- [Show abstract] [Hide abstract] ABSTRACT: To determine the number of participants required in controlled clinical trials investigating the progression of osteoarthritis (OA) of the hip as evaluated by the joint space width (JSW) on radiographs and to evaluate the reproducibility of the JSW measurement methods. Anteroposterior radiographs of hip were taken from 13 healthy volunteers and from 18 subjects with radiographic hip OA. The reproducibility of the JSW was determined from four segments using digital caliper measurements performed on film radiographs and using semiautomatic computerized image analysis of digitized images. Pearson correlation coefficient, coefficient of variability [CV (%)], and sample size values were calculated. It was found that 20 was a typical number of patients for a sufficiently powered study. The highest sample size was found in subjects with OA in the lateral segment. The reproducibility of the semiautomatic computerized method was not significantly better than the digital caliper method. The number of study subjects required to detect a significant joint space narrowing in follow-up studies is influenced by the baseline hip joint OA severity. The JSW measurements with computerized image analysis did not improve the reproducibility and thus performing JSW measurements with a digital caliper is acceptable.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: The aim of this study was to undertake a stereological analysis to quantify the dimensions of the collagen network in the repair tissue of porcine joints after they had been subjected to autologous chondrocyte transplantation (ACT). ACT was used to repair cartilage lesions in knee joints of pigs. Electron-microscopic stereology, immunostaining for type II collagen, and quantitative polarized-light microscopy were utilized to study the collagen fibrils in the repair tissue 3 and 12 months after the operation. The collagen volume density (V(V)) was lower in the repair tissue than in normal cartilage at 3 months (20.4 vs. 23.7%) after the operation. The collagen surface density (S(V), 1.5·10(-2) vs. 3.1·10(-2) nm(2)/nm(3)) and V(V) increased with time in the repair tissue (20.4 vs. 44.7%). Quantitative polarized-light microscopy detected a higher degree of collagen parallelism in the repair tissue at 3 months after the operation (55.7 vs. 49.7%). In contrast, 1 year after the operation, fibril parallelism was lower in the repair tissue than in the control cartilage (47.5 vs. 69.8%). Following ACT, V(V) and S(V) increased in the repair tissue with time, reflecting maturation of the tissue. One year after the operation, there was a lower level of fibril organization in the repair tissue than in the control cartilage. Thus, the newly synthesized collagen fibrils in the repair tissue appeared to form a denser network than in the control cartilage, but the fibrils remained more randomly oriented.
- [Show abstract] [Hide abstract] ABSTRACT: Reasons for performing study: Training at a very young age may influence the characteristics of the collagen network of articular cartilage extracellular matrix (ECM) in horses.Objectives: To investigate whether increasing workload of foals results in significant changes in the biochemical composition of articular cartilage ECM.Methods: Thoroughbred foals (n = 33) were divided into 2 different exercise groups from age 10 days-18 months. One group (PASTEX; n = 15) was reared at pasture; the other (CONDEX; n = 18) underwent a specific additional training programme that increased workload by 30%. At mean age 18 months, 6 animals from each group were subjected to euthanasia. The proximal articular surface of the proximal phalanx of the right hindlimb was examined for the presence of damage using the cartilage degeneration index (CDI). Samples were taken from 2 sites with known different loading patterns. Slices were analysed for DNA, glycosaminoglycans (GAG), collagen and post translational modifications of collagen (formation of hydroxylysylpyridinoline [HP] and pentosidine crosslinks, and hydroxylysine [Hyl]), and exercise groups and different sites compared.Results: There were no differences in CDI between PASTEX and CONDEX animals, indicating the absence of extra joint damage due to the exercise regimen. There were site-related differences for most biochemical variables, corroborating earlier reports. All biochemical variables showed differences between PASTEX and CONDEX groups at one of the sites, and some at both. GAG and collagen levels were lower in the CONDEX group whereas Hyl, HP crosslinks and pentosidine crosslinks were higher.Conclusions and potential relevance: A measurable effect of the conditioning exercise was demonstrated. The margin between too much and too little work when training foals may be narrower than intuitively presumed.
