New Approaches in Biomedical Spectroscopy - American Chemical

Chapter 23

Downloaded by COLUMBIA UNIV on August 6, 2012 | http://pubs.acs.org Publication Date: August 2, 2007 | doi: 10.1021/bk-2007-0963.ch023

Prediction of Bovine Cartilage Proteoglycan Content Using Energy Dispersive X-ray Analysis or Optical Absorbance and a Multivariate Techniques-Fourier Transform Infrared Microspectroscopy Model 1

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J. C. Bowden , L . Rintoul , T. Bostrom , J. M. Pope , and E. Wentrup-Byrne 1,2,*

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Schools of Chemical and Physical Sciences and Tissue Repair and Regeneration, Institute of Health and Biomedical Innovation, Queensland University of Technology, GPO Box 2434 Brisbane, Queensland 4001, Australia

Two analytical techniques: optical absorbance of Safranin-O stained cartilage sections and energy dispersive X-ray analysis have been used to construct partial least squares models from Fourier transform infra red spectral data. These models can be used to semi-quantitatively predict proteoglycan content in cartilage sections. Valuable information about the spatial distribution of the constituents in native, degraded or even engineered cartilage, can be obtained when these are used in conjunction with infra red imaging techniques.

© 2007 American Chemical Society

In New Approaches in Biomedical Spectroscopy; Kneipp, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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As part of our larger study on the changes in articular cartilage associated with the development of osteoarthritis (OA), a suite of analytical and spectroscopic techniques are proving useful. Our long-term goal is to develop a spectroscopic-based diagnostic technique for the monitoring and analysis of the structural and biochemical changes occurring during the course of O A disease. A better understanding of the inter-relationships between different factors affecting cartilage function is essential for this goal to be realised. For example, diffusion tensor magnetic resonance imaging (MRI) has been used to observe differences in the magnitude and anisotropy of water diffusion in cartilage (1). Data from a combination of multivariate and spectroscopic techniques, polarized Fourier transform infra-red microspectroscopy (FTIRM) and polarized light microscopy (PLM), are being systematically compared and correlated with M R I data from the same cartilage samples to ascertain changes in the collagen fibers (2). In addition, this multi-techniques approach has made it possible to develop an easily accessible method combining multivariate statistical analysis and F T I R M data to accurately predict the proteoglycan (PG) content using data obtained from energy dispersive X-ray microanalysis (EDX) and optical absorbance data from Safranin-0 stained specimens (2). Articular cartilage consists of an extracellular matrix comprised mainly of a three-dimensional hydrated network of collagen fibres in which are embedded chondrocytes and PGs. Proteoglycan is the name given to a broad range of macromolecular, heterogeneous compounds formed by combining polymeric sugars called glycosaminoglycans (GAGs) (Figure 1) with a protein (3). Damage to the collagen network has been postulated as a key initiating event in the development of O A and many studies concentrate on examining changes to this collagen matrix (4) since they ultimately lead to changes in the mechanical behaviour and ability of the cartilage tissue to function. Proteoglycans, (Figure 1) as the other major component of the intercellular cartilage matrix, are responsible for the resilience and elasticity of the tissue (5-6). Changes in the P G content in diseased cartilage have been associated with concomitant structural changes in the collagen fibrils (7-8). Hence, quantification of the P G content in cartilage samples should prove useful in monitoring not only molecular changes but also changes in the collagen architecture during the cartilage degradation process. Because of the lack of availability of cartilage samples possessing a range of degradation states for in vitro studies, it is common practice to use enzymes to create models of degraded articular cartilage. The intention is that the model samples produced resemble cartilage with early-stage O A including loss of PGs and changes in the collagen fibres (9-10). In theory, the degree of artificial degradation can be controlled by judicious choice of enzymes and conditions. In practice, however, there are some issues with respect to the consistency of the


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samples produced (10). The suite of samples analysed in this study consisted of "normal" bovine articular cartilage as well as a series of trypsin-degraded samples.

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Figure LA. Cartilage GAG sugar monomer: hyaluronic acid (HA), chondroitin sulphate (CS), keratin sulphate (KS). B. Aggrecan proteoglycan monomer showing protein core and areas of GAG attachment. C. Aggrecan monomers bound to HA.

