Simultaneous changes in the mechanical properties, quantitative collagen organization, and proteoglycan concentration of articular cartilage following canine meniscectomy.

The mechanical properties and microstructure of articular cartilage from the canine tibial plateau were studied 12 weeks after total medial meniscectomy. The organization of the birefringent collagen network was measured with quantitative polarized light microscopy to determine the thickness and the degree of organization of the superficial and deep zones. The zonal concentration of sulfated glycosaminoglycan was quantified with digital densitometry of safranin-O staining. Equilibrium compressive and shear properties, as well as dynamic shear properties, were measured at sites adjacent to those of microstructural analysis. The results evinced significant loss of cartilage function following meniscectomy, with decreases of 20-50% in the compressive and shear moduli. There was no evidence of alterations in the degree of collagen fibrillar organization, although a complete loss of the surface zone was seen in 60% of the samples that underwent meniscectomy. Meniscectomy resulted in a decreased concentration of sulfated glycosaminoglycan, and significant positive correlations were found between the equilibrium compressive modulus and the glycosaminoglycan content. Furthermore, the shear properties of cartilage correlated directly with collagen fibrillar organization measured at the superficial zone of corresponding sites. These findings demonstrate that meniscectomy leads to impaired mechanical function of articular cartilage, with significant evidence of quantitative correlations between cartilage microstructure and mechanics.     

Variation of collagen fiber alignment in a joint surface: a scanning electron microscope study of the tibial plateau in dog, rabbit, and man

To determine if articular cartilage collagen fiber organization differs with location on the tibial plateau, specimens from dogs, humans, and rabbits were studied by scanning electron microscopy. Joint surfaces were fixed, dehydrated, and fractured radially so that the periphery could be compared with the center on single specimens. Generally, fibers were more tightly packed in the lateral side than in the medial and the periphery as compared with the center, where the cartilage was consistently thicker and the radial zone was dominant and composed of straight vertical fibers. In the periphery, the tangential and transitional zones were better developed and contributed up to 50% of the cartilage depth in comparison to only 5% centrally. The soft, dull, malacic appearance of the center results from lack of a true surface layer of tangential collagen fibers.     

Prediction of collagen orientation in articular cartilage by a collagen remodeling algorithm

OBJECTIVE: Tissue engineering is a promising method to treat damaged cartilage. So far it has not been possible to create tissue-engineered cartilage with an appropriate structural organization. It is envisaged that cartilage tissue engineering will significantly benefit from knowledge of how the collagen fiber orientation is directed by mechanical conditions. The goal of the present study is to evaluate whether a collagen remodeling algorithm based on mechanical loading can be corroborated by the collagen orientation in healthy cartilage. METHODS: According to the remodeling algorithm, collagen fibrils align with a preferred fibril direction, situated between the positive principal strain directions. The remodeling algorithm was implemented in an axisymmetric finite element model of the knee joint. Loading as a result of typical daily activities was represented in three different phases: rest, standing and gait. RESULTS: In the center of the tibial plateau the collagen fibrils run perpendicular to the subchondral bone. Just below the articular surface they bend over to merge with the articular surface. Halfway between the center and the periphery, the collagen fibrils bend over earlier, resulting in a thicker superficial and transitional zones. Near the periphery fibrils in the deep zone run perpendicular to the articular surface and slowly bend over to angles of -45 degrees and +45 degrees with the articular surface. CONCLUSION: The collagen structure as predicted with the collagen remodeling algorithm corresponds very well with the collagen structure in healthy knee joints. This remodeling algorithm is therefore considered to be a valuable tool for developing loading protocols for tissue engineering of articular cartilage.     

