Prediction of compressive stiffness of articular cartilage using Fourier transform infrared spectroscopy.

Unique biomechanical behavior of articular cartilage is a result of its structure and composition. Interrelationships of tissue constituents (collagen, proteoglycans (PGs) and water) and tissue biomechanical parameters have been studied, but it is evident that no constituent alone explains the tissue mechanics. Fourier transform infrared (FT-IR) spectra can provide detailed information about the biochemical composition of articular cartilage. In this study, a chemometric approach to predict the biomechanical behavior of articular cartilage directly from the FT-IR spectra, i.e., without converting the data into collagen and PG information, was investigated. Partial least squares regression (PLSR) was used to predict equilibrium modulus (n=32) and dynamic modulus (n=24) of bovine cartilage samples from their average FT-IR spectra. The linear correlation coefficients between the reference and predicted values of Young's modulus and dynamic modulus were r=0.866 (p<0.001) and r=0.898 (p<0.001), respectively. When the compressive biomechanical behavior of AC is predicted, the present study indicates that similar or improved results can be obtained with FT-IR spectroscopy as compared to those of traditional biochemical methods.

Application of second derivative spectroscopy for increasing molecular specificity of Fourier transform infrared spectroscopic imaging of articular cartilage.

OBJECTIVE: Fourier transform infrared (FT-IR) spectroscopic imaging is a promising method that enables the analysis of spatial distribution of biochemical components within histological sections. However, analysis of FT-IR spectroscopic data is complicated since absorption peaks often overlap with each other. Second derivative spectroscopy is a technique which enhances the separation of overlapping peaks. The objective of this study was to evaluate the specificity of the second derivative peaks for the main tissue components of articular cartilage (AC), i.e., collagen and proteoglycans (PGs). MATERIALS AND METHODS: Histological bovine AC sections were measured before and after enzymatic removal of PGs. Both formalin-fixed sections (n = 10) and cryosections (n = 6) were investigated. Relative changes in the second derivative peak heights caused by the removal of PGs were calculated for both sample groups. RESULTS: The results showed that numerous peaks, e.g., peaks located at 1202 cm(-1) and 1336 cm(-1), altered less than 5% in the experiment. These peaks were assumed to be specific for collagen. In contrast, two peaks located at 1064 cm(-1) and 1376 cm(-1) were seen to alter notably, approximately 50% or more. These peaks were regarded to be specific for PGs. The changes were greater in cryosections than formalin-fixed sections. CONCLUSIONS: The results of this study suggest that the second derivative spectroscopy offers a practical and more specific method than routinely used absorption spectrum analysis methods to obtain compositional information on AC with FT-IR spectroscopic imaging.

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.

OBJECTIVE: To evaluate the specificity of the current Fourier transform infrared imaging spectroscopy (FT-IRIS) methods for the determination of depthwise proteoglycan (PG) content in articular cartilage (AC). In addition, curve fitting was applied to study whether the specificity of FT-IRIS parameters for PG determination could be improved. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Changes in spatial collagen content and collagen network architecture in porcine articular cartilage during growth and maturation.

OBJECTIVES: The present study was designed to reveal changes in the collagen network architecture and collagen content in cartilage during growth and maturation of pigs. METHODS: Femoral groove articular cartilage specimens were collected from 4-, 11- and 21-month-old domestic pigs (n=12 in each group). The animal care conditions were kept constant throughout the study. Polarized light microscopy was used to determine the collagen fibril network birefringence, fibril orientation and parallelism. Infrared spectroscopy was used to monitor changes in the spatial collagen content in cartilage tissue. RESULTS: During growth, gradual alterations were recorded in the collagen network properties. At 4 months of age, a major part of the collagen fibrils was oriented parallel to the cartilage surface throughout the tissue. However, the fibril orientation changed considerably as skeletal maturation progressed. At 21 months of age, the fibrils of the deep zone cartilage ran predominantly at right angles to the cartilage surface. The collagen content increased and its depthwise distribution changed during growth and maturation. A significant increase of the collagen network birefringence was observed in the deep tissue at the age of 21 months. CONCLUSIONS: The present study revealed dynamic changes of the collagen network during growth and maturation of the pigs. The structure of the collagen network of young pigs gradually approached a network with the classical Benninghoff architecture. The probable explanation for the alterations is growth of the bone epiphysis with simultaneous adaptation of the cartilage to increased joint loading. The maturation of articular cartilage advances gradually with age and offers, in principle, the possibility to influence the quality of the tissue, especially by habitual joint loading. These observations in porcine cartilage may be of significance with respect to the maturation of human articular cartilage.