The primary aggregating proteoglycan found in human cartilage is aggrecan. Aggrecan contains about 85% w/w sulphated G A G molecules (Figure 1) mainly chondroitin sulphate (CS) and keratin sulphate (KS). The negatively charged sulphate groups allow specific binding of positively charged dyes such as Safranin-0 in a quantitative manner. Safranin-0 is commonly used as a quantitative stain since it was found to have consistent spectral characteristics at various concentrations. However, one of the long-recognised dangers of using this technique is the presence of metrachromasia (7/) which can affect the quantification of the stain. Metachromasia is the term given to the shifting of the absorbance maximum (usually) to shorter wavelengths through dye-dye interactions and generally occurs when a dye is at higher concentrations. The resulting separate populations of dye molecules will affect quantification due to loss of the dye molecule whose extinction coefficient was of interest. Thus, it is essential that a consistent staining and mounting methodology of the cartilage sections to minimise metachromasia is used if Safranin-0 is to be used qualitatively in conjunction with digital photomicroscopy (11). This allows for



380 relative amounts of the stain to be observed, along with its distribution within a cartilage section. Quantification between cartilage sections requires a more rigorous procedure such as the use of microspectrophotometry or absorbance measurements. Optical absorbance measurements using absorbance at a single wavelength allow for accurate determination of stain concentration, providing the stain does not show metachromasia. Although microspectrophotometry allows for the complete spectral characteristics of the dye to be measured and has been shown to accurately quantify amounts of Safranin-0 within different cartilage sections (32), it does require specialised equipment such as a monochromator or a high-resolution filter set. One of the advantages of using FTIR microspectroscopy is that the problems associated with metachromasia can be avoided. Although the individual constituents of cartilage such as collagen (14-15) and PGs (16) have been extensively examined by infra red spectroscopy it was not until more recently that FTIR microscopic (FTIRM) imaging was used to study intact cartilage (17). Because the spectra of the individual constituents displayed considerable overlap the authors used mixtures of collagen and PGs to determine quantitative values. They emphasise the potential of F T I R M imaging techniques in detecting changes in cartilage - more specifically in collagen and PGs - in O A . In addition, the fact that various IR techniques can be coupled with fibre optic probes is of great clinical interest (75). In recent years F T I R M in conjunction with multivariate analysis has been shown to be a powerful tool in the study of various animal and human tissues including cartilage (19). Although IR techniques lack the specificity of immunohistochemical techniques, they do allow for the estimation of component ratios and the spatial distribution of the components of complex tissues such as cartilage, at different stages of the disease process. This in turn may prove extremely valuable in establishing the aetiology of OA. In order that the full clinical potential of these IR techniques be realised, the strengths and limitations of the combined techniques approach must be examined in order to establish the reliability of the quantitative data generated. In this paper we outline our results using the combined tool of IR and multivariate techniques to develop a model to predict P G content in bovine cartilage sections by E D X and absorbance photo-microscopy.

Experimental Methods: Sample Preparation Nine "normal" intact bovine knee patellae from animals aged 18-30 months were harvested from a local abattoir within 24h of slaughter. Samples were maintained prior to analysis in Minimum Essential Medium with Earle's salts


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(Sigma, M-0275) supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma, P4458), 70 μg/mL L-ascorbic acid (Sigma, G3126), and 200mM L-glutamine (Sigma, A4034). Three cartilage sections on bone were taken from each knee and prepared according to the specific protocol required for each of the experimental techniques. In six of the nine knees two sections were trypsin-treated and the third was examined as a "normal" sample. Sections from the final three knees were examined as "normal" cartilage. Trypsin degradation: Degraded samples were prepared by enzymatic treatment with trypsin (Sigma, T4665, 1 g/L) for 14 hours on cartilage harvested as above, at 37 °C in an orbital incubator. Samples were M R I scanned (/) then frozen until required for F T I R M and histology.

Sample sectioning: A rectangular piece of cartilage was isolated and freed from the soft tissue but left attached at one end to the bone. This was mounted onto a microtome stub and the bone end embedded in "optimal cutting temperature" (OCT) medium and frozen in liquid nitrogen. Care was taken that the cartilage itself did not come into contact with the OCT. Serial 15μπι thick slices were microtomed at -14° C. Slices were placed on tin oxide-coated IR reflective transparent slides (MirrlR, Kevley Technologies, O H U S A ) and stored in a constant humidity environment. Edges of the cut sections were fixed with wax to reduce lifting and moving of samples during analysis. Safranin-0 samples for absorbance measurements: After cryomicrotoming the FTIR samples were fixed in 95% ethanol. Samples were then stained using a standard procedure with 0.1% w/v Safranin-O. (10) Post-staining images of the same areas were processed to obtain absorbance images. EDX analysis: The tissue sections were coated with a thin layer of carbon in a high vacuum carbon evaporator and mounted on the stage of a scanning electron microscope (SEM) for elemental analysis.