- [Show abstract] [Hide abstract] ABSTRACT: The aim of this study was to record growth-related changes in collagen network organization and proteoglycan distribution in intermittently peak-loaded and continuously lower-level-loaded articular cartilage. Cartilage from the proximal phalangeal bone of the equine metacarpophalangeal joint at birth, at 5, 11 and 18 months, and at 6-10 years of age was collected from two sites. Site 1, at the joint margin, is unloaded at slow gaits but is subjected to high-intensity loading during athletic activity; site 2 is a continuously but less intensively loaded site in the centre of the joint. The degree of collagen parallelism was determined with quantitative polarized light microscopy and the parallelism index for collagen fibrils was computed from the cartilage surface to the osteochondral junction. Concurrent changes in the proteoglycan distribution were quantified with digital densitometry. We found that the parallelism index increased significantly with age (up to 90%). At birth, site 2 exhibited a more organized collagen network than site 1. In adult horses this situation was reversed. The superficial and intermediate zones exhibited the greatest reorganization of collagen. Site 1 had a higher proteoglycan content than site 2 at birth but here too the situation was reversed in adult horses. We conclude that large changes in joint loading during growth and maturation in the period from birth to adulthood profoundly affect the architecture of the collagen network in equine cartilage. In addition, the distribution and content of proteoglycans are modified significantly by altered joint use. Intermittent peak-loading with shear seems to induce higher collagen parallelism and a lower proteoglycan content in cartilage than more constant weight-bearing. Therefore, we hypothesize that the formation of mature articular cartilage with a highly parallel collagen network and relatively low proteoglycan content in the peak-loaded area of a joint is needed to withstand intermittent stress and shear, whereas a constantly weight-bearing joint area benefits from lower collagen parallelism and a higher proteoglycan content.
- [Show abstract] [Hide abstract] ABSTRACT: We investigated the effects of exercise-induced loading on the collagen network of equine articular cartilage. Collagen fibril architecture at a site (1) subjected to intermittent high-intensity loading was compared with that of an adjacent site (2) sustaining continuous low-level load. From horses exposed to forced exercise (CONDEX group) or not (PASTEX group), the spatial parallelism of fibrils and the orientation angle between fibrils and the surface at depths 9 microm apart through cartilage from surface to tidemark were determined using polarized light microscopy, and expressed as parallelism index (PI) and orientation index (OI). PI was significantly higher in site 2 than 1 in CONDEX and PASTEX groups. PI was significantly higher in forced exercised horses at site 2 but not site 1. OI was significantly greater (more perpendicular to the surface) in the superficial and deep cartilage of site 2 than 1 in both CONDEX and PASTEX groups. Superficial zone OI was higher in exercised horses at site 1 but not at site 2. Exercise increased collagen parallelism and affected orientation. The site differences in OI indicate that Benninghoff's classic predominantly perpendicular arcades appear not to be a consistent architectural feature, but adapt to local forces sustained.
- [Show abstract] [Hide abstract] ABSTRACT: There is ample evidence on topographical heterogeneity of the principal biochemical components of articular cartilage over the surface of the joint and the influence of loading thereon, but no information on depth-related zonal variation in horses. To study depth-related zonal variation in proteoglycan (PG) and collagen content in equine articular cartilage. Two techniques (safranin-O densitometry and Fourier transform infrared spectroscopy) were applied to sections of articular cartilage from the proximal phalangeal bone of the metacarpophalangeal joint of 18-month-old Thoroughbreds that had been raised at pasture from age 0-18 months without (PASTEX) and with (CONDEX) additional exercise. Two sites were investigated: site 1 at the joint margin that is unloaded at rest or at slow gaits, but subjected to high-intensity loading during athletic activity; and site 2, a continuously, but less intensively, loaded site in the centre of the joint. Proteoglycan values increased from the surface to the deep layers of the cartilage, collagen content showed a reverse pattern. PG content was significantly higher at site 2 in both PASTEX and CONDEX animals without an effect of exercise. In the PASTEX animals collagen content was significantly higher at site 1, but in the CONDEX group the situation was reversed, due to a significant exercise effect on site 1, leading to a reduced collagen content. Collagen and PG content gradients agree with findings in other species. The observations on PG levels suggest that the exercise level was not strenuous. The collagen results in the PASTEX group confirmed earlier findings, the lower levels at site 1 in the CONDEX group being possibly due to an advancement of the physiological maturation process of collagen remodelling. This study confirms earlier observations that even moderate variations in exercise level in early age may have significant effects on the collagen network of articular cartilage.