Estimation of mechanical properties of articular cartilage with MRI - dGEMRIC, T2 and T1 imaging in different species with variable stages of maturation.

BACKGROUND: Magnetic resonance imaging (MRI) is one of the most potential methods for non-invasive diagnosis of cartilage disorders. Several methods have been established for clinical use; T(1) relaxation time imaging with negatively charged contrast agent (delayed gadolinium enhanced MRI of cartilage, dGEMRIC) has been shown to be sensitive to proteoglycan (PG) content while T(2) relaxation time has been demonstrated to express properties of the collagen fibril network. The use of native T(1) relaxation time has received less attention. OBJECTIVE: In the present study, magnetic resonance (MR) parameters of different types of patellar cartilage were studied with respect to the mechanical properties of the tissue. The general usefulness of the parameters to predict mechanical properties was investigated using cartilage from different species and stages of maturation. METHODS: dGEMRIC, T(2) and native T(1) relaxation times of healthy mature human, juvenile porcine and juvenile bovine articular cartilage samples were measured at 9.4T at 25 degrees C. Mechanical properties (Young's modulus and dynamic modulus) of the samples were measured in unconfined compression using a material testing device. The relationships between MRI and mechanical parameters and potential differences between different types of tissues were tested statistically. RESULTS: Significant, but varying relationships were established between T(1) or T(2) relaxation time and mechanical properties, depending on tissue type. The values of mechanical parameters were in line with the results previously reported in the literature. Unexpectedly, dGEMRIC showed no statistically significant association with the mechanical properties. Variation in the assumption of native T(1) value did not induce significant differences in the calculated contrast agent concentration, and consequently did not affect prediction of mechanical properties. CONCLUSION: For patellae, a complex variation in the relationships between T(2) and mechanical properties in different groups was revealed. The results support the conclusion that juvenile animal tissue, exhibiting a highly complex collagenous architecture, may not always serve as a realistic model for mature human tissue with a typical three-zone network organization, and other than bulk metrics are required for the analysis of cartilage T(2). As the multilayered collagen network can strongly control the mechanical characteristics of juvenile tissue, it may mask the mechanical role of PGs and explain why dGEMRIC could not predict mechanical parameters in patellar cartilage.

T(2) relaxation time mapping reveals age- and species-related diversity of collagen network architecture in articular cartilage.

OBJECTIVE: The magnetic resonance imaging (MRI) parameter T(2) relaxation time has been shown to be sensitive to the collagen network architecture of articular cartilage. The aim of the study was to investigate the agreement of T(2) relaxation time mapping and polarized light microscopy (PLM) for the determination of histological properties (i.e., zone and fibril organization) of articular cartilage. METHODS: T(2) relaxation time was determined at 9.4 T field strength in healthy adult human, juvenile bovine and juvenile porcine patellar cartilage, and related to collagen anisotropy and fibril angle as measured by quantitative PLM. RESULTS: Both T(2) and PLM revealed a mutually consistent but varying number of collagen-associated laminae (3, 3-5 or 3-7 laminae in human, porcine and bovine cartilage, respectively). Up to 44% of the depth-wise variation in T(2) was accounted for by the changing anisotropy of collagen fibrils, confirming that T(2) contrast of articular cartilage is strongly affected by the collagen fibril anisotropy. A good correspondence was observed between the thickness of T(2)-laminae and collagenous zones as determined from PLM anisotropy measurements (r=0.91, r=0.95 and r=0.91 for human, bovine and porcine specimens, respectively). CONCLUSIONS: According to the present results, T(2) mapping is capable of detecting histological differences in cartilage collagen architecture among species, likely to be strongly related to the differences in maturation of the tissue. This diversity in the MRI appearance of healthy articular cartilage should also be recognized when using juvenile animal tissue as a model for mature human cartilage in experimental studies.

Proteoglycan and collagen sensitive MRI evaluation of normal and degenerated articular cartilage.