Anatomic variation of depth‐dependent mechanical properties in neonatal bovine articular cartilage

Articular cartilage has well known depth‐dependent structure and has recently been shown to have similarly non‐uniform depth‐dependent mechanical properties. Here, we study anatomic variation of the depth‐dependent shear modulus and energy dissipation rate in neonatal bovine knees. The regions we specifically focus on are the patellofemoral groove, trochlea, femoral condyle, and tibial plateau. In every sample, we find a highly compliant region within the first 500 µm of tissue measured from the articular surface, where the local shear modulus is reduced by up to two orders of magnitude. Comparing measurements taken from different anatomic sites, we find statistically significant differences localized within the first 50 µm. Histological images reveal these anatomic variations are associated with differences in collagen density and fiber organization. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 686–691, 2013     

Three-dimensional collagen architecture in bovine articular cartilage

The three-dimensional architecture of bovine articular cartilage collagen and its relationship to split lines has been studied with scanning electron microscopy. In the middle and superficial zones, collagen was organised in a layered or leaf-like manner. The orientation was vertical in the intermediate zone, curving to become horizontal and parallel to the articular surface in the superficial zone. Each leaf consisted of a fine network of collagen fibrils. Adjacent leaves merged or were closely linked by bridging fibrils and were arranged according to the split-line pattern. The surface layer (lamina splendens) was morphologically distinct. Although ordered, the overall collagen structure was different in each plane (anisotropic) a property described in previous morphological and biophysical studies. As all components of the articular cartilage matrix interact closely, the three-dimensional organisation of collagen is important when considering cartilage function and the processes of cartilage growth, injury and repair.     

Nonlinear optical microscopy of articular cartilage

OBJECTIVE: To assess the ability of nonlinear optical microscopy (NLOM) to image ex vivo healthy and degenerative bovine articular cartilage. METHOD: Fresh bovine femoral-tibial joints were obtained from an abattoir. Articular cartilage specimens were harvested from the tibial plateau. Normal and degenerative specimens were imaged by NLOM and subsequently fixed and processed for histological examination. RESULTS: NLOM provided high resolution images of articular cartilage at varying depths with high sensitivity to tissue morphology and high specificity to tissue components without fixing, sectioning or staining. Spectroscopic segmentation of nonlinear optical signals isolated the collagen matrix from the chondron (chondrocyte and non-collagen pericellular matrix). Images from the superficial zone were consistent with the presence of a matrix composed of both elastin-like and collagen fibers distributed in a depth-dependent morphological arrangement, whereas only collagen was demonstrated in the middle and deep zones. Alterations of collagen matrix associated with advanced degenerative joint disease (fibrocartilage) were observed with NLOM. Individual chondrocytes were imaged and demonstrated intracellular fluorescence consistent with the presence of products of intracellular biochemical processes. CONCLUSION: Thin images of living articular cartilage using NLOM may be obtained with (sub-)cellular resolution at varying depths without fixing, sectioning or staining. Extracellular matrical collagen and chondron may be imaged separately in native tissue using spectrally distinct, endogenous, nonlinear optical signals. NLOM was sensitive to macromolecular composition and pathologic changes in articular cartilage matrix. Advances in instrumentation may lead to the application of NLOM to study articular cartilage in vivo.     

Topographical variations in the morphology and biochemistry of adult canine tibial plateau articular cartilage

Topographically, there are both morphological and biochemical differences in the articular cartilage of the tibial plateau of normal adult dogs when the cartilage covered by the meniscus is compared with that more centrally placed and not covered by meniscus. Histologically, differences are present in the surface morphology, in intra- and extracellular lipid content, and in the morphology of the mineralization front. Electron microscopy shows, in the covered cartilage, variability in collagen fiber size, with evenly spaced fibers apparently randomly distributed and an orderly relationship between the proteoglycans and collagen, whereas in the uncovered area, the collagen is aggregated into bundles and appears to be dissociated in large part from the proteoglycans. The most striking feature in the biochemistry of the two regions is an increased water content in the uncovered cartilage, as compared with the covered. In addition, there is an increased amount of proteoglycans that can be extracted in the uncovered cartilage. The heterogeneity of the cartilage on the tibial plateau should be taken into account when considering both the histologic and biochemical variations found in osteoarthritic cartilage; and when reflecting on the pathogenesis of osteoarthritis.     

