Biotribological properties at the stem–cement interface lubricated with different media

Biotribological properties at the stem–cement interface lubricated with different media

journal of the mechanical behavior of biomedical materials 20 (2013) 209–216 Available online at www.sciencedirect.com www.elsevier.com/locate/jmbbm...

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journal of the mechanical behavior of biomedical materials 20 (2013) 209–216

Available online at www.sciencedirect.com

www.elsevier.com/locate/jmbbm

Research Paper

Biotribological properties at the stem–cement interface lubricated with different media H.Y. Zhanga,n, J.B. Luoa, M. Zhoua, Y. Zhangb, Y.L. Huangc a

State Key Laboratory of Tribology, Department of Precision Instruments and Mechanology, Tsinghua University, Beijing 100084, China School of Material and Mechanical Engineering, Beijing Technology and Business University, Beijing 100048, China c Jinghang Biomedicine Engineering Division, Beijing Institute of Aeronautical Material, Beijing 100095, China b

art i cle i nfo

ab st rac t

Article history:

Background: Debonding of the stem–cement interface occurs inevitably in-vivo under physio-

Received 5 July 2012

logical loading, and pseudo-synovial fluid is subsequently pumped into this interface, serving

Received in revised form

as the lubricant. However, the influence of protein adsorption onto the femoral stem surface

4 January 2013

has not been well taken into consideration in previous in vitro studies.

Accepted 6 January 2013

Materials and methods: The biotribological properties at the stem–cement interface were

Available online 16 January 2013

investigated through a series of fretting frictional tests using polished stainless steel 316L

Keywords:

stem and smooth bone cement, lubricated by three different media at body temperature, i.e.

Stem–cement interface

100% calf serum, 25% calf serum, and 0.9% saline solution. The surface characterization of the

Biotribology

femoral stem was evaluated sequentially using optical microscope, optical interferometer,

Surface characterization

scanning electron microscope, and Raman spectroscopy.

Protein adsorption

Results: The friction coefficient generally kept stable during the test, and the minimum value (0.254) was obtained when 100% calf serum was used as the lubricant. Slight scratches were detected within the contact area for the stainless steel 316L stems lubricated by 100% calf serum and 25% calf serum, which was further surrounded by the adsorbed protein film with alveolate feature. Additionally, a wear scar was present within the contact area when 0.9% saline solution was used as the lubricant. Conclusions: Protein adsorption onto the stainless steel 316L stem surface affected the biotribological properties at the stem–cement interface under oscillatory fretting mechanism. Generation of wear debris at the stem–cement interface may be postponed by modification of physicochemical properties of the femoral stem to promote protein adsorption. & 2013 Elsevier Ltd. All rights reserved.

1.

Introduction

The primary reason for failure of cemented total hip replacement has consistently been attributed to aseptic loosening, which is further caused by generation of wear debris through wear of the femoral components (Herberts and Malchau, 2000).

n

Corresponding author. Tel.: þ86 10 62783968; fax: þ86 10 62781379. E-mail address: [email protected] (H.Y. Zhang).

1751-6161/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jmbbm.2013.01.001

Recently, wear at the stem–cement interface is showing an increasing significance due to the great progress made to reduce wear at the head–cup interface (Zhang et al., 2008a; Blunt et al., 2009). The stem–cement interface has long been cited as a weak link, and it has been indicated from both clinical and experimental studies that debonding of this

