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A novel technique for evaluation of articular cartilage lubrication based on the surface plasmon resonance
Life Cycle Tribology D. Dowson et al. (Editors) © 2005 Elsevier B.V. All rights reserved
389
A novel technique for evaluation of articular cartilage lubrication based on the surface plasmon resonance M. H. Naka a , Y. Arima a , H. Iwata a , M. Hasuob, Y. Fuwac, Y. Moritad, K. Ikeuchi a a
Institute Frontier for Medical Sciences, Kyoto University 53 Kawahara-cho Shogoin Sakyo-ku Kyoto, 606-8507, Japan
Department of Engineering Physics and Mechanics, Kyoto University Yoshida-Honmachi Sakyo-ku Kyoto, 606-8501, Japan c
Toyota Motor Corporation, Japan 1 Toyota-cho Toyota Aichi, 471-8572, Japan
^ Department of Electronics, Information and Communication, Osaka Institute of Technology 5-16-1 Omiya Asahi-ku Osaka, 535-8585, Japan Evaluation of lubrication in articular cartilage has been developed with various techniques in order to obtain reasonable explanations about this superb mechanism. However, a definitive theory about this mechanism of lubrication is not clear until present time. One of the most relevant restrictions for the comprehension of this mechanism is the difficulty to detect and to evaluate the surface layer of articular cartilage. Acquisition of images from the conventional techniques are not sufficient for the analysis of the surface layer conditions because the most of methods require the dehydration of specimens which change their physiological state. In addition, methods that allow the evaluation of articular cartilage in the aqueous environment do not provide sufficient and clear data for a definitive evaluation. In this work, we present the application of Surface Plasmon Resonance (SPR) principle in the evaluation of articular cartilage surface. In our apparatus, it is possible to acquire images of articular cartilage surface during the lubrication experiments and in the aqueous environment. Variations in the reflectance were used in order to identify the substances present in the surface layer, since the intensity of light depends on the chemical characteristics of the substances located on the articular cartilage surface. The correlation of friction behavior with the possible substances present in the surface layer of articular cartilage can contribute to clarify the mechanism of lubrication of articular cartilage. 1. INTRODUCTION Lubrication in synovial joints has been investigated by several researches with different approaches. Roles of substances present in the synovial joints (proteins and lipids) [l,2], synovial fluid (natural lubricant of synovial joints) [3], mechanical functions [4] and superficial conditions [5] of articular
cartilage are some of factors investigated. Basically, the most important elements for the lubrication in synovial joints are articular cartilage (bearing surface of joints) and synovial fluid (natural lubricant of joints). In this work, the role of articular cartilage in the lubrication is emphasized, with more attention to the role of the articular surface in the lubrication performance.
390
The evaluation of superficial conditions of articular cartilage during lubrication requires the use of special techniques due to inherent difficulties found in the living tissues. One of the major obstacles for this evaluation is the high content of water. Presence of water increases the difficulty in the focus regulation in the capturing of images from articular cartilage. Methods that involve dehydration process can change the natural aspect of articular surface through the degradation owing to the exudation of water. Other limitations, such as the translucent appearance of articular cartilage, also increase the difficulty for evaluation of articular surface. Furthermore, living tissues suffers fast degradation after they are removed from their natural and physiological environment. In this situation, the time between the obtainment of specimens and the experimental measurement needs to be optimized. A method that involves the simultaneous examination of the superficial condition of articular cartilage and the lubrication performance, may improve the evaluation of tribological properties of articular cartilage. For this reason, one apparatus that allow the capturing of images from the articular surface during friction tests is presented in this work. In order to estimate the distribution of substances in the articular surface, the apparatus was developed with the principle of Surface Plasmon Resonance (SPR). This principle, described in the following section, provides important information based on the optical properties of substances found in the articular surface. 1.1. Background When a light reaches an interface between two mediums, one part of light will be reflected and the other refracted. If the angle of incidence is higher than a specific angle known as the critical angle, the light is fully reflected. That is, total internal reflection (TIR) occurs. The critical angle depends on the refractive indices of both the mediums and it is calculated by the following equation:
d
critical =
s m
-i
(1)
where ri\ and n2 are the refractive indices of mediums with small and large values, respectively, as shown in Fig. 1. During the TIR, one electromagnetic field is generated in the interface and propagates into the medium with lower refractive index. This field is known as evanescent waves field [6]. As the distance from interface increases, the magnitude of the evanescent waves field decreases exponentially as illustrated in Fig. 1.
Evanescent Field Intensity
Figure 1. Decay of evanescent field in relation to the distance from the interface, m and m are the refractive index of both medias of interface, 0mc is the angle of incidence. The energy balance transported by the evanescent waves is zero [7], however, if some perturbation occurs in the region where these waves are propagating, the intensity of reflected light can suffer alterations. This principle is widely used in the sensor applications and the procedures for recognition of biological environments [8" 10]. The penetration depth limits the evanescent waves, since the intensity of evanescent field decreases exponentially as the distance to the interface increases. The following equation represents the penetration depth of evanescent waves:
391
(2) 2
6mc
-n\
interface, and the contrast of images obtained from the reflected light may be useful for the evaluation of superficial conditions of specimens placed on the metal layer.