Deformation of loaded articular cartilage prepared for scanning electron microscopy with rapid freezing and freeze-substitution fixation

To investigate the effect of joint loading on collagen fibers in articular cartilage, 45 knees of adult rabbits were examined by scaning electron microscopy. The knees were loaded at the patella with a simulated “quadriceps force” of 0.5-4 times body weight for 0.5 or 25 minutes, plunge-frozen, and fixed by freezesubstitution with aldehydes. Six knees were loaded for 3 hours and then fixed conventionally. Fixed tibial plateaus were examined and then freeze-fractured through the area of tibiofemoral contact, dried, coated, and examined by scanning electron microscopy to assess the overall deformation of the tibial articular surface and matrix collagen fibers. With tissue prepared by conventional fixation used as a standard, the quality of fixation was graded by light and transmission electron microscopy of patellar cartilage taken from half of the freeze-fixed knees. In loaded specimens, an indentation was present where the femur contacted the tibial plateau. The diameter and apparent depth of the dent were proportional to the magnitude and duration of the load; no dent was seen in the controls. The thickness of the cartilage at the center of the indentation was reduced 15-80%. Meniscectomy always produced larger deformations in otherwise equivalent conditions. Icecrystal damage to cells was evident by transmission electron microscopy and scanning electron microscopy, but at magnifications as high as ×30,000 the collagen fibrils prepared by freeze-substitution and conventional aqueous methods were identical. In loaded regions, the collagen matrix of the tibial cartilage was deformed in two ways: (a) radial collagen fibers exhibited a periodic crimp, and (b) in regions where an indentation was created by the femoral condyle, the radial fibers were bent, in effect creating tangential zone where none had existed before. The radial fibers apparently are loaded axially and buckle under normal loads.     

The effect of detachment of the articular cartilage from its calcified zone on the cartilage microstructure, assessed by 2H-spectroscopic double quantum filtered MRI.

Most studies on articular cartilage properties have been conducted after detachment of the cartilage from the bone. In the present work we investigated the effect of detachment on collagen fiber architecture. We used one-dimensional (2)H double quantum filtered MRI on cartilage bone plugs equilibrated in deuterated saline. The quadrupolar splittings observed in the different zones were related to the degree of order and the density of the collagen fibers. The method is non-destructive, allowing for measurements on the same plug without the need for fixation, dehydration, sectioning and decalcification. Detachment of the radial from the calcified zone resulted in swelling of the cartilage plug in physiological saline and a concomitant decrease in the quadrupolar splitting. The effect of mechanical pressure on the (2)H quadrupolar splittings for the detached cartilage and for the calcified zone-bone plugs were compared with those of the same zones in the intact cartilage-bone plug. The splitting in the radial zone of the detached cartilage collapsed at much smaller loads compared to the intact cartilage-bone plug. The effect of the load on the size of the cartilage was also greater for the detached plug. These results indicate that anchoring of the cartilage to the bone through the calcified zone plays an important role in retaining the order of the collagen fibers. The water (2)H quadrupolar splitting in intact and proteoglycan-depleted cartilage was the same, indicating that the proteoglycans do not contribute to the ordering of the collagen fibers.     

Remobilization does not fully restore immobilization induced articular cartilage atrophy.

The recovery of articular cartilage from immobilization induced atrophy was studied. The right hind limbs of 29-week-old beagle dogs were immobilized for 11 weeks and then remobilized for 50 weeks. Cartilage from the immobilized knee was compared with tissue from age matched control animals. After the immobilization period, uncalcified articular cartilage glycosaminoglycan concentration was reduced by 20% to 23%, the reduction being largest (44%) in the superficial zone. The collagen fibril network showed no significant changes, but the amount of collagen crosslinks was reduced (13.5%) during immobilization. After remobilization, glycosaminoglycan concentration was restored at most sites, except for in the upper parts of uncalcified cartilage in the medial femoral and tibial condyles (9% to 17% less glycosaminoglycans than in controls). The incorporation of 35SO4 was not changed, and remobilization also did not alter the birefringence of collagen fibrils. Remobilization restored the proportion of collagen crosslinks to the control level. The changes induced by joint unloading were reversible at most sites investigated, but full restoration of articular cartilage glycosaminoglycan concentration was not obtained in all sites, even after remobilization for 50 weeks. This suggests that lengthy immobilization of a joint can cause long lasting articular cartilage proteoglycan alterations at the same time as collagen organization remains largely unchanged. Because proteoglycans exert strong influence on the biomechanical properties of cartilage, lengthy immobilization may jeopardize the well being of articular cartilage.     