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interface may be inevitable in-vivo for almost all stem designs under physiological loading (Schmalzried et al., 2000; Zhang et al., 2008b,c). Once debonding occurs, pseudo-synovial fluid is subsequently pumped into the stem–cement interface, serving as potential lubricant (Geringer et al., 2005). The composition of pseudo-synovial fluid is similar to that of normal synovial fluid, and the proteins, in particular, are considered to play an important part in contributing to the biotribological mechanisms of the total joint system as it is quite easy for them to adsorb onto the surfaces of artificial hip implant (Widmer et al., 2001; Chen et al., 2008). In 1991, Jasty et al. (Jasty et al, 1991) reported that a layer of collagenous and fibrinous tissue was clinically observed surrounding the metal prostheses, which may be related to the adsorbed proteins on the implant. Thereafter, protein adsorption onto the acetabular components (e.g. ultra high molecular weight polyethylene (UHMWPE), Al2O3 ceramic and CoCrMo alloy) has been well studied in literature (Karuppiah et al., 2006; Mavraki and Cann, 2009). However, relatively little attention has been paid to protein adsorption onto the femoral components, e.g. stainless steel, Ti6Al4V alloy. Additionally, most previous research performed to investigate abrasive/fretting wear at the stem–cement interface just employed saline solution or Ringer’s solution as the lubricant (Brown et al., 2007; Zhang et al., 2009a,b). Consequently, the effect of protein adsorption has not sufficiently been taken into consideration (Zhang et al., 2012). This present study therefore aims to gain a better understanding on protein adsorption mechanism at the stem–cement interface and its influence on the biotribological properties through a series of frictional tests using polished stainless steel 316L stem and smooth bone cement.

2.

Materials and methods

2.1.

Preparation of experimental samples

A number of medical-grade stainless steel 316L cylindrical pins (length: 10 mm; diameter: 4 mm) were manufactured to

simulate the femoral stem, with one spherically shaped end highly polished to a surface roughness value of 30 nm (scan area: 0.17  0.13 mm2). These pins were mounted to the holder of a universal materials tester (UMT-III, Centre for Tribology Inc., Campbell, California, USA) as the upper specimen. The polymethylmethacrylate bone cement (Synthetic Material Research Institute, Tianjin, China) was hand-mixed according to the manufacturer’s instructions, and delivered into a custom-made container using a syringe. A metallic cover made of medical-grade stainless steel 316L with one planar surface highly polished to similar surface roughness as that of the pins, was connected to the container and stabilized by screws. The bone cement was fully cured in situ after an hour, and then detached from the metallic cover. Accordingly, smooth bone cement disks (diameter: 30 mm; thickness: 5 mm) were obtained as the lower specimen with a surface roughness value of 0.3 mm (scan area: 0.86  0.64 mm2).

2.2.

Tribological experiment

The bone cement disk was firmly fixed into the circular groove of a custom-made fixture, which was designed to connect with the precision linear stage of the UMT-III tester and hold the lubricant. Three different media were tested, i.e. 100% calf serum (Sijiqing Biological Engineering Materials Co. Ltd., Hangzhou, China), 25% calf serum, and 0.9% saline solution. Calf serums with different concentrations were used to investigate whether this will influence the biotribological behavior at the stem–cement interface, while 0.9% saline solution without any proteins was tested as a comparison with the calf serum. The calf serum lubricants were thawed before use, and no additives were added to minimize bacterial contamination during the test. A temperature control of the lubricants at 3771 1C was achieved through using a heating rod and a thermocouple as the temperature feedback, Fig. 1. The tribological experiment was performed in a reciprocating mode (oscillation amplitude: 0.25 mm) at 1.0 Hz for a duration of 3 h utilizing the UMT-III tester. A normal load of

Fig. 1 – Equipment for the tribological experiment between simulated femoral stem and bone cement with a ball-on-flat configuration and temperature control: (1) heating rod; (2) thermocouple and (3) stainless steel 316L pin and bone cement disk immersed in 100% calf serum lubricant.

journal of the mechanical behavior of biomedical materials 20 (2013) 209 –216

0.98 N was applied, which was equivalent to an apparent maximum contact pressure of 64.9 MPa for the contact between stainless steel 316L and bone cement. This was calculated based on the following equations for the contact pressure of ball-on-flat configuration, according to the Hertz theory (Wen and Huang, 2011). The contact 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 (Norman et al., 2001).    2=3 1 3w 1=3 E0 ð1Þ P¼ p 2 R   1 1 1m1 2 1m2 2 þ 0 ¼ E 2 E1 E2

ð2Þ

where P is the apparent maximum contact pressure, w is normal load (0.98 N), R is the radius of the ball (2 mm), E1 and m1 are the elastic modulus and Poisson’s ratio of stainless steel 316L (210 GPa and 0.3, respectively), and E2 and m2 are the elastic modulus and Poisson’s ratio of bone cement (2.3 GPa and 0.23, respectively). A total of four tests were performed for each lubricant to enable statistical validity. Before each test, the specimens including the metallic pin and the bone cement disk were cleaned in an ultrasonic bath using consecutively petroleum ether, acetone and deionized water for 15 min each followed by drying with an N2 gas jet. After each test, the metallic pin and the bone cement disk were removed from the testing apparatus, dried with the N2 gas jet.