- [Show abstract] [Hide abstract] ABSTRACT: Subchondral bone provides structural support to overlying articular cartilage and plays an important biomechanical role in osteochondral diseases. Mechanical features of bone correlate strongly with bone mineral density, which is directed by the loading conditions to which the tissue is subjected. To investigate the influence of physical activity levels on subchondral bone mineral density (sBMD) in foals during early development. Three groups of foals were subjected to different physical activity levels from birth until age 5 months. A proportion of these foals were subjected to euthanasia at 5 months while remaining foals were subjected to similar physical activity levels for 6 months until euthanasia at 11 months. Osteochondral specimens were collected for measurement of sBMD with peripheral quantitative computed tomography at 2 differently loaded anatomical sites of the proximal phalangeal bone at 1, 2, 3, 4 and 5 mm depth from the osteochondral junction. Growth significantly increased sBMD but by a different amount depending on anatomical location and physical activity level. Significantly higher sBMD was found at the habitually loaded central area in comparison to the intermittently peak loaded marginal site. Exercise increased sBMD throughout the whole depth of analysed tissue, but changes were generally more obvious at a depth of 2 mm. Interestingly, foals subjected to additional sprint training preserved the exercise-induced sBMD increase at the habitually loaded central area during the 6 months of the second phase of the study. Habitual low-intensity loading elicits a greater response in sBMD in quantitative terms than high-intensity low-frequency loading at the sites investigated in this study. Future sBMD may be influenced by means of well-tailored exercise regimens at young age. Specific physical activity levels during early development may potentially reduce the prevalence of osteochondral injury later in life.
- [Show abstract] [Hide abstract] ABSTRACT: The present study was designed to reveal changes in the collagen network architecture and collagen content in cartilage during growth and maturation of pigs. 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. 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. 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.
- [Show abstract] [Hide abstract] ABSTRACT: The effects of growth and maturation on the mineral deposition and the collagen framework of equine subchondral bone (SCB) were studied. Osteochondral specimens (diameter 6 mm) from the left metacarpophalangeal joint of 5-(n=8), 11-(n=8) and 18-month-old (n=6) horses were investigated at two differently loaded sites (Site 1 (S1): intermittent peak loading; Site 2 (S2): habitual loading). The SCB mineral density (BMD) was measured with peripheral quantitative computer tomography (pQCT), and the data were adjusted against the volume fraction (Vv) of the bone extracellular matrix (ECM). Polarised light microscopy (PLM) was used to analyze the Vv, the collagen fibril parallelism index and the orientation angle distribution in two fractions (1 mm/fraction) beneath the osteochondral junction of the SCB. PLM analysis was made along two randomly selected perpendicularly oriented vertical sections to measure the tissue anisotropy in the x-, y-, and z-directions. The BMD of SCB at S1 and S2 increased significantly during maturation. At the same time, the Vv of the ECM increased even more. This meant that the Vv-adjusted BMD decreased. There were no significant differences between sites. The basic collagen fibril framework of SCB seems to be established already at the age of 5 months. During maturation, the extracellular matrix underwent a decrease in collagen fibril parallelism but no changes in collagen orientation. The variation was negligible in the collagen network estimates in the two section planes. Growth and maturation induce significant changes in the equine SCB. The BMD increase in SCB is primarily due to the growth of bone volume and not to any increase in mineral deposition. An increase in weight-bearing appears to greatly affect the BMD and the volume of the extracellular matrix. Growth and maturation induce a striking change in collagen fibril parallelism but not in fibril orientation. The structural anisotropy of the subchondral bone is significant along the vertical (x-y) direction but not in the transversal (z) direction.
- [Show abstract] [Hide abstract] ABSTRACT: Polarized light microscopy is a traditional method for visualizing the collagen network architecture of articular cartilage. Articular cartilage repair and tissue engineering studies have raised new demands for techniques capable of quantitative characterization of the scar and repair tissues, including properties of the collagen network. Modern polarized light microscopy can be used to measure collagen fibril orientation, parallelism, and birefringence. New commercial instruments are computer controlled and the measurements are easy to perform. However, often the interpretation of results causes difficulties, even errors, because the theoretical aspects of the technique are demanding. The aim of this study was to describe the instrumentation and properties of a modern polarized light microscope, to point out some sources of error in the interpretation of the results, and to recall the theoretical background of the polarized light microscopy.