where X. is the wavelength of the light and Qim. is the angle of incidence. In according to the Eq. (2), it is possible to perceive that the effect of evanescent waves only occurs within the nanometers region from the interface. In other words, alterations in the reflected light due to the perturbation of evanescent field correspond to alterations that occur in the regions nearly from the interface. When one nano-layer of metal is placed on the interface where the light is reflected, a surface plasma wave (SPW) can be generated. A SPW is an electromagnetic wave, which propagates along the boundary between a dielectric and a metal surfaces and behaves like quasi-free electron plasma [11,12]. At a specific angle of incidence, the SPW resonates with the energy of the incident light. In this case, a decrease in the intensity of the reflected light is observed. This phenomenon is known as the Surface Plasmon Resonance (SPR) and this angle of incident is called the SPR angle. The curve of the SPR reflectance is a function of the light wavelength, the angle of incident, the refractive indices and the thickness of the metal layer. In Fig. 2, the theoretical curves of the SPR reflectance for 4 kinds of mediums, found in the articular cartilage surface, are presented. The metal for the simulation is gold with 49nm of thickness. The refractive indices of materials are presented in Table 1. The medium, in which the light is derived and reflected, is assumed as glass (BK/7). From these curves, it is possible to observe that the reflectance intensity is useful as an important parameter for the evaluation of superficial conditions because it varies with the refractive index of the medium where the evanescent waves propagate. Alterations in the refractive index of medium can be correlated to substances presents near the
• Water ° Saline » CS 2.0mg/ml * Collagen 2.0mg/ml
70
75
80
85
Angle of Incidence
Figure 2. Theoretical reflectance as a function of the angle of incidence for 4 kinds of substances present on the gold layer: water, saline, chondroitin sulphate (CS) AC — 2.0mg/ml (dissolved in PBS), collagen type II lmg/ml (dissolved in distilled water with 0.25% of acetic acid 0.5M). Table 1. Refractive indices (n) at 24°C Substance Gold (*) BK-7 Water Saline CS (Chondroitin Sulphate AC) Collagen Type II
n 0.153+/3.53 1.515 1.331 1.341 1.339 1.356
(*) for calculus the reflectance with the SPW effect, it is necessary to consider the complex refractive index of the metal layer. 2. MATERIALS AND METHODS The experimental methods consisted basically by the friction tests with simultaneous capturing of images from the articular surface with the SPR apparatus. Alterations in the intensity of reflectance observed in the images are correlated to the friction behavior in the transition tests.
392 392
2.1. Materials Specimens of articular cartilage were obtained from the lateral femoral condyles of matured and healthy pigs (about six months old and 100kg). The diameter of the specimens was 5mm with mean thickness of 1.4mm. In order to examine the role of articular surface in the lubrication, the specimens were divided into three groups (three specimens were used for each group): normal, wiped and enzymatic degraded. In the normal condition, the surface was not touched in order to keep its natural characteristics. In the wiped condition, the articular surface was wiped with tissue paper imbibed with saline. The aim of this procedure is to minimize the action of substances presents or adsorbed in the articular surface during lubrication tests, such as proteins, lipids and others. In the enzymatic degradation, the specimen was digested in solutions of collagenase type II with concentration of lmg/ml. The digestion time was 1 hour at 37°C.
Gold Layer Class(BK7)Layer Mating Oil
2.2. Experimental Apparatus and Methods A schematic illustration of the SPR apparatus for the image capturing is presented in Fig. 3. The light source was HeNe laser of 632.8nm wavelength and 5mW power (Melles Griot). A cube polarizer polarizes the laser light in the p-polarize plane, where the surface plasmon can be excited. The material of the prism was BK/7 glass with refractive index of 1.515. The beamspliter divides the laser beam (before and after the prism) in two parts : one travels to a photodiode detector and the other travels to a CCD camera. A previous calibration was done before the experiments in order to estimate the real values of the intensity of incident and reflected. The photodiode detector is used in order to detect undesired variations derived of the laser light. The variations detected in the CCD camera and photodiode reference 2
- Reservoir Articular ^^-^ Cartilage
-
Cube Polarizer S\jr^
Lens —
Laser
^ - v ^ ^ v . ^ Beamspliter
Prism \ \Beamspliter
^V*"'^^ / ~f ^ ~ " \ » . M
>HML.\
CCD Camera 1
7
Photodiode
^/\K.
(I
ft Photod iode Refere ice 1 | )
V 1 •
Reference2 / ^
CCD Camera 2
f
/
Acquisition of Data
Figure 3. SPR apparatus for the capturing of images. In the top-right, description of layers on the prism surface is presented. cannot correspond necessarily to the changes cartilage and the prism surface. These in the interface between the articular variations may be associated with the
393
fluctuations of the electricity and the temperature, noise and other factors that affect the performance of the light source. For incite the surface plasmon resonance, one substrate was set on the prism surface. This substrate is a glass plate (BK7) with 25x25mm of dimension and lmm of thickness. In the one side of the glass plate, one layer of chromium was deposited with lnm of thickness in order to assure a good coating of gold (49nm of thickness) on glass. The thickness of coating layers was determined by the limitation of the penetration depth of the evanescent waves, which is previously mentioned (section l.l). The substrate was rinsed three times with propanol and distilled water in order to eliminate the probable contaminations. Mating oil was used for an adequate coupling between the prism and the substrate, which avoid effects of micro-separations and discontinuity at the interface owing to the roughness of both the surfaces. The mating oil was provided from American Optical Co., with refractive index of 1.5150, which was the same as that of the prism and the substrate. The probe used in the friction experiment is illustrated in Fig. 4. The deflection of upper and lower leave springs is detected with laser light displacement sensor and is converted to the friction force and the normal load, respectively. The images were analyzed through the comparison between the images captured with the CCD cameras 1 and 2. Each image was acquired in the BMP-8 bits pattern, which has a resolution of the light intensity in an arbitrary range from 0 to 255 (28). The alterations between the images are related to the changes in the surface interface conditions. The experimental parameters used in the SPR apparatus and the friction tests are listed in Table 2. The angle of incidence was chosen based on the method presented by Lyon et al. [13] that optimized the contrast of the reflectance for the substances involved in the experiment (See more details in the Discussion section).