Instrumentation FTIRM: A Nicolet Contim^m microscope, with an M C T / A detector, attached to a Nexus 870 spectrometer was used in line scan mode. The analysis aperture was set at 75x150 μπι. Spectra were taken in 75 μιη steps from the bone to the articular surface: 16scans at a resolution of 4cm" were ratioed with a 128 scan background using Happ-Genzel apodization. The 650-4000cm* range was used. EDX: E D X spectra were acquired using an E D A X microanalysis system fitted to an FEI Quanta 200 S E M . Line scans perpendicular to the articular 1

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382 surface were made using a defocussed beam to minimise electron beam-induced sample damage and to average each area of analysis. The sample working distance was 10.0 mm and the focal distance 7.0mm. The scans approximately corresponded to those made using F T I R M . Net element peak counts at each point on the scan were collected at an accelerating voltage of 20kV, using beam spot size 5.0, 60 s dwell time per analysis point, and an amplifier time constant of 17 ms. Absorbance measurements: A Nikon Labophot-pol microscope using the 5x objective and fitted with a blue 1W L E D light source (λ = 480 nm, ) λ i n 25 nm, LumiLED, U S A ) and 10 bit digital camera (SV Micro, Sound Vision, Framingham, M A ) was used. Images were taken both before and after staining. These images were then processed (ImageJ vl.34s, N I H U S A ) according to our published protocol (10). =


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Data Analysis Multivariate techniques: Partial Least Squares (PLS) regression was carried out on collected data using T h e Unscrambled multivariate analysis package (v7.5, 1999, C A M O ASA). Spectral data was mean centred and the 800-1550 cm" region analysed. Sample sets: Optical absorbance, 15 sections, 265 spectra; E D X S/C model, 15 sections, 264 spectra. 1

Results and Discussion Some of the advantages of using modern IR techniques such as mapping and imaging - as opposed to classic histological and bulk chemical analysis methods - are: their minimally destructive capabilities, the possibility of visualising the spatial distribution of the principal constituents, the clinically important option of coupling with fibre optic probes plus the fact that when used in combination with other techniques it becomes possible to obtain semi-quantitative data. Spencer et al (19) determined the relative concentrations of chondroitin sulphate and collagen in tissue sections using multivariate least-squares analysis on their spectral sets on the assumption that each spectrum was a linear combination of these two constituents. They found that the CS to collagen ratios were lower than their biochemical results obtained previously (20). They attributed this to the non-specificity of the FTIR technique to different types of proteins and suggest that by including other minor matrix constituents it may be possible to better differentiate and quantify them. In our approach we have applied PLS techniques to IR data in conjunction with E D X and optical absorbance data to develop a model which makes it possible to predict sulphur content, ie P G content, in both native and trypsindigested cartilage. We are currently extending the use of this model to IR


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imaging data. Figure 2 shows the infra red spectra of representative (A) untreated (UT) and (B) trypsin-treated (TT) cartilage specimens. As can be seen from the spectra, spectral differences are subtle even when P G content is depleted as a result of trypsin treatment. In tissues such as cartilage, there is

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Figure 2. Typical infrared spectra of A: untreated cartilage; B: trypsin-treated cartilage.

significant spectral overlap of the constituents of interest, even in the native state, which is why more complex approaches are needed to extract semiquantitative data from the spectra. Figure 3 shows results for the PLS-predicted vs measured optical absorbance data for the series of untreated cartilage samples. As a visual aid the perfect 1 to 1 calibration is shown as a solid line. From the data it can be seen that the model reasonably predicts absorbance values up to about 2.5 absorbance units but at higher values the model underpredicts. This, we believe, could be attributed to the fact that at higher concentrations the Safranin-0 stain may be subject to metachromasia-type artifacts. Calibration gave an R of 0.87 and a Standard Error of Calibration (SEC) of 0.35 using eleven PCs. Figure 4 shows the wavenumber dependence of the coefficient weightings, sometimes referred to as the correlation spectrum (B), from the PLS calibration. There is good agreement between the peaks in the reference CS spectrum (A) and the regions of high weighting in the correlation spectrum. The CS spectrum 2


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Actual A _ Figure 3. Predicted vs Actual Optical Absorbance from the PLS model of infrared spectra and optical absorbance data for untreated cartilage

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shows bands due to the sulphate stretch area at 1245 cm" and strong absorption due to sugar C-O-C, C - O H and C-C ring vibrations at 1125-920 cm' (17,19). Thus, we conclude that Safranin-0 optical absorbance measurements are indeed a good measure of G A G and hence P G content and that infra red spectra from any cartilage specimen can be used in conjunction with the PLS model to predict the P G content. Figure 5 shows optical absorbance data from a cartilage section, not included in the calibration set, as a function of distance from the articular surface to the bone. Also shown is the PLS-predicted P G content calculated from the infra red data. There is good agreement except for the under prediction in the higher absorbance region. Although E D X has previously proven useful in the analyses of cartilage (21,22) to the best of our knowledge it has not been used in conjunction with either infra red or multivariate techniques. Figure 6 shows results for the P L S predicted vs measured E D X sulphur to carbon intensity ratios (S/C) for the same untreated cartilage samples. Ratios to carbon were used to minimise variability in peak intensities due to potential variations in beam current and specimen thickness. Using twelve PCs this calibration gave an R of 0.78 and a SEC of 0.013. Figure 7 shows the E D X S/C profile from the same cartilage section as in Fig. 5. Also shown is the PLS-predicted S/C ratio derived from the infra red 1

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New Approaches in Biomedical Spectroscopy - American Chemical

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