Quantitative magnetic resonance imaging (MRI) techniques have earlier been developed to characterize the structure and composition of articular cartilage. Particularly, Gd-DTPA(2-)-enhanced T1 imaging is sensitive to cartilage proteoglycan content, while T2 relaxation time mapping is indicative of the integrity and arrangement of the collagen network. However, the ability of these techniques to detect early osteoarthrotic changes in cartilage has not been demonstrated. In this study, normal and spontaneously degenerated bovine patellar cartilage samples (n=32) were investigated in vitro using the aforementioned techniques. For reference, mechanical, histological and biochemical properties of the adjacent tissue were determined, and a grading system, the cartilage quality index (CQI), was used to score the structural and functional integrity of each sample. As cartilage degeneration progressed, a statistically significant increase in the superficial T2 (r=0.494, p<0.05) and a decrease in superficial and bulk T1 in the presence of Gd-DTPA(2-) (r=-0.681 and -0.688 (p<0.05), respectively) were observed. Gd-DTPA(2-)-enhanced T1 imaging served as the best predictor of tissue integrity and accounted for about 50% of the variation in CQI. The present results reveal that changes in the quantitative MRI parameters studied are indicative of structural and compositional alterations as well as the mechanical impairment of spontaneously degenerated articular cartilage.

Biomechanical properties of knee articular cartilage.

Structure and properties of knee articular cartilage are adapted to stresses exposed on it during physiological activities. In this study, we describe site- and depth-dependence of the biomechanical properties of bovine knee articular cartilage. We also investigate the effects of tissue structure and composition on the biomechanical parameters as well as characterize experimentally and numerically the compression-tension nonlinearity of the cartilage matrix. In vitro mechano-optical measurements of articular cartilage in unconfined compression geometry are conducted to obtain material parameters, such as thickness, Young's and aggregate modulus or Poisson's ratio of the tissue. The experimental results revealed significant site- and depth-dependent variations in recorded parameters. After enzymatic modification of matrix collagen or proteoglycans our results show that collagen primarily controls the dynamic tissue response while proteoglycans affect more the static properties. Experimental measurements in compression and tension suggest a nonlinear compression-tension behavior of articular cartilage in the direction perpendicular to articular surface. Fibril reinforced poroelastic finite element model was used to capture the experimentally found compression-tension nonlinearity of articular cartilage.

Comparison of the equilibrium response of articular cartilage in unconfined compression, confined compression and indentation.

At mechanical equilibrium, articular cartilage is usually characterized as an isotropic elastic material with no interstitial fluid flow. In this study, the equilibrium properties (Young's modulus, aggregate modulus and Poisson's ratio) of bovine humeral, patellar and femoral cartilage specimens (n=26) were investigated using unconfined compression, confined compression, and indentation tests. Optical measurements of the Poisson's ratio of cartilage were also carried out. Mean values of the Young's modulus (assessed from the unconfined compression test) were 0.80+/-0.33, 0.57+/-0.17 and 0.31+/-0.18MPa and of the Poisson's ratio (assessed from the optical test) 0.15+/-0.06, 0.16+/-0.05 and 0.21+/-0.05 for humeral, patellar, and femoral cartilages, respectively. The indentation tests showed 30-79% (p<0.01) higher Young's modulus values than the unconfined compression tests. In indentation, values of the Young's modulus were independent of the indenter diameter only in the humeral cartilage. The mean values of the Poisson's ratio, obtained indirectly using the mathematical relation between the Young's modulus and the aggregate modulus in isotropic material, were 0.16+/-0.06, 0.21+/-0.05, and 0.26+/-0.08 for humeral, patellar, and femoral cartilages, respectively. We conclude that the values of the elastic parameters of the cartilage are dependent on the measurement technique in use. Based on the similar values of Poisson's ratios, as determined directly or indirectly, the equilibrium response of articular cartilage under unconfined and confined compression is satisfactorily described by the isotropic elastic model. However, values of the isotropic Young's modulus obtained from the in situ indentation tests are higher than those obtained from the in vitro unconfined or confined compression tests and may depend on the indenter size in use.

Ultrasonic characterization of articular cartilage.

