Infrared microspectroscopic determination of collagen cross-links in articular cartilage

Collagen forms an organized network in articular cartilage to give tensile stiffness to the tissue. Due to its long half-life, collagen is susceptible to cross-links caused by advanced glycation end-products. The current standard method for determination of cross-link concentrations in tissues is the destructive high-performance liquid chromatography (HPLC). The aim of this study was to analyze the cross-link concentrations nondestructively from standard unstained histological articular cartilage sections by using Fourier transform infrared (FTIR) microspectroscopy. Half of the bovine articular cartilage samples (n = 27) were treated with threose to increase the collagen cross-linking while the other half (n = 27) served as a control group. Partial least squares (PLS) regression with variable selection algorithms was used to predict the cross-link concentrations from the measured average FTIR spectra of the samples, and HPLC was used as the reference method for cross-link concentrations. The correlation coefficients between the PLS regression models and the biochemical reference values were r = 0.84 (p <0.001), r = 0.87 (p <0.001) and r = 0.92 (p <0.001) for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), and pentosidine (Pent) cross-links, respectively. The study demonstrated that FTIR microspectroscopy is a feasible method for investigating cross-link concentrations in articular cartilage. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.Peer reviewe

Fourier Transform Infrared Spectroscopic Imaging and Multivariate Regression for Prediction of Proteoglycan Content of Articular Cartilage

Fourier Transform Infrared (FT-IR) spectroscopic imaging has been earlier applied for the spatial estimation of the collagen and the proteoglycan (PG) contents of articular cartilage (AC). However, earlier studies have been limited to the use of univariate analysis techniques. Current analysis methods lack the needed specificity for collagen and PGs. The aim of the present study was to evaluate the suitability of partial least squares regression (PLSR) and principal component regression (PCR) methods for the analysis of the PG content of AC. Multivariate regression models were compared with earlier used univariate methods and tested with a sample material consisting of healthy and enzymatically degraded steer AC. Chondroitinase ABC enzyme was used to increase the variation in PG content levels as compared to intact AC. Digital densitometric measurements of Safranin O –stained sections provided the reference for PG content. The results showed that multivariate regression models predict PG content of AC significantly better than earlier used absorbance spectrum (i.e. the area of carbohydrate region with or without amide I normalization) or second derivative spectrum univariate parameters. Increased molecular specificity favours the use of multivariate regression models, but they require more knowledge of chemometric analysis and extended laboratory resources for gathering reference data for establishing the models. When true molecular specificity is required, the multivariate models should be used

Effects of Articular Cartilage Constituents on Phosphotungstic Acid Enhanced Micro-Computed Tomography

Contrast-enhanced micro-computed tomography (CE mu CT) with phosphotungstic acid (PTA) has shown potential for detecting collagen distribution of articular cartilage. However, the selectivity of the PTA staining to articular cartilage constituents remains to be elucidated. The aim of this study was to investigate the dependence of PTA for the collagen content in bovine articular cartilage. Adjacent bovine articular cartilage samples were treated with chondroitinase ABC and collagenase to degrade the proteoglycan and the collagen constituents in articular cartilage, respectively. Enzymatically degraded samples were compared to the untreated samples using CE mu CT and reference methods, such as Fourier-transform infrared imaging. Decrease in the X-ray attenuation of PTA in articular cartilage and collagen content was observed in cartilage depth of 0-13% and deeper in tissue after collagen degradation. Increase in the X-ray attenuation of PTA was observed in the cartilage depth of 13- 39% after proteoglycan degradation. The X-ray attenuation of PTA-labelled articular cartilage in CE mu CT is associated mainly with collagen content but the proteoglycans have a minor effect on the X-ray attenuation of the PTA-labelled articular cartilage. In conclusion, the PTA labeling provides a feasible CE mu CT method for 3D characterization of articular cartilage.Peer reviewe

Critical-sized cartilage defects in the equine carpus

Aim: The horse joint, due to its similarity with the human joint, is the ultimate model for translational articular cartilage repair studies. This study was designed to determine the critical size of cartilage defects in the equine carpus and serve as a benchmark for the evaluation of new cartilage treatment options. Material and Methods: Circular full-thickness cartilage defects with a diameter of 2, 4, and 8 mm were created in the left middle carpal joint and similar osteochondral (3.5 mm in depth) defects in the right middle carpal joint of 5 horses. Spontaneously formed repair tissue was examined macroscopically, with MR and mu CT imaging, polarized light microscopy, standard histology, and immunohistochemistry at 12 months. Results: Filling of 2 mm chondral defects was good (77.8 +/- 8.5%), but proteoglycan depletion was evident in Safranin-O staining and gadolinium-enhanced MRI (T-1Gd). Larger chondral defects showed poor filling (50.6 +/- 2.7% in 4 mm and 31.9 +/- 7.3% in 8 mm defects). Lesion filling in 2, 4, and 8 mm osteochondral defects was 82.3 +/- 3.0%, 68.0 +/- 4.6% and 70.8 +/- 15.4%, respectively. Type II collagen staining was seen in 9/15 osteochondral defects but only in 1/15 chondral defects. Subchondral bone pathologies were evident in 14/15 osteochondral samples but only in 5/15 chondral samples. Although osteochondral lesions showed better neotissue quality than chondral lesions, the overall repair was deemed unsatisfactory because of the subchondral bone pathologies. Conclusion: We recommend classifying 4 mm as critical osteochondral lesion size and 2 mm as critical chondral lesion size for cartilage repair research in the equine carpal joint model.Peer reviewe