Collagen network primarily controls Poisson's ratio of bovine articular cartilage in compression.

The equilibrium Young's modulus of articular cartilage is known to be primarily determined by proteoglycans (PGs). However, the relation between the Poisson's ratio and the composition and structure of articular cartilage is more unclear. In this study, we determined Young's modulus and Poisson's ratio of bovine articular cartilage in unconfined compression. Subsequently, the same samples, taken from bovine knee (femoral, patellar and tibial cartilage) and shoulder (humeral cartilage) joints, were processed for quantitative microscopic analysis of PGs, collagen content, and collagen architecture. The Young's modulus, Poisson's ratio, PG content (estimated with optical density measurements), collagen content, and birefringence showed significant topographical variation (p < 0.05) among the test sites. Experimentally the Young's modulus was strongly determined by the tissue PG content (r = 0.86, p < 0.05). Poisson's ratio revealed a significant negative linear correlation (r = -0.59, p < 0.05) with the collagen content, as assessed by the Fourier transform infrared imaging. Finite element analyses, conducted using a fibril reinforced biphasic model, indicated that the mechanical properties of the collagen network strongly affected the Poisson's ratio. We conclude that Poisson's ratio of articular cartilage is primarily controlled by the content and organization of the collagen network.     

Implementation of subject‐specific collagen architecture of cartilage into a 2D computational model of a knee joint—data from the osteoarthritis initiative (OAI)

A subject‐specific collagen architecture of cartilage, obtained from T₂ mapping of 3.0 T magnetic resonance imaging (MRI; data from the Osteoarthritis Initiative), was implemented into a 2D finite element model of a knee joint with fibril‐reinforced poroviscoelastic cartilage properties. For comparison, we created two models with alternative collagen architectures, addressing the potential inaccuracies caused by the nonoptimal estimation of the collagen architecture from MRI. Also two models with constant depth‐dependent zone thicknesses obtained from literature were created. The mechanical behavior of the models were analyzed and compared under axial impact loading of 846N. Compared to the model with patient‐specific collagen architecture, the cartilage model without tangentially oriented collagen fibrils in the superficial zone showed up to 69% decrease in maximum principal stress and fibril strain and 35% and 13% increase in maximum principal strain and pore pressure, respectively, in the superficial layers of the cartilage. The model with increased thickness for the superficial and middle zones, as obtained from the literature, demonstrated at most 73% increase in stress, 143% increase in fibril strain, and 26% and 23% decrease in strain and pore pressure, respectively, in the intermediate cartilage. The present results demonstrate that the computational model of a knee joint with the collagen architecture of cartilage estimated from patient‐specific MRI or literature lead to different stress and strain distributions. The findings also suggest that minor errors in the analysis of collagen architecture from MRI, for example due to the analysis method or MRI resolution, can lead to alterations in knee joint stresses and strains. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:10–22, 2012     

Mechanical properties of articular cartilage covered by the meniscus

OBJECTIVE: To investigate the mechanical properties and morphological characteristics of articular cartilage on the tibial plateau of human knees, including the region covered by the meniscus. DESIGN: Using a 1-mm diameter flat-ended cylindrical probe to apply a constant load (0.6 MPa) at specific sites on the tibial plateau, the mechanical properties of articular cartilage were studied using seven cadaver knees. Comparison was made between data obtained by the cartilage covered by the meniscus and that not covered. This was done for both the medial and lateral plateaus. Histological sections of the articular cartilage were also performed to study differences between cartilage from these regions of the tibial plateau. RESULTS: Compared to cartilage that was not covered by the meniscus, the articular cartilage beneath the meniscus showed a significantly (P<0.05) larger modulus by as much as 70%, and was less thick by about 40%. Also, the subchondral bone quantity and calcified layer thickness were observed to be significantly lesser in the regions covered by the meniscus. CONCLUSIONS: Our findings revealed a significant difference between the mechanical properties and associated structures of articular cartilage in the region covered by the meniscus compared with the articular cartilage not covered by the meniscus.     