Reaction of Normal Force
MS-2
Figure 4. Probe of the friction test. LDS1 is the laser light displacement sensor used for measurement of normal force and LDS2 was used for the measurement of friction force. MS-1 is the micro-stage used for the setting of normal force applied on the articular cartilage and MS-2 is used for the setting of sliding motion. Table 2. Experimental parameters Diameter of Laser Beam 10mm Angle of Incidence 71.9° Normal Load 0.03N Speed of sliding O.lmm/s Displacement of Sliding 2mm Solution Saline 24° C Temperature 3. RESULTS In Fig. 5, the results of the friction coefficient in relation to the time are presented. The normal condition has the lowest friction coefficient throughout the friction test. At the beginning of the test, the wiped condition has the highest friction coefficient, while the normal and the enzymatic degraded conditions have the same level of friction. After 5 minutes from the start, the friction for the enzymatic degraded condition shows a gradual increase and after 10 minutes, the level of the friction is practically the same as that of the wiped condition.
394
However, this situation is improbable because represents the ideal case, where the light is fully reflected without loses. The normal condition has the highest reflectance, while the enzymatic degraded condition has the lowest one. In Fig. 7 is presented the correlation between the friction coefficient and the reflectance. In Table 3, the correlation coefficients for the normal, wiped and enzymaturdegraded conditions are presented.
Normal • Wiped A Collagenase O 'S 015
4 o
o O
§ o
0.1
Li_
0.05 0 Time (minute)
0.9
Figure 5. Friction coefficients with respect to the time for the normal, wiped and enzymatic degraded (collagenase) specimens.
0.89
A-S A
A
0.88
I
Normal • Wiped A Collagenase O
0.87
qz
The correlation of reflectance with the time of sliding during friction test is presented in Fig. 6 for all superficial conditions.
01
0.86 0.85 0.84
0.9
0.05
0.1
0.15
Friction Coefficient
0.89
Figure 7. Correlation between the reflectance and the friction coefficient for the normal, wiped and enzymatic degraded (collagenase) specimens.
g 0.88 Normal • Wiped A Collagenase O
£ 0.87 <*• 0.86
0.85
o
o
o
o
o
0
0.84 Time (minute)
Figure 6. Reflectance intensities with respect to the time for the normal, wiped and enzymatic degraded (collagenase) specimens. The reflectance intensity is calculated by the following equation^ R=
'reft
(3)
inc
where lrejj is the intensity of the reflected light and Ilm is the intensity of the incident light. According to Eq. (3), the maximum reflectance occurs when the R is equal to one.
Table 3. Correlation coefficient between the reflectance and the friction coefficient Condition R2 Normal 0.875 Wiped 0.033 Enzymatic-degraded 0.579 The normal condition presented the highest correlation coefficient, while the wiped condition has presented an absence of systematic relationship. The degradedenzymatic condition presented a reasonable correlation. In order to verify if the reflectance has a correlation with the increase in contact area of the articular cartilage against the gold layer, graphs that correlate the variation of (a) the reflectance in relation to the area of contact and the (b) friction coefficient in relation to the area of contact were plotted in Fig. 8.
395 0.06 -
After 10 minutes
After test (unloading)
•g 0.055 »= 0.05 v 0 0.045 •
1 ° 04 ' "• 0.035
•
0.03 1000
2000
3000
4000
5000
I
Contact Area (pixels )
0.8982 0.898 0.8978 « 0.8976
|
0.8974
g 0.8972
%
0.897
0.8964 0.8962 2000
3000
4000
5000
Contact Area (pixels)
Figure 8. Correlation between the contact area and (a) the reflectance and (b) the friction coefficient for the normal condition of the articular cartilage. The contact area was calculated from the captured images. The images was filtered with FFT (Fast Fourier Transform) filter and a particle analyzer was used to identify the contact area. The correlation coefficients between the reflectance and the contact area, and between friction coefficient and the contact area were 0.065 and 0.183, respectively. In Fig. 9, the images of the articular surface in the normal condition before and after the friction test are shown. The profiles of both the surfaces are also shown. It is noted that these images do not correspond to the real alterations that occur during the friction test because they were captured directly from the CCD camera without corrections due to the fluctuations of light intensity. For the analysis, the images were calibrated with the variations of the light intensity captured with the detectors, as mentioned previously in the Material and Methods section.