- [Show abstract] [Hide abstract] ABSTRACT: Training at a very young age may influence the characteristics of the collagen network of articular cartilage extracellular matrix (ECM) in horses. To investigate whether increasing workload of foals results in significant changes in the biochemical composition of articular cartilage ECM. Thoroughbred foals (n = 33) were divided into 2 different exercise groups from age 10 days-18 months. One group (PASTEX; n = 15) was reared at pasture; the other (CONDEX; n = 18) underwent a specific additional training programme that increased workload by 30%. At mean age 18 months, 6 animals from each group were subjected to euthanasia. The proximal articular surface of the proximal phalanx of the right hindlimb was examined for the presence of damage using the cartilage degeneration index (CDI). Samples were taken from 2 sites with known different loading patterns. Slices were analysed for DNA, glycosaminoglycans (GAG), collagen and post translational modifications of collagen (formation of hydroxylysylpyridinoline [HP] and pentosidine crosslinks, and hydroxylysine [Hyl]), and exercise groups and different sites compared. There were no differences in CDI between PASTEX and CONDEX animals, indicating the absence of extra joint damage due to the exercise regimen. There were site-related differences for most biochemical variables, corroborating earlier reports. All biochemical variables showed differences between PASTEX and CONDEX groups at one of the sites, and some at both. GAG and collagen levels were lower in the CONDEX group whereas Hyl, HP crosslinks and pentosidine crosslinks were higher. A measurable effect of the conditioning exercise was demonstrated. The margin between too much and too little work when training foals may be narrower than intuitively presumed.
- [Show abstract] [Hide abstract] ABSTRACT: To determine the interrelations between degenerative changes in articular cartilage and underlying trabecular bone during development of osteoarthritis and to test the ability of quantitative magnetic resonance imaging (MRI) to detect those changes. Human cadaver patellae were investigated with quantitative MRI methods, T(2) and dGEMRIC, at 1.5T. Same measurements for isolated cartilage samples were performed at 9.4T. Bone samples, taken at sites matched with cartilage analyses, were measured with MRI and peripheral quantitative computed tomography (pQCT). Mechanical and quantitative microscopic methods were also utilized for both cartilage and bone samples. Significant differences were found between the samples with different stages of degeneration in mechanical properties, T(2) at 1.5T and proteoglycan (PG) content of articular cartilage. dGEMRIC at 9.4T discerned samples with advanced degeneration from the others. Bone variables measured with pQCT discerned samples with no or minimal and advanced degeneration, and mechanical properties of trabecular bone discerned samples with no or minimal degeneration from the others. Significant linear correlations were found between the bone and cartilage parameters. Characteristically, associations between variables were stronger within the samples with no or minimal degeneration compared to all samples. Quantitative MRI variables, especially T(2) relaxation time of articular cartilage, may be feasible surrogate markers for early and advanced osteoarthritic changes in joint tissues, including decreased elastic moduli, PG and collagen contents of cartilage and mineral density and volume fraction of trabecular bone. Further work is required to resolve the relaxation mechanisms at clinically applicable field strengths.
- [Show abstract] [Hide abstract] ABSTRACT: Articular cartilage injuries cause a major clinical problem because of the negligible repair capacity of cartilage. Autologous chondrocyte transplantation is a surgical method developed to repair cartilage lesions. In the operation, cartilage defect is covered with a periosteal patch and the suspension of cultured autologous chondrocytes is injected into the lesion site. The method can form good repair tissue, but new techniques are needed to make the operation easier and to increase the postoperative biomechanical properties of the repair tissue. In this study, we investigated poly-L,D-lactic acid (PLDLA) scaffolds alone or seeded with autologous chondrocytes in the repair of circular 6-mm cartilage lesions in immature porcine knee joints. Spontaneous repair was used as a reference. Histologic evaluation of the repair tissue showed that spontaneous repair exhibited higher scores than either PLDLA scaffold group (with or without seeded chondrocytes). The scaffold material was most often seen embedded in the subchondral bone underneath the defect area, probably because of the hardness of the PLDLA material. However, some of the cell-seeded and nonseeded scaffolds contained cartilaginous tissue, suggesting that invasion of mesenchymal cells inside nonseeded scaffolds had occurred. Hyaluronan deposited in the scaffold had possibly acted as a chemoattractant for the cell recruitment. In conclusion, the PLDLA scaffold material used in this study was obviously mechanically too hard to be used for cartilage repair in immature animals.
- [Show abstract] [Hide abstract] ABSTRACT: Osteoarthritis (OA) is characterised by progressive erosion of articular cartilage with a number of associated degenerative processes within the joint. Animal models of OA provide the only feasible way to systematically study the development and progression of OA, in order to understand the molecular events, and to develop tools for prevention and therapy of OA. Gene manipulation techniques have provided opportunities to generate transgenic mouse models for OA. In heterozygous Dell mice, incorporation of Col2a1 transgenes with a short deletion mutation results in production of shortened proalpha1 (II) collagen chains and a phenotype resembling human OA. This chapter describes techniques and practical aspects of preparation and processing of skeletal samples for radiological, histological, and molecular biologic analyses that have been used to monitor the development of knee OA in Dell mice. A simple histological grading system to evaluate the progression of OA lesions, and examples of other degenerative alterations in the knee joint structures are presented. Semiquantitative microscopic techniques are described for the analysis of proteoglycan distribution based on safranin O staining of glycosaminoglycans, and for the analysis of collagen matrix based on birefringence of polarized light. Reference is also made to an experimental setup for correlating voluntary running activity of mice with OA score.