Induction of a neoarthrosis by precisely controlled motion in an experimental mid-femoral defect.

Bone regeneration during fracture healing has been demonstrated repeatedly, yet the regeneration of articular cartilage and joints has not yet been achieved. It has been recognized however that the mechanical environment during fracture healing can be correlated to the contributions of either the endochondral or intramembranous processes of bone formation, and to resultant tissue architecture. Using this information, the goal of this study was to test the hypothesis that induced motion can directly regulate osteogenic and chondrogenic tissue formation in a rat mid-femoral bone defect and thereby influence the anatomical result. Sixteen male Sprague Dawley rats (400 +/- 20 g) underwent production of a mid-diaphyseal, non-critical sized 3.0 mm segmental femoral defect with rigid external fixation using a custom designed four pin fixator. One group of eight animals represented the controls and underwent surgery and constant rigid fixation. In the treatment group the custom external fixator was used to introduce daily interfragmentary bending strain in the eight treatment animals (12 degree angular excursion), with a hypothetical symmetrical bending load centered within the gap. The eight animals in the treatment group received motion at 1.0 Hz, for 10 min a day, with a 3 days on, one day off loading protocol for the first two weeks, and 2 days on, one day off for the remaining three weeks. Data collection included histological and immunohistological identification of tissue types, and mean collagen fiber angles and angular conformity between individual fibers in superficial, intermediate, and deep zones within the cartilage. These parameters were compared between the treatment group, rat knee articular cartilage, and the control group as a structural outcome assessment. After 35 days the control animals demonstrated varying degrees of osseous union of the defect with some animals showing partial union. In every individual within the mechanical treatment group the defect completely failed to unite. Bony arcades developed in the experimental group, capping the termini of the bone segments on both sides of the defect in four out of six animals completing the study. These new structures were typically covered with cartilage, as identified by specific histological staining for Type II collagen and proteoglycans. The distribution of collagen within analogous superficial, intermediate, and deep zones of the newly formed cartilage tissue demonstrated preferred fiber angles consistent with those seen in articular cartilage. Although not resulting in complete joint development, these neoarthroses show that the induced motion selectively controlled the formation of cartilage and bone during fracture repair, and that it can be specifically directed. They further demonstrate that the spatial organization of molecular components within the newly formed tissue, at both microanatomical and gross levels, are influenced by their local mechanical environment, confirming previous theoretical models.     

Deformation of articular cartilage collagen structure under static and cyclic loading

Relatively little is known about the morphology of articuar cartilage under conditions of normal use. yet a more profound knowledge is both critical to the understanding of cartilage function and helpful for the validation of tissue-engineered cartilage. In this study, the deformation of the articular cartilage of the tibial plateau under compressive static and cyclic loading is characterized. Whole knee joints of rabbits were loaded ex vivo while the knee was held statically or allowed to move against resistance. Load magnitudes of quadriceps were maintained at either three (high) or one (low) times body weight for 30 minutes. For cyclic loading, the tibia was flexed between 70 and 150° relative to the femur at 1 Hz with either a cyclic or constant force. The recovery of cartilage after unloading was examined for each loading condition. At the end of the loading, specimens were cryofixed while under load, freeze-substituted, and prepared for scanning electron microscopy. Morphological examination demonstrated significantly higher deformation of the collagen structure throughout all cartilage zones under static loading conditions compared with cyclic loading condition's in which deformation was limited to the superficial regions. The minimum thickness of the cartilage that remained after loading was dependent on the magnitude of load and was significantly smaller with static loads (54% of the thickness of the unloaded controls) than after cyclic loading or constant-force cyclic loading (78 or 66% of the thickness of the unloaded controls, p < 0.05). Acute bending of the collagen fibers was observed under both loading conditions: in the superficial half of the articular cartilage after static loading and in the superficial quarter after cyclic loading. Complete recovery of all deformation occurred within 30 minutes but was significantly faster after cyclic loading. These data suggest that the structure of the collagen of articular cartilage exhibits a zone-specific deformation that is dependent on the magnitude and type of load.     