Figure 9. At top, images captured from the normal condition of the articular cartilage during the friction test. At bottom, the reflectance profile obtained from the image analysis. 4. DISCUSSION The increases of the friction coefficient, in the normal and wiped conditions, as the time evolves, shows similar results to our previous researches [14], in spite of the uncommon counter face used (gold). However, the degraded specimens with collagenase presented two distinct behaviors: similar to the normal condition in the short time of the sliding, and similar to the wiped condition in the long time of the sliding. Different levels of the reflectance were observed for the wiped, normal and enzymatic-degraded conditions. These differences may be related to the substances present in the articular surface during the lubrication. In Fig. 10, the reflectance curve of collagen, water and chondroitin sulphate (most probable substances found in the articular surface) were subtracted to the reflectance curve of saline (reflectance based on the graph of Fig. 2). It is possible to observe the intensity of contrast in relation to saline, the solution where the specimens were immersed during the friction test.
396
Angle of Incidence
Figure 10. Contrast of the reflectance of water, chondroitin sulphate AC (CS) and collagen type II in relation to saline. The experiment was realized with the angle of incidence of 71.8°. In this situation, the contrast presented an increase for the collagen, a slightly decrease for the CS and a considerable decrease for water. Since the contrast is strong between water, collagen and CS, this value of the angle of incidence is chosen as mentioned in the previous Materials and Methods section, with the method used by Lyonetal. [13]. 0.02 0.015 A
eta
c
A
*
A • Normal
A
Wiped
A
0.005
t> "K
A A
0.01
O Cotlagenase
0
c -0.005 I & -0.01 <
o
O
0
o
o
o
o
0
o -0.015 C
2
4
6
8
10
12
T me (minute)
Figure 11. Contrast of the reflectance between the images obtained during the test and the images before the contact of specimens with the gold layer. In Fig. 11, the reflectance of all the conditions were subtracted from the reflectance before the start of the friction test and without the contact of specimens, where only saline was in contact with the gold layer. In other words, the reflectance of this graph
shows the images without the saline solution influence. The contrast was only positive for the wiped condition, while negative contrast was observed in the normal and the enzymatic degraded conditions. At first sight, the contrast analysis suggests the presence of collagen in the surface in the wiped condition, the presence of CS and water in the normal condition and the presence of water in the enzymatic degraded condition. However, the range of variation in the contrast of the reflectance was much different for both the graphs (Figs. 10 and 11), which may not allow this affirmation. The calculus of the contrast of the reflectance was obtained with the average of intensity of all pixels of the image (included non contact area), which causes a considerable increase in the standard deviation. On the other hand, this high standard deviation may indicate the presence of the regions where the contrast of the reflectance is considerably high, which allow the accomplishment of the evaluation of the reflectance of specimens in relation to the graph of Fig. 10. Thus, if the accuracy of apparatus is improved, relevant data can be obtained with this system. For example, one analysis based on pixel by pixel could provide important information about the superficial condition of articular cartilage. Moreover, the CS and collagen evaluated in the SPR reflectance curve do not correspond to the real values of the proteoglycans and the collagen contents in the articular cartilage surface, because the concentration can vary in comparison to the values used in the theoretical calculus. From the contact area analysis, the correlation between the reflectance intensity and the contact area was found to be low. On the other hand, the correlation between the contact area and the friction coefficient was higher while it is not sufficient to affirm the existence of a significant correlation. Although the theories of lubrication correlates the friction coefficient with the contact area, the reduced normal load applied (0.03N) on the specimen can be responsible for this low
397
coefficient of the correlation. Thus, the observed variations in the reflectance are almost independent of the increase of the contact area and depend on the superficial conditions. In summary, the results indicated a possibility of high contact of the collagen network with the counter face in the wiped condition, which was suggested from the higher friction than that of the normal condition. In the normal condition, the existence of lubricant layer on the articular cartilage surface was thought to be associated with the low friction. In the enzymaticdegraded condition, it was observed a possible existence of high content of water in the surface, probably due to the collagenase effect, since the degradation of collagen network prejudices the ability of the articular cartilage to resist the swelling tendency [15]. In this situation, the degradation of the collagen network is responsible for the increase in the friction coefficient after the long time of sliding. 5. CONCLUSION The results obtained in this work demonstrated that the application of the principle of SPR in the analysis of lubrication in synovial joints is promissory. Application of contrast techniques based on the refractive index may be a useful alternative for the evaluation of articular surface. In addition, the obtained correlations between the SPR reflectance and the friction coefficient suggest that the superficial condition of articular cartilage play important role during lubrication.