2.3.

Evaluation of the femoral stem surface

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followed by 25% calf serum (0.347) and 0.9% saline solution (0.404). The change of friction coefficient as a function of test duration for the three different lubricants was shown in Fig. 3. It was indicated that although there was certain fluctuation, the friction coefficient basically did not change much after the initial start-up period.

3.2.

Surface characterization of the metallic stem

The optical microscopic graphs and optical interferometeric graphs of the stainless steel 316L pins lubricated by three different media were shown in Fig. 4. With regard to the samples lubricated by 100% calf serum, slight scratches were observed within the contact area, which was further surrounded by several concentric colorful rings. The formation of these rings may be related with protein adsorption onto the surface, indicating a difference in surface height between the rings and the contact area. The 2D line profile across the surface showed that the height difference was about 2.0 mm, which may be the thickness of the adsorbed protein film around the contact area. With regard to the samples lubricated by 25% calf serum, similarly, relatively more severe scratches were present within the contact area. However, the difference in surface height between the colorful rings and the contact area was not prominent, suggesting a much thinner protein film adsorbed onto the surface. With regard to the samples lubricated by 0.9% saline solution, a wear scar was observed within the contact area. It was indicated from the 2D line profile across the surface that the depth of the wear scar was about 2.0 mm. The SEM micrographs of the stainless steel 316L pins were shown in Fig. 5. As for the samples lubricated by 100% calf serum, a continuous solid film was observed covering the

Following completion of all the tribological tests, the contact area of the stainless steel 316L pins were initially evaluated using an optical microscope to detect any evidence of protein adsorption on the surface. Then, the surface topography of the contact area on the metallic pins was measured employing a microXAM-3D optical interferometer (KLA-Tencor Corp, Milpitas, California, USA), through which the thickness of the adsorbed proteins could be obtained. Additionally, the metallic pins were investigated using a Quanta 200 ESEM FEG scanning electron microscope (SEM, FEI, Eindhoven, Netherlands) associated with an energy dispersive X-ray analysis (EDX) to enable subtle observation and comparison of elemental composition for different positions of the stem surface. Furthermore, the metallic pins were characterized through an optical scattering technique employing Raman spectroscopy (LabRAM HR800, Horiba JY, Ville d’Asq, France) to analyze the chemical functional groups of the adsorbed proteins.

3.

Results

3.1.

Tribological experiment

The mean friction coefficient values of the tribological experiment using the three different lubricants were shown in Fig. 2. The minimum friction coefficient (0.254) was obtained when 100% calf serum was used as the lubricant,

Fig. 2 – Average values of friction coefficient for the tribological experiment between polished stainless steel 316L stem and bone cement, lubricated by three different media: 100% calf serum (0.254), 25% calf serum (0.347), and 0.9% saline solution (0.404).

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Fig. 3 – Change of friction coefficient as a function of test duration for the tribological experiment between polished stainless steel 316L stem and bone cement, lubricated by the three different media.

