations [2, 3]. As a consequence, wear at the stem-cement interface shows an increasing significance in the overall wear of cemented THR [4-6]. As a weak link through mechanical bonding, the stem-cement interface inevitably debonds as a result of the cyclical physiological loading in vivo, which has been validated by clinical and experimental studies [7-9]. 1

This project is supported by National Natural Science Foundation of China (Grant No. 51005130), Scientific Research Starting Foundation for Returned Overseas Chinese Scholars, Ministry of Education of China (Grant No. 20121028121). * Corresponding author. E-mail: [email protected]

0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved

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Once debonding occurs, pseudo-synovial fluid will subsequently be pumped into the stem-cement interface, affecting the fretting wear behaviour through modifying the metal surface with protein molecules contained in the fluid [10, 11]. As early as 1991, protein adsorption onto artificial hip joint was reported by Jasty et al., who observed a layer of collagenous and fibrinous tissue surrounding the metal prostheses [12]. Thereafter, this issue was well studied with regard to protein adsorption on CoCrMo alloy, UHMWPE, and Al2O3 ceramic, etc [13, 14]. However, relatively little attention has been paid to the femoral components, i.e. the stem-cement interface. In addition, most previous research investigating the fretting mechanism at the stem-cement interface employed saline solution [15-17], therefore the influence of protein adsorption on the metal surface has not been taken into consideration. Accordingly, in the present study, the influence of protein concentration on the biotribological properties of the stem-cement interface was investigated using polished Ti6Al4V and bone cement, lubricated by protein solutions with different concentrations. 2. Materials and Methods 2.1. Experimental samples Cylindrical pins of medical-grade titanium alloy Ti6Al4V with a length of 10 mm and a diameter of 4 mm were manufactured to simulate the femoral stem. One spherically shaped end was finely polished to a surface roughness value of 45.5 nm (scan area: 0.1x0.12 mm2). The metal pin was mounted tightly to a UMT-III universal materials tester as the upper specimen. Polymethylmethacrylate bone cement was hand-mixed, delivered into a custom-made container, and cured in situ under pressurization by a planar Ti6Al4V cover. This cover was polished to a similar surface roughness value as that of the pins. Therefore, smooth bone cement disks (diameter of 30 mm and thickness of 5 mm) were obtained after detached from the metal cover, with a surface roughness value of 0.3 μm (scan area: 0.5x0.45 mm2). A custom-made fixture was designed to connect with the linear stage of the UMT-III tester, and the bone cement disk was placed on the fixture as the lower specimen. Preparation of the experimental samples is shown in Fig. 1.

Fig. 1. Preparation of experimental samples: (a) Ti6Al4V pin; (b) bone cement disk.

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2.2. Fretting frictional tests Bovine serum albumin (BSA, approx 14x4x4 nm3) was added to 0.9% saline solution to prepare the protein solutions with different concentrations (5%, 30%, and 75%). Temperature control of the lubricants at 37°C was achieved by using a heating rod and a thermocouple. The fretting frictional test was performed in a reciprocating mode (oscillation amplitude: 50 μm) at 2.0 Hz for a duration of 2.0 hours utilizing the UMT-III tester. A normal load of 0.98 N was applied, which was equivalent to an apparent maximum contact pressure of 64 MPa. This was calculated based on the Hertz theory for ball-on-flat configuration [18], Eqs. (1) and (2). This pressure was comparable to the normal contact stress at the stem-cement interface, which would be dominant following debonding of the femoral stem from the cement mantle [19]. 1

2

1 § 3w · 3 § E ' · 3 P= ¨ π © 2 ¸¹ ¨© R ¸¹

(1)

1 1 § 1 − μ12 1 − μ2 2 · = ¨ + ¸ E ' 2 © E1 E2 ¹

(2)

where P is the apparent maximum contact pressure, w is the normal load (0.98 N), R is the radius of ball (2 mm), E1 and 1 are the elastic modulus and Poisson’s ratio of Ti6Al4V (110 GPa and 0.3), and E2 and 2 are the elastic modulus and Poisson’s ratio of bone cement (2.3 GPa and 0.23). Four experiments were conducted for each solution to ensure the statistical validity. Before the tests, the metal pins were cleaned in an ultrasonic bath by petroleum ether, acetone and deionized water for 15 minutes respectively. The bone cement disks were cleaned in the ultrasonic bath by deionized water for 5 minutes. After the tests, the metal pin and bone cement disk were removed from the testing apparatus, dried with N2 gas jet. 2.3. Ti6Al4V surface characterization After all the fretting frictional tests were completed, the contact area of Ti6Al4V pins with bone cement was examined using a Quanta 200 FEG scanning electron microscope (SEM) associated with an energy dispersive X-ray analysis (EDX) in order to facilitate subtle observation and comparison of elemental composition for different positions. Additionally, the Ti6Al4V pins were characterized by a LabRAM HR800 Raman spectroscope to analyze the chemical functional groups of adsorbed proteins. Furthermore, the surface topography of the Ti6Al4V pins was measured employing a micro XAM-3D optical interferometer, through which the thickness of the adsorbed proteins could be obtained. 3. Results 3.1. Fretting frictional tests The difference in the mean friction coefficient for the fretting frictional tests using BSA solutions with different concentrations is not significant. However, a general decreasing trend was obtained with the increase of BSA concentration. The minimum value was 0.126 when 75% BSA solution was used as the lubricant, followed by that of 30% and 5% BSA solutions (0.130 and 0.143) respectively. In spite of certain fluctuation, the friction coefficient did not change much during the test, Fig. 2.

