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.

During maturation, significant modulation of tissue structure, composition and mechanical properties takes place. Importantly, the present study provides insight into the mechanical, chemical and structural interactions that lead to functional properties of mature articular cartilage.

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 5 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 21 ). 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. Microsc. Res. Tech. 73:503-512, 2010. V

Articular cartilage composition and structure are maintained and remodeled by chondrocytes under the influence of loading. Exercise-induced changes in the composition, structure, mechanical properties and tissue integrity of growing and aging hamster articular cartilage were investigated. Articular cartilage samples (n = 191) were harvested from the proximal tibiae of hamsters aged 1, 3, 6, 12 and 15 months. The hamsters were divided into runners and controls. The runners had free access to a running wheel between 1 and 3 months (runner groups 3-, 12-and 15-month-old hamsters) or 1 and 6 months (runner group 6-month-old hamsters) of age. Control animals were subjected to a sedentary lifestyle. Mechanical indentation tests and depth-wise compositional and structural analyses were performed for the cartilage samples. Furthermore, the integrity of articular cartilage was assessed using histological osteoarthritis grading. Exercise affected the collagen network organization after a 5-month exercise period, especially in the middle and deep zones. However, no effect on the mechanical properties was detected after exercise. Before the age of 12 months, the runners showed less osteoarthritis than the controls, whereas at 15 months of age the situation was reversed. It is concluded that, in hamsters, physical exercise at a young age enhances cartilage maturation and alters the depth-wise cartilage structure and composition. This may be considered beneficial. However, exercise at a young age demonstrated adverse effects on cartilage at a later age with a significant increase in the incidence of osteoarthritis.

Fourier transform infrared (FTIR) microspectroscopy has been used to estimate the spatial proteoglycan (PG) and collagen contents in articular cartilage (AC). However, it is not clear whether the results of FTIR analyses are consistent between different species. Our aim was to clarify how three different FTIR PG parameters in use, i.e., the integrated absorbance in the carbohydrate region, the carbohydrate/amide I ratio, and the second derivative peak at 1062  cm-1, can indicate the densitometrically assessed (reference method) spatial PG content in a sample set consisting of osteoarthritic human and bovine AC samples. The results show that all the parameters can accurately reflect the PG content, when the species are analyzed separately. When all samples are pooled, the correlation with the reference method is high (r=0.760, n=104) for the second derivative peak at 1062  cm-1 and is significantly lower (p<0.05) for the carbohydrate region (r=0.587, n=104) and for the carbohydrate/amide I ratio (r=0.579, n=104). Therefore, the analysis of the carbohydrate region may provide inconsistent results, if the cartilage samples from different species are in use. Based on the present study, second derivative analysis yields more consistent results for human and bovine cartilages.

We investigated the effects of lifelong voluntary exercise on articular cartilage of mice. At the age of 4 weeks C57BL mice (n = 152) were divided into two groups, with one group serving as a sedentary control whereas the other was allowed free access to a running wheel from the age of 1 month onward. Mice were euthanized at four different time points (1, 2, 6, and 18 months of age). Articular cartilage samples were gathered from the load-bearing area of the tibial medial plateaus, and osteoarthritis was graded. Additionally, the proteoglycan content distribution was assessed using digital densitometry, collagen fibril orientation, and parallelism with polarized light microscopy, and collagen content using Fourier transform infrared imaging spectroscopy. The incidence of osteoarthritis increased with aging, but exercise had no effect on this trend. Furthermore, the structure and composition revealed significant growth, maturation, and age-dependent properties. Exercise exerted a minor effect on collagen fibril orientation in the superficial zone. Fibril orientation at 2 months of age was more perpendicular to surface (p < 0.05) in controls compared with runners, whereas the situation was reversed at the age of 18 months (p < 0.05). The collagen content of the superficial zone was higher (p < 0.01) at the age of 18 months in controls compared with runners but the proteoglycan content did not display any exercise-dependent changes. In conclusion, growth, maturation, and aging exerted a clear effect on integrity, structure, and composition of medial tibial plateau articular cartilage in mice, whereas lifelong voluntary exercise had only a minor effect on collagen architecture and content.