REFERENCES 1. J.E. Pickard, J. Fisher, E. Ingham and J. Egan, Biomaterials, 19 (1998) 1807. 2. B.A. Hill and R.W. Crawford, J. Arthroplasty, 18 (2003) 499. 3. T. Murakami, H. Higaki, Y. Sawae, N. Ohtsuki, S. Moriyama and Y. Nakanishi, Proc Instn Mech Engr Part H, 212 HI (1998) 23. 4. R. Krishnan, M. Kopacz and G.A. Athesian, J. Orthop Res, 22 (2004) 565. 5. K. Ikeuchi, H. Yoshida, Y. Morita, M.H. Naka, In Proc 4 th Internat Biotrib Forum (2003) 72. 6. C. Henkel, H. Wallis, N. Westbrook, C.I. Westbrook, A. Aspect, K. Sengstock, W.Ertmer, Appl Phys B, 69 (1999) 277. 7. F. de Fornel, Evanescent Waves from Newtonian Optics to Atomic Optics, Springer-Verlag, Germany, 2001. 8. T.I. Ksenevich, A.A. Beloglazov, P.I. Nikitin, N.A. Kalabina and S.Y. Zaitsev, Applied Surf. Science, 92 (1996) 426. 9. K.-F. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, P. Leiderer.U. Weiland and M. Bstmeyer, Biophys J, 76 (1999) 509. 10. E. Hutter, J.H. Fendler and D. Roy, J Appl Phys, 90 (2001) 1978. 11. J. Homola, Anal Bioanal Chem, 337 (2003) 528. 12. G.G. Nenninger, P. Tobiska, J. Homola and S.S. Yee, Sens. Actuators, B 74 (2001) 145. 13. L.A. Lyon, W.D. Holliway and M.J. Natan, Rev Sci Instrum, 70 (1999), 2076. 14. M.H. Naka, Y. Morita and K. Ikeuchi, In Proc 4 th Internat Biotrib Forum (2003) 74. 15. T.K. Langsjo, J. Rieppo, A. Pelttari, N. Oksala, V. Kovanen and H.J. Helminen, Cells Tissues Organs, 172 (2002), 265.
389
A novel technique for evaluation of articular cartilage lubrication based on the surface plasmon resonance M. H. Naka a , Y. Arima a , H. Iwata a , M. Hasuob, Y. Fuwac, Y. Moritad, K. Ikeuchi a a
Institute Frontier for Medical Sciences, Kyoto University 53 Kawahara-cho Shogoin Sakyo-ku Kyoto, 606-8507, Japan
Department of Engineering Physics and Mechanics, Kyoto University Yoshida-Honmachi Sakyo-ku Kyoto, 606-8501, Japan c
Toyota Motor Corporation, Japan 1 Toyota-cho Toyota Aichi, 471-8572, Japan
^ Department of Electronics, Information and Communication, Osaka Institute of Technology 5-16-1 Omiya Asahi-ku Osaka, 535-8585, Japan Evaluation of lubrication in articular cartilage has been developed with various techniques in order to obtain reasonable explanations about this superb mechanism. However, a definitive theory about this mechanism of lubrication is not clear until present time. One of the most relevant restrictions for the comprehension of this mechanism is the difficulty to detect and to evaluate the surface layer of articular cartilage. Acquisition of images from the conventional techniques are not sufficient for the analysis of the surface layer conditions because the most of methods require the dehydration of specimens which change their physiological state. In addition, methods that allow the evaluation of articular cartilage in the aqueous environment do not provide sufficient and clear data for a definitive evaluation. In this work, we present the application of Surface Plasmon Resonance (SPR) principle in the evaluation of articular cartilage surface. In our apparatus, it is possible to acquire images of articular cartilage surface during the lubrication experiments and in the aqueous environment. Variations in the reflectance were used in order to identify the substances present in the surface layer, since the intensity of light depends on the chemical characteristics of the substances located on the articular cartilage surface. The correlation of friction behavior with the possible substances present in the surface layer of articular cartilage can contribute to clarify the mechanism of lubrication of articular cartilage. 1. INTRODUCTION Lubrication in synovial joints has been investigated by several researches with different approaches. Roles of substances present in the synovial joints (proteins and lipids) [l,2], synovial fluid (natural lubricant of synovial joints) [3], mechanical functions [4] and superficial conditions [5] of articular
cartilage are some of factors investigated. Basically, the most important elements for the lubrication in synovial joints are articular cartilage (bearing surface of joints) and synovial fluid (natural lubricant of joints). In this work, the role of articular cartilage in the lubrication is emphasized, with more attention to the role of the articular surface in the lubrication performance.
390
The evaluation of superficial conditions of articular cartilage during lubrication requires the use of special techniques due to inherent difficulties found in the living tissues. One of the major obstacles for this evaluation is the high content of water. Presence of water increases the difficulty in the focus regulation in the capturing of images from articular cartilage. Methods that involve dehydration process can change the natural aspect of articular surface through the degradation owing to the exudation of water. Other limitations, such as the translucent appearance of articular cartilage, also increase the difficulty for evaluation of articular surface. Furthermore, living tissues suffers fast degradation after they are removed from their natural and physiological environment. In this situation, the time between the obtainment of specimens and the experimental measurement needs to be optimized. A method that involves the simultaneous examination of the superficial condition of articular cartilage and the lubrication performance, may improve the evaluation of tribological properties of articular cartilage. For this reason, one apparatus that allow the capturing of images from the articular surface during friction tests is presented in this work. In order to estimate the distribution of substances in the articular surface, the apparatus was developed with the principle of Surface Plasmon Resonance (SPR). This principle, described in the following section, provides important information based on the optical properties of substances found in the articular surface. 1.1. Background When a light reaches an interface between two mediums, one part of light will be reflected and the other refracted. If the angle of incidence is higher than a specific angle known as the critical angle, the light is fully reflected. That is, total internal reflection (TIR) occurs. The critical angle depends on the refractive indices of both the mediums and it is calculated by the following equation:
d
critical =
s m
-i
(1)
where ri\ and n2 are the refractive indices of mediums with small and large values, respectively, as shown in Fig. 1. During the TIR, one electromagnetic field is generated in the interface and propagates into the medium with lower refractive index. This field is known as evanescent waves field [6]. As the distance from interface increases, the magnitude of the evanescent waves field decreases exponentially as illustrated in Fig. 1.
