Imaging of collagen and proteoglycan in cartilage sections using Fourier transform infrared spectral imaging

Abstract

Objective To test the hypothesis that Fourier transform infrared (FTIR) spectral imaging, coupled with multivariate data processing techniques, can image the spatial distribution of matrix constituents in native and engineered cartilage samples. Methods Tissue sections from native and trypsin‐digested bovine nasal cartilage (BNC) and from engineered cartilage, generated by chick sternal chondrocytes grown in a hollow fiber bioreactor, were placed either on calcium fluoride windows for FTIR analysis or gelatinized microscope slides for histologic analysis. Based on the assumption that cartilage is predominantly chondroitin sulfate (CS) and type II collagen, chemical images were extracted from FTIR spectral imaging data sets using 2 multivariate methods: the Euclidean distance algorithm and a least‐squares approach. Results Least‐squares analysis of the FTIR data of native BNC yielded a collagen content of 54 ± 13% and a CS content of 37 ± 16% (mean ± SD). Euclidean distance analysis of measurements made on trypsin‐digested BNC demonstrated only trace amounts of CS. For engineered cartilage, the CS content was significantly lower (15 ± 5%), while the collagen content (73 ± 6%) was significantly higher than biochemically determined values (CS 34%, collagen 5%, protein 61%). These differences are due to the fact that the dimethylmethylene blue assay overestimated the CS content of the tissue because it is not specific for CS, while the FTIR spectral imaging technique overestimated the collagen content because it lacks specificity for different proteins. Conclusion FTIR spectral imaging combines histology‐like spatial localization with the quantitative capability of bulk chemical analysis. For molecules with a unique spectral signature, such as CS, the FTIR technique coupled with multivariate analysis can define a unique spatial distribution. However, for some applications, the lack of specificity of this technique for different types of proteins may be a limitation.

Stress–relaxation of human patellar articular cartilage in unconfined compression: Prediction of mechanical response by tissue composition and structure

“…the average value of the depth-wise amide I absorbance profile was first scaled to match with the measured bulk collagen solid mass fraction and the depth-dependent values were then calculated. The depth-wise PG distribution was determined similarly from the biochemical PG content and the depth-dependent FT-IRIS data (Bi et al, 2006(Bi et al, , 2007Camacho et al, 2001;Potter et al, 2001;Rieppo et al, 2004). Volume fractions for the collagen and PG distributions were similar to their mass fractions, as constant solid tissue density (r S ¼ 1.4338 g/ml) was assumed (Basser et al, 1998;Shapiro et al, 2001;Wilson et al, 2007).…”

Postnatal development of depth-dependent collagen density in ovine articular cartilage

“…FTIRμS was first applied to biological tissues when it became possible to use a microscope in the light path [49]. The first applications of FTIRμS for AC were investigated at the start of the current century [50,51]. Biochemical analysis of hydroxyproline content is an alternative and well-established technique to quantify collagen density in AC.…”

“…integrated peak areas [50], (partial) least square fits [52], Euclidean distance analysis [51] and deconvolution approaches [39]. Analysis with integrated peak areas is the most straightforward, and with our calibration sequence we estimated an error of 5% for collagen densities for a 4:1 ratio of collagen to chondroitin sulphate [1,3].…”

Collagen Network of Articular Cartilage Modulates Fluid Flow and Mechanical Stresses in Chondrocyte

“…Fourier transform infrared imaging spectroscopy was used to qualitatively determine the structure and composition of articular cartilage from microscopic cross-sections (Camacho et al 2001;Potter et al 2001;Rieppo et al 2004Rieppo et al , 2005. A full-thickness human cartilage sample without subchondral bone was prepared and embedded into Tissue Tek OCT embedding media (Sakura Finetek, Torrence, CA, USA) and frozen.…”

“…Light microscopy combined with histological staining techniques offers a tool to investigate the proteoglycan distribution around the cells, but until recently a method for the quantitation of the collagen distribution has been lacking. Fourier transform infrared imaging spectroscopy (FT-IRIS) can be used to analyze the spatial distribution of the proteoglycans and collagen network around the chondrocytes during a single imaging session (Camacho et al 2001;Potter et al 2001;Rieppo et al 2004Rieppo et al , 2005. As some theoretical models of articular cartilage differentiate between the mechanical roles of the proteoglycans and collagen fibrils (Cohen et al 1998;Korhonen et al 2003a;Li et al 1999;Soltz and Ateshian 2000;Wilson et al 2004), the incorporation of realistic cartilage microstructure and composition from FT-IRIS into a model may provide an important step towards further understanding the role of the cell mechanics in the adaptation and degeneration of articular cartilage.…”