surface, which may be caused by adsorption of proteins. It was interesting to note that the contact area and the surrounding regions seemed to be protected through a film with alveolate feature, where the underneath metallic surface appeared relatively smooth. Additionally, the elemental composition obtained from the EDX analysis showed a significantly increased content of C from the solid film surface associated with a great decrease of the substrate compositions (e.g. Fe, Cr, and Ni). Some new elements such as O, N, K, Na, and Cl were also detected, which were derived from the calf serum lubricant. As for the samples lubricated by 25% calf serum, similarly, a continuous solid film was present on the stem surface without covering the contact area. With regard to the samples lubricated by 0.9% saline solution, the contact area was damaged with the formation of a wear scar and the deposition of NaCl crystal on the surface. In order to validate the chemical functional groups of the solid film adsorbed on the stainless steel 316L pins, the solid film was evaluated utilizing Raman spectroscopic analysis, through which the intense Raman-active vibrations of a molecule could be indicated from the Raman shift shown on the spectrum. It was indicated from Fig. 6 that the spectrum was primarily characterized by five peaks at 1310 cm1, 1445 cm1, 1660 cm1, 2930 cm1 and 3330 cm1, which corresponded to the vibration modes of amide III bands, C–H bending, amide I bands, C–H stretching and amide A, respectively. This could be regarded as evidence to confirm that the solid film was indeed adsorbed proteins from the calf serum lubricant as amide bond (–CONH–) was the basic unit of protein. Furthermore, a line scan of Raman signal at the highest peak, i.e. 2930 cm1, was performed across the surface as shown in Fig. 4(a), with different intensities obtained at twelve positions. It was shown from Fig. 7 that the intensity of Raman signal initially decreased (from point 1 to point 6) until a minimum value (almost zero) was reached at the contact area, and then increased back again (from point 7 to point 12) when the scan position was away from the contact area. Additionally, the thickness of the continuous solid protein film covering the stem surface was characterized through a vertical scanning methodology based on Raman spectroscopy, i.e. the focal spot of the laser beam moved automatically from a certain depth below a surface to a certain height above the surface with pre-determined scan interval. It was shown from Fig. 8 that the intensity of the

C–H stretching vibration at 2930 cm1 initially started to increase from 6 mm below the protein film until a maximum was reached, and then the intensity decreased back to nearly zero again at 8 mm above the protein film. This indicated that the thickness of the solid protein film was about 14 mm.

4.

Discussion

It has been accepted that debonding of the stem–cement interface is evitable (Zhang et al., 2011a,b), and pseudo-synovial fluid could be pumped into this interface under physiological loading. Thus, various proteins contained in the fluid would adsorb onto the femoral stem surface, affecting the fretting mechanism at the stem–cement interface. Calf serum has been by far the most widely used lubricant for in vitro biotribological studies (Brown and Clarke, 2006; Zhang et al., in press), with albumin as the major protein in addition to other components such as lipoprotein, cations (Naþ, Kþ) and anions (Cl–). It was indicated from previous in-vitro wear simulations that protein adsorption onto the acetabular components, i.e. CoCrMo alloy and UHMWPE, could protect the contacting surfaces from rubbing each other (Scholes and Unsworth, 2006). It was demonstrated from the present study that, when 100% calf serum was used as the lubricant, the friction coefficient was the lowest (0.254) and only slight scratches were detected within the contact area of the polished stainless steel 316L stem, which may be related with protein adsorption onto the stem surface. Regarding with the tests lubricated by saline solution, an increase in friction coefficient was obtained (0.404) and a wear scar was observed within the contact area of the stem surface. Although there is no general relationship between friction and wear, it is quite useful to perform frictional studies in order to predict the wear behavior of materials. Therefore, it is considered clinically beneficial for the femoral stem surface to promote protein adsorption in order to postpone generation of wear debris at the stem–cement interface and further aseptic loosening of the total joint system. It was shown from the SEM micrographs of the stainless steel 316L pins lubricated by calf serum that the surface was covered by a continuous solid film, but the contact area and the surrounding regions were characterized by formation of a film with alveolate feature, which may be caused by cavitation during separation of the contact surfaces or due to evaporation of the

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213

Fig. 4 – Surface characterization of the polished stainless steel 316L pins lubricated by different media: (a) 100% calf serum, optical microscopic graph showing slight scratches within the contact area and optical interferometeric measurement (an area surrounded by the white dotted rectangle) showing the 2D surface profile along the line across the scratches; (b) 25% calf serum, optical microscopic graph showing relatively more severe scratches within the contact area and optical interferometeric measurement (an area surrounded by the white dotted rectangle) showing the 2D surface profile along the line across the scratches and (c) 0.9% saline solution, optical microscopic graph showing wear scar within the contact area and optical interferometeric measurement showing the 2D surface profile along the line across the wear scar.