Evanescent Field Intensity
Figure 1. Decay of evanescent field in relation to the distance from the interface, m and m are the refractive index of both medias of interface, 0mc is the angle of incidence. The energy balance transported by the evanescent waves is zero [7], however, if some perturbation occurs in the region where these waves are propagating, the intensity of reflected light can suffer alterations. This principle is widely used in the sensor applications and the procedures for recognition of biological environments [8" 10]. The penetration depth limits the evanescent waves, since the intensity of evanescent field decreases exponentially as the distance to the interface increases. The following equation represents the penetration depth of evanescent waves:
391
(2) 2
6mc
-n\
interface, and the contrast of images obtained from the reflected light may be useful for the evaluation of superficial conditions of specimens placed on the metal layer.
where X. is the wavelength of the light and Qim. is the angle of incidence. In according to the Eq. (2), it is possible to perceive that the effect of evanescent waves only occurs within the nanometers region from the interface. In other words, alterations in the reflected light due to the perturbation of evanescent field correspond to alterations that occur in the regions nearly from the interface. When one nano-layer of metal is placed on the interface where the light is reflected, a surface plasma wave (SPW) can be generated. A SPW is an electromagnetic wave, which propagates along the boundary between a dielectric and a metal surfaces and behaves like quasi-free electron plasma [11,12]. At a specific angle of incidence, the SPW resonates with the energy of the incident light. In this case, a decrease in the intensity of the reflected light is observed. This phenomenon is known as the Surface Plasmon Resonance (SPR) and this angle of incident is called the SPR angle. The curve of the SPR reflectance is a function of the light wavelength, the angle of incident, the refractive indices and the thickness of the metal layer. In Fig. 2, the theoretical curves of the SPR reflectance for 4 kinds of mediums, found in the articular cartilage surface, are presented. The metal for the simulation is gold with 49nm of thickness. The refractive indices of materials are presented in Table 1. The medium, in which the light is derived and reflected, is assumed as glass (BK/7). From these curves, it is possible to observe that the reflectance intensity is useful as an important parameter for the evaluation of superficial conditions because it varies with the refractive index of the medium where the evanescent waves propagate. Alterations in the refractive index of medium can be correlated to substances presents near the
• Water ° Saline » CS 2.0mg/ml * Collagen 2.0mg/ml
70
75
80
85
Angle of Incidence
Figure 2. Theoretical reflectance as a function of the angle of incidence for 4 kinds of substances present on the gold layer: water, saline, chondroitin sulphate (CS) AC — 2.0mg/ml (dissolved in PBS), collagen type II lmg/ml (dissolved in distilled water with 0.25% of acetic acid 0.5M). Table 1. Refractive indices (n) at 24°C Substance Gold (*) BK-7 Water Saline CS (Chondroitin Sulphate AC) Collagen Type II
n 0.153+/3.53 1.515 1.331 1.341 1.339 1.356
(*) for calculus the reflectance with the SPW effect, it is necessary to consider the complex refractive index of the metal layer. 2. MATERIALS AND METHODS The experimental methods consisted basically by the friction tests with simultaneous capturing of images from the articular surface with the SPR apparatus. Alterations in the intensity of reflectance observed in the images are correlated to the friction behavior in the transition tests.
392 392
2.1. Materials Specimens of articular cartilage were obtained from the lateral femoral condyles of matured and healthy pigs (about six months old and 100kg). The diameter of the specimens was 5mm with mean thickness of 1.4mm. In order to examine the role of articular surface in the lubrication, the specimens were divided into three groups (three specimens were used for each group): normal, wiped and enzymatic degraded. In the normal condition, the surface was not touched in order to keep its natural characteristics. In the wiped condition, the articular surface was wiped with tissue paper imbibed with saline. The aim of this procedure is to minimize the action of substances presents or adsorbed in the articular surface during lubrication tests, such as proteins, lipids and others. In the enzymatic degradation, the specimen was digested in solutions of collagenase type II with concentration of lmg/ml. The digestion time was 1 hour at 37°C.
Gold Layer Class(BK7)Layer Mating Oil
2.2. Experimental Apparatus and Methods A schematic illustration of the SPR apparatus for the image capturing is presented in Fig. 3. The light source was HeNe laser of 632.8nm wavelength and 5mW power (Melles Griot). A cube polarizer polarizes the laser light in the p-polarize plane, where the surface plasmon can be excited. The material of the prism was BK/7 glass with refractive index of 1.515. The beamspliter divides the laser beam (before and after the prism) in two parts : one travels to a photodiode detector and the other travels to a CCD camera. A previous calibration was done before the experiments in order to estimate the real values of the intensity of incident and reflected. The photodiode detector is used in order to detect undesired variations derived of the laser light. The variations detected in the CCD camera and photodiode reference 2
- Reservoir Articular ^^-^ Cartilage
-
Cube Polarizer S\jr^
Lens —
Laser
^ - v ^ ^ v . ^ Beamspliter
Prism \ \Beamspliter
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>HML.\
CCD Camera 1
7
Photodiode
^/\K.