Preprocessing Strategies for Sparse Infrared Spectroscopy: A Case Study on Cartilage Diagnostics

The aim of the study was to optimize preprocessing of sparse infrared spectral data. The sparse data were obtained by reducing broadband Fourier transform infrared attenuated total reflectance spectra of bovine and human cartilage, as well as of simulated spectral data, comprising several thousand spectral variables into datasets comprising only seven spectral variables. Different preprocessing approaches were compared, including simple baseline correction and normalization procedures, and model-based preprocessing, such as multiplicative signal correction (MSC). The optimal preprocessing was selected based on the quality of classification models established by partial least squares discriminant analysis for discriminating healthy and damaged cartilage samples. The best results for the sparse data were obtained by preprocessing using a baseline offset correction at 1800 cm−1, followed by peak normalization at 850 cm−1 and preprocessing by MSC.publishedVersio

3D morphometric analysis of calcified cartilage properties using micro-computed tomography

OBJECTIVE: Our aim is to establish methods for quantifying morphometric properties of calcified cartilage (CC) from micro-computed tomography (muCT). Furthermore, we evaluated the feasibility of these methods in investigating relationships between osteoarthritis (OA), tidemark surface morphology and open subchondral channels (OSCCs). METHOD: Samples (n = 15) used in this study were harvested from human lateral tibial plateau (n = 8). Conventional roughness and parameters assessing local 3-dimensional (3D) surface variations were used to quantify the surface morphology of the CC. Subchondral channel properties (percentage, density, size) were also calculated. As a reference, histological sections were evaluated using Histopathological osteoarthritis grading (OARSI) and thickness of CC and subchondral bone (SCB) was quantified. RESULTS: OARSI grade correlated with a decrease in local 3D variations of the tidemark surface (amount of different surface patterns (rs = -0.600, P = 0.018), entropy of patterns (EP) (rs = -0.648, P = 0.018), homogeneity index (HI) (rs = 0.555, P = 0.032)) and tidemark roughness (TMR) (rs = -0.579, P = 0.024). Amount of different patterns (ADP) and EP associated with channel area fraction (CAF) (rp = 0.876, P < 0.0001; rp = 0.665, P = 0.007, respectively) and channel density (CD) (rp = 0.680, P = 0.011; rp = 0.582, P = 0.023, respectively). TMR was associated with CAF (rp = 0.926, P < 0.0001) and average channel size (rp = 0.574, P = 0.025). CC topography differed statistically significantly in early OA vs healthy samples. CONCLUSION: We introduced a mu-CT image method to quantify 3D CC topography and perforations through CC. CC topography was associated with OARSI grade and OSCC properties; this suggests that the established methods can detect topographical changes in tidemark and CC perforations associated with OA

Vibrational spectroscopy of articular cartilage

Articular cartilage is a connective tissue that is located at the ends of long bones. Type II collagen, proteoglycans, water, and chondrocytes are the main constituents of articular cartilage. Osteoarthritis, the most common joint disease in the world, causes degenerative changes in articular cartilage tissue. Fourier transform infrared, Raman, and near infrared spectroscopic techniques offer versatile tools to assess biochemical composition and quality of articular cartilage. These vibrational spectroscopic techniques can be used to broaden our understanding about the compositional changes during osteoarthritis, and they also hold promise in disease diagnostics. In this article, the current literature of articular cartilage spectroscopic studies is reviewed

Scatter plots between optical density of safranin O and different univariate FT-IR PG parameters.

<p>A) Integrated area of carbohydrate region (984–1140 cm⁻¹). B) Integrated area of carbohydrate region normalized with amide I (1584–1720 cm⁻¹). C) Intensity of ^2nd derivative peak located at 1374 cm⁻¹. D) Intensity of 2^nd derivative peak located at 1062 cm⁻¹. Statistical significance of <i>p</i><0.001 is indicated by two asterisks (**).</p

Mean absorption spectrum and second derivative spectrum of the data set.

<p>A) Mean infrared absorption spectrum of AC and B) mean second derivative spectrum of AC.</p