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Fig. 2. The change of friction coefficient as a function of test duration.

3.2. Ti6Al4V surface characterization The SEM micrographs of the Ti6Al4V pins are shown in Fig. 3. As for the samples lubricated by 5% BSA solution, the surface was covered mainly by NaCl crystal, and the presence of slight scratch within the contact area was detected following the removal of NaCl crystal by ultrasonic cleaning with deionized water. When 30% BSA solution was used as the lubricant, the Ti6Al4V pins demonstrated a solid film with alveolate feature. This phenomenon is more distinctive for those samples lubricated by 75% BSA solution. The elemental composition obtained from EDX analysis shows a significantly increased content of C from the solid film in comparison with the original Ti6Al4V pins, as shown in Fig. 4. This can be attributed to BSA adsorption on the metal surface, which forms a solid film covering the contact area. In order to validate the chemical functional groups of the solid film, the Ti6Al4V pins lubricated by 75% BSA solution were evaluated utilizing Raman spectroscopic analysis, through which the intense Raman-active vibrations of a molecule can be indicated from the Raman shift. It is indicated from Fig. 5 that the spectrum is characterized by four peaks at 1445 cm-1, 1660 cm-1, 2930 cm-1 and 3330 cm-1, which correspond to the vibration modes of C-H bending, amide I bands, C-H stretching and amide A, respectively. This is considered as further evidence to confirm that the solid film is indeed adsorbed protein from the BSA solutions as amide bond (-CONH-) is the basic unit of protein.

Fig. 3. The Ti6Al4V pins lubricated by BSA solutions with different concentrations: (a) 5%; (b) 30%; (c) 75%.

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Fig. 4. Comparison of elemental composition: (a) original Ti6Al4V; (b) BSA adsorption film.

Fig. 5. Raman spectroscopy of the adsorbed BSA on Ti6Al4V pins.

The three-dimensional (3D) optical interferometric micrograph and 2D line profile of the Ti6Al4V pins lubricated by 75% BSA solution is demonstrated in Fig. 6. The thickness of adsorbed BSA was calculated as the difference in surface height between the solid film and the metal substrate (2.5 μm).

Fig. 6. The Ti6Al4V pins lubricated by 75% BSA solution: (a) 3D interferometric micrograph; (b) 2D line profile.

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4. Discussion Under cyclical physiological loading, the integrity of the stem-cement interface eventually fails and the pseudo-synovial fluid is pumped into this interface, serving as the lubricant [20, 21]. The proteins contained in the fluid can influence the fretting wear behavior at the stem-cement interface due to rapid adsorption onto the femoral stem surface. Previous studies mainly focused on protein adsorption onto acetabular components, and concluded that the contacting surfaces could be protected from rubbing each other [22]. With regard to the in vitro simulations, BSA solution is a commonly used lubricant as albumin is the major protein in synovial fluid. In the present study, a series of fretting frictional tests were performed using polished Ti6Al4V and bone cement, lubricated by BSA solutions with different concentrations. It was shown that when 75% BSA solution was used, the friction coefficient was the lowest, and the contact area of the Ti6Al4V pin with bone cement was covered by a layer of protein film, protecting the metal substrate from fretting damage. It is considered that frictional test is a useful method to predict the wear behavior of materials, and fretting wear at the stem-cement interface can be postponed by promotion of protein adsorption on metal surface [23, 24]. The presence of a solid film with alveolate feature on the Ti6Al4V samples lubricated by solutions of higher BSA concentrations was clearly demonstrated by the SEM micrographs. This phenomenon could be caused by evaporation of the water molecules in the lubricant as a result of the accumulated heat in the local contact area during the test. The EDX and Raman spectroscopic analyses of the solid film confirmed that this film was adsorbed BSA on the metal surface, with the detection of an increase in the content of C and the basic unit of protein, i.e. amide bond. In the present study, the introduction of Raman spectroscopic characterization is considered as a more feasible method in comparison with other techniques, e.g. radiolabeling and fluorescent labeling [25, 26], especially when evaluating the physically adsorbed proteins on metal surface. From a physiochemical point of view, protein adsorbs onto biomaterials mainly by hydrophobic interaction and electrostatic interaction. BSA is a globular protein with a hydrophobic core and hydrophilic surface, and tends to adsorb on all surfaces owing to a gain in conformational entropy [27]. Additionally, it is indicated from previous studies that protein adsorption is dependent on pH value, ionic strength, and protein concentration of the solution [28]. Therefore, it is considered that the use of 75% BSA solution would result in a relatively thicker BSA film on Ti6Al4V pins, protecting the metal substrate more effectively. 5. Conclusions In the present study, the influence of protein concentration on the biotribological properties of the stem-cement interface was investigated. In general, protein solution with higher bovine serum albumin concentration results in lower friction coefficient and thicker protein film, which is considered to be beneficial for the reduction of fretting wear at the stem-cement interface. References [1] [2]

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The influence of protein concentration on the biotribological properties of the stem-cement interface.

The stem-cement interface in total hip replacement experiences fretting wear following debonding under cyclical physiological loading. However, the in...
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