(I
ft Photod iode Refere ice 1 | )
V 1 •
Reference2 / ^
CCD Camera 2
f
/
Acquisition of Data
Figure 3. SPR apparatus for the capturing of images. In the top-right, description of layers on the prism surface is presented. cannot correspond necessarily to the changes cartilage and the prism surface. These in the interface between the articular variations may be associated with the
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fluctuations of the electricity and the temperature, noise and other factors that affect the performance of the light source. For incite the surface plasmon resonance, one substrate was set on the prism surface. This substrate is a glass plate (BK7) with 25x25mm of dimension and lmm of thickness. In the one side of the glass plate, one layer of chromium was deposited with lnm of thickness in order to assure a good coating of gold (49nm of thickness) on glass. The thickness of coating layers was determined by the limitation of the penetration depth of the evanescent waves, which is previously mentioned (section l.l). The substrate was rinsed three times with propanol and distilled water in order to eliminate the probable contaminations. Mating oil was used for an adequate coupling between the prism and the substrate, which avoid effects of micro-separations and discontinuity at the interface owing to the roughness of both the surfaces. The mating oil was provided from American Optical Co., with refractive index of 1.5150, which was the same as that of the prism and the substrate. The probe used in the friction experiment is illustrated in Fig. 4. The deflection of upper and lower leave springs is detected with laser light displacement sensor and is converted to the friction force and the normal load, respectively. The images were analyzed through the comparison between the images captured with the CCD cameras 1 and 2. Each image was acquired in the BMP-8 bits pattern, which has a resolution of the light intensity in an arbitrary range from 0 to 255 (28). The alterations between the images are related to the changes in the surface interface conditions. The experimental parameters used in the SPR apparatus and the friction tests are listed in Table 2. The angle of incidence was chosen based on the method presented by Lyon et al. [13] that optimized the contrast of the reflectance for the substances involved in the experiment (See more details in the Discussion section).
Reaction of Normal Force
MS-2
Figure 4. Probe of the friction test. LDS1 is the laser light displacement sensor used for measurement of normal force and LDS2 was used for the measurement of friction force. MS-1 is the micro-stage used for the setting of normal force applied on the articular cartilage and MS-2 is used for the setting of sliding motion. Table 2. Experimental parameters Diameter of Laser Beam 10mm Angle of Incidence 71.9° Normal Load 0.03N Speed of sliding O.lmm/s Displacement of Sliding 2mm Solution Saline 24° C Temperature 3. RESULTS In Fig. 5, the results of the friction coefficient in relation to the time are presented. The normal condition has the lowest friction coefficient throughout the friction test. At the beginning of the test, the wiped condition has the highest friction coefficient, while the normal and the enzymatic degraded conditions have the same level of friction. After 5 minutes from the start, the friction for the enzymatic degraded condition shows a gradual increase and after 10 minutes, the level of the friction is practically the same as that of the wiped condition.
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However, this situation is improbable because represents the ideal case, where the light is fully reflected without loses. The normal condition has the highest reflectance, while the enzymatic degraded condition has the lowest one. In Fig. 7 is presented the correlation between the friction coefficient and the reflectance. In Table 3, the correlation coefficients for the normal, wiped and enzymaturdegraded conditions are presented.
Normal • Wiped A Collagenase O 'S 015
4 o
o O
§ o
0.1
Li_
0.05 0 Time (minute)
0.9
Figure 5. Friction coefficients with respect to the time for the normal, wiped and enzymatic degraded (collagenase) specimens.
0.89
A-S A
A
0.88
I
Normal • Wiped A Collagenase O
0.87
qz
The correlation of reflectance with the time of sliding during friction test is presented in Fig. 6 for all superficial conditions.
01
0.86 0.85 0.84
0.9
0.05
0.1
0.15
Friction Coefficient
0.89
Figure 7. Correlation between the reflectance and the friction coefficient for the normal, wiped and enzymatic degraded (collagenase) specimens.
g 0.88 Normal • Wiped A Collagenase O
£ 0.87 <*• 0.86
0.85
o
o
o
o
o
0
0.84 Time (minute)
Figure 6. Reflectance intensities with respect to the time for the normal, wiped and enzymatic degraded (collagenase) specimens. The reflectance intensity is calculated by the following equation^ R=
'reft
(3)
inc
where lrejj is the intensity of the reflected light and Ilm is the intensity of the incident light. According to Eq. (3), the maximum reflectance occurs when the R is equal to one.
Table 3. Correlation coefficient between the reflectance and the friction coefficient Condition R2 Normal 0.875 Wiped 0.033 Enzymatic-degraded 0.579 The normal condition presented the highest correlation coefficient, while the wiped condition has presented an absence of systematic relationship. The degradedenzymatic condition presented a reasonable correlation. In order to verify if the reflectance has a correlation with the increase in contact area of the articular cartilage against the gold layer, graphs that correlate the variation of (a) the reflectance in relation to the area of contact and the (b) friction coefficient in relation to the area of contact were plotted in Fig. 8.