Zonal and topographical differences in articular cartilage gene expression.

Articular cartilage is composed of phenotypically different zones. In young articular cartilage, there are only two distinct zones: superficial and growth. The zones have different mechanical properties and play specific roles within functional cartilage tissue. In small animal models, it is difficult to separate the zones quickly and efficiently using only a dissecting microscope. Surface abrasion is a method that has been developed to harvest cells from articular cartilage to produce highly purified samples in a simple, reproducible process. Using this harvesting technique, the superficial zone has been separated from the underlying growth zone. Superficial cells comprised approximately 4% of the total cells obtained. Superficial and growth zone chondrocytes from articular cartilage were analyzed using real-time RT-PCR. Expressed superficial zone protein was 3-fold greater in the superficial zone population than in the growth zone population (p < 0.01). This, along with histological evidence, indicates that surface abrasion is successful as a zonal separation technique. Additionally, type II collagen was expressed 8-fold more abundantly in the growth zone than in the superficial zone (p < 0.005). There was no difference in aggrecan expression between the two zones. Regional variations among the femoral groove and medial and lateral condyles were also examined. No significant variations in SZP, type II collagen, or aggrecan were found, which makes the pooling of zonal cells from different regions an acceptable option for tissue engineering studies.     

Maturation-dependent change and regional variations in acoustic stiffness of rabbit articular cartilage: an examination of the superficial collagen-rich zone of cartilage

OBJECTIVE: The purpose of the study was to investigate maturation-dependent changes of acoustic (ultrasound) stiffness and other ultrasound features of articular cartilage in healthy rabbit knees. METHODS: Five groups of rabbits of various ages (3 weeks, 8 weeks, 6 months, 1 year, 2.5 years) consisting of five rabbits per group were examined. Signal intensity (index of stiffness), signal duration (index of surface irregularity) and interval between signals (index of thickness) of the ultrasound reflection from articular cartilage were examined at four sites: posterior lateral femoral condyle, posterior medial femoral condyle, lateral tibial plateau, and medial tibial plateau. The sites were observed macroscopically and microscopically with a light microscope and a polarized light microscope. RESULTS: At the lateral and medial condyles and the lateral tibial plateau, signal intensity was least in 3-week-old specimens. The intensity increased until 6 months or 1 year of age. At these sites, the signal durations and intervals between signals were least at the ages of 6 months or 1 year. At the medial tibial plateau, the intensity was the least at 2.5 years of age and the interval between signals was least at 3 weeks of age; there was no effect of age on signal duration. Cartilage surfaces of all specimens were smooth and no degenerative changes were macroscopically or microscopically evident. The surface brightness of cartilage under the polarized light microscope was consistent with signal intensity values. CONCLUSIONS: The response of articular cartilage to ultrasound was maturation-dependent. Acoustic properties differed from mechanical stiffness properties, which were determined using indentation. Ultrasound may detect properties of the surface collagen of the articular cartilage.     