Osteoarthrosis is the most important joint disease that threatens health of the musculoskeletal system of elderly people. Today, there is a need for sensitive, quantitative diagnostic methods for successful and early diagnosis of the disorder. In the present study, we aimed at evaluating the applicability of ultrasound for quantitative assessment of cartilage structure and properties. Bovine articular cartilage was investigated both in vitro and in situ using high frequency ultrasound. Cartilage samples were also tested mechanically in vitro to reveal relationships between acoustic and mechanical parameters of the tissue. The collagen organization and proteoglycan content of cartilage samples were mapped, using quantitative polarized light microscopy and digital densitometry, respectively, to reveal their effect on the acoustic properties of tissue. The high frequency pulse-echo ultrasound (20-30 MHz) technique proved to be sensitive in detecting the degeneration of the superficial collagen-rich cartilage zone. In addition, ultrasound was found to be a potential tool for measuring cartilage thickness. When the results from biomechanical indentation measurements and ultrasound measurements of normal and enzymatically degraded articular cartilage were combined, collagen or proteoglycan degradation in the tissue could be sensitively and specifically differentiated from each other. To conclude, high frequency ultrasound is a useful tool for evaluation of the quality of superficial articular cartilage as well as for the measurement of cartilage thickness. Therefore, ultrasound appears to be a valuable supplement to the mechanical measurements of articular cartilage stiffness.

T2 relaxation reveals spatial collagen architecture in articular cartilage: a comparative quantitative MRI and polarized light microscopic study.

It has been suggested that orientational changes in the collagen network of articular cartilage account for the depthwise T2 anisotropy of MRI through the magic angle effect. To investigate the relationship between laminar T2 appearance and collagen organization (anisotropy), bovine osteochondral plugs (N = 9) were T2 mapped at 9.4T with cartilage surface normal to the static magnetic field. Collagen fibril arrangement of the same samples was studied with polarized light microscopy, a quantitative technique for probing collagen organization by analyzing its ability to rotate plane polarized light, i.e., birefringence (BF). Depthwise variation of safranin O-stained proteoglycans was monitored with digital densitometry. The spatially varying cartilage T2 followed the architectural arrangement of the collagen fibril network: a linear positive correlation between T2 and the reciprocal of BF was established in each sample, with r = 0.91 +/- 0.02 (mean +/- SEM, N = 9). The current results reveal the close connection between the laminar T2 structure and the collagen architecture in histologic zones.

Quantitative MR microscopy of enzymatically degraded articular cartilage.

Structural changes in bovine patellar articular cartilage, induced by component selective enzymatic treatments, were investigated by measuring tissue T(2) relaxation at 9.4 T. This MRI parameter was compared with Young's modulus, a measure of elastic stiffness and loadbearing ability of cartilage tissue. Collagenase was used to digest the collagen network and chondroitinase ABC to remove proteoglycans. Polarized light microscopy and digital densitometry were used to assess enzyme penetration after 44 hr of enzymatic digestion. T(2) relaxation in superficial cartilage increased significantly only in samples treated with collagenase. A statistically significant decrease in Young's modulus was observed in both enzymatically treated sample groups. These results confirm that T(2) of articular cartilage is sensitive to the integrity of collagen in the extracellular matrix. Nonetheless, it does not appear to be an unambiguous indicator of cartilage stiffness, which is significantly impaired in osteoarthrosis.

Characterization of enzymatically induced degradation of articular cartilage using high frequency ultrasound.

Ultrasound may provide a quantitative technique for the characterization of cartilage changes typical of early osteoarthrosis. In this study, specific changes in bovine articular cartilage were induced using collagenase and chondroitinase ABC, enzymes that selectively degrade collagen fibril network and digest proteoglycans, respectively. Changes in cartilage structure and properties were quantified using high frequency ultrasound, microscopic analyses and mechanical indentation tests. The ultrasound reflection coefficient of the physiological saline-cartilage interface (R1) decreased significantly (-96.4%, p < 0.01) in the collagenase digested cartilage compared to controls. Also a significantly lower ultrasound velocity (-6.2%, p < 0.01) was revealed after collagenase digestion. After chondroitinase ABC digestion, a new acoustic interface at the depth of the enzyme penetration front was detected. Cartilage thickness, as determined with ultrasound, showed a high, linear correlation (R = 0.943, n = 60, average difference 0.073 mm (4.0%)) with the thickness measured by the needle-probe method. Both enzymes induced a significant decrease in the Young's modulus of cartilage (p < 0.01). Our results indicate that high frequency ultrasound provides a sensitive technique for the analysis of cartilage structure and properties. Possibly ultrasound may be utilized in vivo as a quantitative probe during arthroscopy.