395 0.06 -
After 10 minutes
After test (unloading)
•g 0.055 »= 0.05 v 0 0.045 •
1 ° 04 ' "• 0.035
•
0.03 1000
2000
3000
4000
5000
I
Contact Area (pixels )
0.8982 0.898 0.8978 « 0.8976
|
0.8974
g 0.8972
%
0.897
0.8964 0.8962 2000
3000
4000
5000
Contact Area (pixels)
Figure 8. Correlation between the contact area and (a) the reflectance and (b) the friction coefficient for the normal condition of the articular cartilage. The contact area was calculated from the captured images. The images was filtered with FFT (Fast Fourier Transform) filter and a particle analyzer was used to identify the contact area. The correlation coefficients between the reflectance and the contact area, and between friction coefficient and the contact area were 0.065 and 0.183, respectively. In Fig. 9, the images of the articular surface in the normal condition before and after the friction test are shown. The profiles of both the surfaces are also shown. It is noted that these images do not correspond to the real alterations that occur during the friction test because they were captured directly from the CCD camera without corrections due to the fluctuations of light intensity. For the analysis, the images were calibrated with the variations of the light intensity captured with the detectors, as mentioned previously in the Material and Methods section.
Figure 9. At top, images captured from the normal condition of the articular cartilage during the friction test. At bottom, the reflectance profile obtained from the image analysis. 4. DISCUSSION The increases of the friction coefficient, in the normal and wiped conditions, as the time evolves, shows similar results to our previous researches [14], in spite of the uncommon counter face used (gold). However, the degraded specimens with collagenase presented two distinct behaviors: similar to the normal condition in the short time of the sliding, and similar to the wiped condition in the long time of the sliding. Different levels of the reflectance were observed for the wiped, normal and enzymatic-degraded conditions. These differences may be related to the substances present in the articular surface during the lubrication. In Fig. 10, the reflectance curve of collagen, water and chondroitin sulphate (most probable substances found in the articular surface) were subtracted to the reflectance curve of saline (reflectance based on the graph of Fig. 2). It is possible to observe the intensity of contrast in relation to saline, the solution where the specimens were immersed during the friction test.
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Angle of Incidence
Figure 10. Contrast of the reflectance of water, chondroitin sulphate AC (CS) and collagen type II in relation to saline. The experiment was realized with the angle of incidence of 71.8°. In this situation, the contrast presented an increase for the collagen, a slightly decrease for the CS and a considerable decrease for water. Since the contrast is strong between water, collagen and CS, this value of the angle of incidence is chosen as mentioned in the previous Materials and Methods section, with the method used by Lyonetal. [13]. 0.02 0.015 A
eta
c
A
*
A • Normal
A
Wiped
A
0.005
t> "K
A A
0.01
O Cotlagenase
0
c -0.005 I & -0.01 <
o
O
0
o
o
o
o
0
o -0.015 C
2
4
6
8
10
12
T me (minute)
Figure 11. Contrast of the reflectance between the images obtained during the test and the images before the contact of specimens with the gold layer. In Fig. 11, the reflectance of all the conditions were subtracted from the reflectance before the start of the friction test and without the contact of specimens, where only saline was in contact with the gold layer. In other words, the reflectance of this graph
shows the images without the saline solution influence. The contrast was only positive for the wiped condition, while negative contrast was observed in the normal and the enzymatic degraded conditions. At first sight, the contrast analysis suggests the presence of collagen in the surface in the wiped condition, the presence of CS and water in the normal condition and the presence of water in the enzymatic degraded condition. However, the range of variation in the contrast of the reflectance was much different for both the graphs (Figs. 10 and 11), which may not allow this affirmation. The calculus of the contrast of the reflectance was obtained with the average of intensity of all pixels of the image (included non contact area), which causes a considerable increase in the standard deviation. On the other hand, this high standard deviation may indicate the presence of the regions where the contrast of the reflectance is considerably high, which allow the accomplishment of the evaluation of the reflectance of specimens in relation to the graph of Fig. 10. Thus, if the accuracy of apparatus is improved, relevant data can be obtained with this system. For example, one analysis based on pixel by pixel could provide important information about the superficial condition of articular cartilage. Moreover, the CS and collagen evaluated in the SPR reflectance curve do not correspond to the real values of the proteoglycans and the collagen contents in the articular cartilage surface, because the concentration can vary in comparison to the values used in the theoretical calculus. From the contact area analysis, the correlation between the reflectance intensity and the contact area was found to be low. On the other hand, the correlation between the contact area and the friction coefficient was higher while it is not sufficient to affirm the existence of a significant correlation. Although the theories of lubrication correlates the friction coefficient with the contact area, the reduced normal load applied (0.03N) on the specimen can be responsible for this low
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coefficient of the correlation. Thus, the observed variations in the reflectance are almost independent of the increase of the contact area and depend on the superficial conditions. In summary, the results indicated a possibility of high contact of the collagen network with the counter face in the wiped condition, which was suggested from the higher friction than that of the normal condition. In the normal condition, the existence of lubricant layer on the articular cartilage surface was thought to be associated with the low friction. In the enzymaticdegraded condition, it was observed a possible existence of high content of water in the surface, probably due to the collagenase effect, since the degradation of collagen network prejudices the ability of the articular cartilage to resist the swelling tendency [15]. In this situation, the degradation of the collagen network is responsible for the increase in the friction coefficient after the long time of sliding. 5. CONCLUSION The results obtained in this work demonstrated that the application of the principle of SPR in the analysis of lubrication in synovial joints is promissory. Application of contrast techniques based on the refractive index may be a useful alternative for the evaluation of articular surface. In addition, the obtained correlations between the SPR reflectance and the friction coefficient suggest that the superficial condition of articular cartilage play important role during lubrication.
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