The structural adaptations in compressed articular cartilage by microscopic MRI (microMRI) T(2) anisotropy

OBJECTIVES: To detect structural adaptations of collagen fiber matrix in compressed articular cartilage in individual sub-tissue zones by microscopic magnetic resonance imaging (microMRI) at 23 microm in-depth resolution. DESIGN: Each of the six beagle humeral cartilage specimens was placed in a specially built nonmetallic compression device inside an in-situ rotation device, and was imaged four times at 7T: without and during static compression, and at two orientations: 0 degrees and 55 degrees . Proton intensity images and quantitative T(2) maps were constructed and analyzed. RESULTS: Upon compression at 55 degrees (the magic angle), rather than appearing homogenous, T(2)-weighted intensity images of cartilage showed a distinct laminar appearance and the T(2) profiles exhibited two distinctive peaks. At both 0 degrees and 55 degrees orientations, lower values of T(2) were observed in compressed tissue. A significant correlation was established between changes in tissue T(2) at 55 degrees and a thickness reduction due to compression. At a mean of 20% strain value, modifications in cartilage structure were studied at each histological zone. We found that the percentage of superficial zone was significantly doubled, the percentage of radial zone was significantly decreased by 10%, and no significant change in the transitional zone. CONCLUSIONS: External loading can induce a new kind of laminar appearance at the magic angle. microMRI T(2) anisotropy can be used to analyze the zone-specific alterations in collagen fibril organization in articular cartilage in response to mechanical compression.     

Mechanical and biochemical changes in the superficial zone of articular cartilage in canine experimental osteoarthritis

The changes in the tensile mechanical properties and biochemical composition of the superficial zone of articular cartilage were examined in a canine model of early osteoarthritis generated by transection of the anterior cruciate ligament. Sixteen weeks following ligament transection, the tensile stiffness of the articular cartilage was decreased by 44% and the ion-induced stress relaxation of the tissue was increased by 57% compared with the contralateral control. Biochemical analyses indicated that the water content of the experimental tissue was increased by 13%, which was reflected as an apparent 37% decrease in the proteoglycan content and a 36% decrease in the collagen content (expressed per wet weight). The hydroxypyridinium crosslink density was decreased in the experimental tissue by 11%. A significant negative correlation was found between the ion-induced stress relaxation and the hydroxypyridinium crosslink density in both control tissue (R = ?0.56) and experimental tissue (R = ?0.70). No correlation was noted between the tensile stiffness and the biochemical composition of the tissue. These results suggest that, in the superficial zone of articular cartilage, the structure of the tissue may play a more important role than the composition in the determination of its mechanical properties. A major event observed in the model of early osteoarthritis appears to be the disruption and remodeling of the collagen network in the superficial zone of the articular cartilage.     

Site-specific immunostaining for type X collagen in noncalcified articular cartilage of canine stifle knee joint

Type X collagen is a short-chain collagen that is strongly expressed in hypertrophic chondrocytes. In this study, we used an immunohistochemical technique exploiting a prolonged hyaluronidase unmasking of type X collagen epitopes to show that type X collagen is not restricted to calcified cartilage, but is also present in normal canine noncalcified articular cartilage. A 30° valgus angulation procedure of the right tibia was performed in 15 dogs at the age of 3 months, whereas their nonoperated sister dogs served as controls. Samples were collected 7 and 18 months after the surgery and immunostained for type X collagen. The deposition of type X collagen increased during maturation from age 43 weeks to 91 weeks. In the patella, most of the noncalcified cartilage stained for type X collagen, whereas, in the patellar surface of the femur, it was present mainly in the femoral groove close to cartilage surface. In femoral condyles, the staining localized mostly in the superficial cartilage on the lateral and medial sides, but not in the central weight-bearing area. In tibial condyles, type X collagen was often observed close to the cartilage surface in medial parts of the condyles, although staining could also be seen in the deep zone of the cartilage. Staining for type X collagen appeared strongest at sites where the birefringence of polarized light was lowest, suggesting a colocalization of type X collagen with the collagen fibril arcades in the intermediate zone. No significant difference in type X collagen immunostaining was observed in lesion-free articular cartilage between controls and dogs that underwent a 30° valgus osteotomy. In osteoarthritic lesions, however, there was strong immunostaining for both type X collagen and collagenase-induced collagen cleavage products. The presence of type X collagen in the transitional zone of cartilage in the patella, femoropatellar groove, and in tibial cartilage uncovered by menisci suggests that it may involve a modification of collagen fibril arrangement at the site of collagen fibril arcades, perhaps providing additional support to the collagen network.