- [Show abstract] [Hide abstract] ABSTRACT: Chondroitin sulfate is the major constituent of cartilage. Inadequate sulfate availability results in the production of undersulfated proteoglycans. In osteoarthritis, there is a net loss of articular cartilage proteoglycans. Theoretically, it is possible that during the progress of disease undersulfated glycosaminoglycans are synthesized producing proteoglycans with poorer biological properties. In this study, we tested whether in early human osteoarthritic articular cartilage (Mankin's score of 2 and 3) or more advanced disease (Mankin's score over 3), there are proteoglycans that contain a higher relative amount of nonsulfated chondroitin disaccharide isomer in their chondroitin sulfate chains by analyzing the molar ratios of chondroitin sulfate disaccharide isoforms with fluorophore-assisted carbohydrate electrophoresis. Our results indicated that the nonsulfated disaccharide of chondroitin sulfate formed in average only 1-2% of the total chondroitin sulfate. More important, the molar ratio of nonsulfated disaccharide did not appear to be increased in the osteoarthritic articular cartilage. We conclude that undersulfation of articular cartilage proteoglycans is not present in the human osteoarthritic joint.
- [Show abstract] [Hide abstract] ABSTRACT: Osteoarthritis (OA) is characterised by progressive erosion of articular cartilage with a number of associated degenerative processes within the joint. Animal models of OA provide the only feasible way to systematically study the development and progression of OA, in order to understand the molecular events, and to develop tools for prevention and therapy of OA. Gene manipulation techniques have provided opportunities to generate transgenic mouse models for OA. In heterozygous Del1 mice, incorporation of Col2a1 transgenes with a short deletion mutation results in production of shortened proα1(II) collagen chains and a phenotype resembling human OA. This chapter describes techniques and practical aspects of preparation and processing of skeletal samples for radiological, histological, and molecular biologic analyses that have been used to monitor the development of knee OA in Del1 mice. A simple histological grading system to evaluate the progression of OA lesions, and examples of other degenerative alterations in the knee joint structures are presented. Semiquantitative microscopic techniques are described for the analysis of proteoglycan distribution based on safranin O staining of glycosaminoglycans, and for the analysis of collagen matrix based on birefringence of polarized light. Reference is also made to an experimental setup for correlating voluntary running activity of mice with OA score. Key WordsTransgenic mouse–osteoarthritis–cartilage–bone–type II collagen–proteoglycan–mRNA–safranin O–polarized light microscopy–joint loading–knee joint degeneration–animal model
- [Show abstract] [Hide abstract] ABSTRACT: The purpose of this study was to find out how deep chondral lesions heal in growing animals spontaneously and after autologous chondrocyte transplantation. A 6mm deep chondral lesion was created in the knee joints of 57 immature pigs and repaired with autologous chondrocyte transplantation covered with periosteum or muscle fascia, with periosteum only, or left untreated. After 3 and 12 months, the repair tissue was evaluated with International Cartilage Repair Society (ICRS) macroscopic grading, modified O'Driscoll histological scoring, and staining for collagen type II and hyaluronan, and with toluidine blue and safranin-O staining for glycosaminoglycans. The repair tissue structure was also examined with quantitative polarized light microscopy and indentation analysis of the cartilage stiffness. The ICRS grading indicated nearly normal repair tissue in 65% (10/17) after the autologous chondrocyte transplantation and 86% (7/8) after no repair at 3 months. At 1 year, the repair tissue was nearly normal in all cases in the spontaneous repair group and in 38% (3/8) in the chondrocyte transplantation group. In most cases, the cartilage repair tissue stained intensely for glycosaminoglycans and collagen type II indicating repair tissue with true constituents of articular cartilage. There was a statistical difference in the total histological scores at 3 months (P=0.028) with the best repair in the spontaneous repair group. A marked subchondral bone reaction, staining with toluidine blue and collagen type II, was seen in 65% of all animals. The spontaneous repair ability of full thickness cartilage defects of immature pigs is significant and periosteum or autologous chondrocytes do not bring any additional benefits to the repair.
University of Kuopio
Kuopio, Northern Savo, Finland
- Department of Anatomy