Quantitative analysis of spatial proteoglycan content in articular cartilage with FTIRIS: Critical evaluation of analysis methods and specificity of the parameters

Quantitative analysis of spatial proteoglycan content in articular cartilage with Fourier transform infrared imaging spectroscopy: Critical evaluation of analysis methods and specificity of the parameters

Microscopy Research and Technique 2010, 73, 503-512
L. Rieppo, S. Saarakkala, T. Narhi, J. Holopainen, M. Lammi, H.J. Helminen, J.S. Jurvelin, J. Rieppo

Abstract: Current Fourier transform infrared imaging spectroscopy (FT-IRIS) methods for the determination of depthwise proteoglycan (PG) content in articular cartilage (AC) were evaluated. In addition, curve fitting was applied to study whether the specificity of FT-IRIS parameters for PG determination could be improved. Two sample groups from the steer AC were prepared for the study (n = 8 samples/group). In the first group, chondroitinase ABC enzyme was used to degrade the PGs from the superficial cartilage, while the samples in the second group served as the controls. Samples were examined with FT-IRIS and analyzed using previously reported direct absorption spectrum techniques and multivariate methods and, in comparison, by curve fitting. Safranin O-stained sections were measured with digital densitometry to obtain a reference for depthwise PG distribution. Carbohydrate region-based absorption spectrum methods showed a statistically weaker correlation with the PG reference distributions than the results of the curve fitting (subpeak located approximately at 1,060 cm^-1). Furthermore, the shape of the depthwise profiles obtained using the curve fitting was more similar to the reference profiles than with the direct absorption spectrum analysis. Results suggest that the current FT-IRIS methods for PG analysis lack the specificity for quantitative measurement of PGs in AC. The curve fitting approach demonstrated that it is possible to improve the specificity of the PG analysis. However, the findings of the present study suggest that further development of the FT-IRIS analysis techniques is still needed.