water molecules in the lubricant as a result of the accumulated heat in the local contact area during the test. The associated EDX analysis and Raman spectroscopy of the solid film confirmed that the solid film was indeed adsorbed proteins on the metallic surface, with the detection of basic unit of protein, namely amide bond (–CONH–). Previously, X-ray photoelectron spectroscopy (XPS), radiolabeling (I125), and fluorescent labelling, were used to evaluate protein adsorption onto artificial materials (Gispert et al., 2006; Crockett et al., 2009). The introduction of Raman spectroscopy in the present study is considered to be a

more feasible method, especially when characterizing physically adsorbed proteins on the metallic surface. It was further indicated from the line scan of Raman signal across the contact area that there was almost no protein film covering the contact area, which may subsequently be removed due to the oscillatory fretting mechanism at the stem–cement interface. The protein film around the contact area of the stem surface (as shown in Fig. 4(a)) was relatively thinner compared with the continuous solid protein film covering the stem surface (as shown in Fig. 8), which may be caused by the removal of the

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Fig. 5 – SEM micrographs of the polished stainless steel 316L pins lubricated by the three different media: (a) 100% calf serum; (b) 100% calf serum, EDX analysis showing comparison of elemental composition between area A and area B; (c) 25% calf serum and (d) 0.9% saline solution.

Fig. 6 – Characterization of the solid film adsorbed on the polished stainless steel 316L pins using Raman spectroscopy. The detection of amide III bands and amide I bands peaks could confirm the presence of protein film on the surface.

protein film under fretting mechanism at the stem–cement interface. It was considered that the protein film formed on the metallic pins correspond to a multi-layer physical adsorption. From a physiochemical point of view, protein adsorbs onto biomaterials through various intermolecular interactions, e.g. hydrophobic interaction and electrostatic interaction (Nakanishi et al., 2001). Albumin, as the most abundant protein

in calf serum, is a globular protein characterized by a hydrophobic core and hydrophilic surface. It tends to adsorb on all surfaces irrespective of electrostatic interaction, owing to a gain in conformational entropy (Roba et al., 2009). In addition, both the stainless steel 316L and bovine serum albumin are negatively charged in the calf serum because the pH value of the solution is larger than the point of zero charge (PZC) of stainless

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Fig. 7 – Line scan of Raman signal at 2930 cm1 across the contact area of the polished stainless steel 316L pins lubricated by 100% calf serum, the intensity initially decreases when the position is towards the contact area (from point 1 to point 6), and then increases back again when the position is away from the contact area (from point 7 to point 12).

Fig. 8 – Characterization of the thickness of the continuous protein film adsorbed onto the surface of stainless steel 316L pins using a vertical scanning methodology based on Raman spectroscopy.

steel 316L and the isoelectric point (PI) of albumin (pHcalf serum ¼ 7.88, PZCstainless steel 316Lo7, PIbovine serum albumino6) (Hossain and Gao, 2008). The presence of cation ions in the calf serum would act as a bridge between the negatively charged groups through electrostatic interaction as it was previously reported that the amount of adsorbed albumin onto stainless steel surface increased by one order of magnitude when Ca2þ cation was present in the aqueous solution (Pradier et al., 2002). Furthermore, it is accepted that protein adsorption is strongly dependent on pH value, ionic strength, and protein concentration of the solution (Malmsten, 1995). Consequently, the high protein concentration (38.5 mg/ml) of calf serum used in the present study also promoted formation of a multi-layer protein adsorption onto the stainless steel 316L surface.

5.

Conclusions

In the present study, the protein adsorption mechanism and biotribological properties at the stem–cement interface under fretting were investigated through a series of frictional tests. The following conclusions can be drawn from this work: 1. The analyses of elemental composition and chemical functional groups of the solid film on polished stainless steel 316L stems obtained using EDX and Raman spectrum confirmed that the solid film was attributed to protein adsorption onto the stem surface. 2. Proteins from the calf serum lubricant protected the contact area of the polished stainless steel 316L stems from severe fretting damage through adsorption onto the stem surface.

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Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant nos. 51005130, 51027007), the Scientific Research Starting Foundation for the Returned Overseas Chinese Scholars, Ministry of Education of China (Grant no. 20121028121), and the Research Fund of the State Key Laboratory of Tribology, Tsinghua University (Grant no. SKLT12B06). The authors would like to thank Mr. Guoyue Du, Jinghang Biomedicine Engineering Division, Beijing Institute of Aeronautical Material, for his great help with the manufacturing of the specimens.

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