Search

Objective: To determine the feasibility of detecting the structural orientation in cartilage with Diffraction Enhanced X-Ray Imaging. Design: Human tali and femoral head specimens were Diffraction Enhanced X-Ray Imaged (DEI) at the SYRMEP beamline at Elettra at various energy levels to detect the architectural arrangement of collagen within cartilage. DEI utilizes a monochromatic and highly collimated beam, with an analyzer crystal that selectively weights out photons according to the angle they have been deviated with respect to the original direction. This provides images of very high contrast, and with the rejection of X-ray scatter. Results: DEI allowed the visualization of articular cartilage and a structural orientation, resembling arcades, within. Conclusion: Our diffraction enhanced images represent the first radiographic detection of the structural orientation in cartilage. Our data are in line with previous studies on the structural organization of joint cartilage. They confirm the model of a vaulting system of collagen fiber bundles interrupted by proteoglycan aggregates.

Objective Superficial articular chondrocytes display distinct spatial remodeling processes in response to the onset of distant osteoarthritis (OA). Such processes may be used to diagnose early events before manifest OA results in tissue destruction and clinical symptoms. Using a novel method of spatial quantification by calculating the angles between a chondrocyte and its surrounding neighbors, we compared maturational and degenerative changes of the cellular organizations in rat and human cartilage specimens. Methods The nuclei of superficial chondrocytes obtained from intact rat cartilage and from human knee cartilage, as well as from cartilage with focal and severe OA, were digitally recorded in top-down views. Their Cartesian coordinates were used to determine the nearest neighbor for each chondrocyte and the angle between these 2 cells and a reference. These angles, cellularity, nearest neighbor distances, and aggregation were analyzed as a function of location and OA severity. Results Neighboring rat chondrocytes exhibited intricate angular patterns with 4 dominant angles that were maintained during maturation and during the onset and progression of OA. Within intact cartilage, human chondrocytes demonstrated 1 dominant angle and, thus, a significantly different angular organization. With early OA onset, human chondrocytes that were located within intact cartilage displayed an increased occurrence of 4 angles; the resulting angular patterns were indistinguishable from those observed in rats. The angular remodeling was associated with location- and OA severity–dependent changes in cellularity and aggregation. Conclusion This study is the first to identify the presence of angular characteristics of spatial chondrocyte organization and species-specific remodeling processes correlating with OA onset. The appearance of distinct angular and spatial patterns between neighboring chondrocytes can identify the onset of distant OA prior to microscopically visible tissue damage and possibly before clinical onset. With further development, this novel concept may become suitable for the diagnosis and followup of patients susceptible to OA.

A better understanding of the unique cellular and functional properties of the superficial zone of articular cartilage may aid current strategies in tissue engineering which attempts a layered design for the repair of cartilage lesions to avert or postpone the onset of osteoarthritis. However, data pertaining to the cellular organization of non-degenerated superficial zone of articular cartilage is not available for most human joints. The present study analyzed the arrangement of chondrocytes of non-degenerated human joints (shoulder, elbow, knee, and ankle) by using fluorescence microscopy of the superficial zone in a top-down view. The resulting horizontal chondrocyte arrangements were tested for randomness, homogeneity or a significant grouping via point pattern analysis and were correlated with the joint type in which they occurred. The present study demonstrated that human superficial chondrocytes occurred in four distinct patterns of strings, clusters, pairs or single chondrocytes. Those patterns represented a significant grouping (p < 0.0001) with horizontal alignment. Each articular joint surface was dominated by only one of these four patterns (p < 0.001). Specific patterns correlated with specific diarthrodial joint types (p < 0.001). Further studies need to establish whether these organizational patterns are a consequence of their surrounding environment or whether they are linked to a functional purpose.

SummaryVariations among joints in the initiation and progression of degeneration may be explained, in part, by metabolic, biochemical and biomechanical differences. Compared to the cartilage in the knee joint, ankle cartilage has a higher content of proteoglycans and water, as well as an increased rate of proteoglycan turnover and synthesis, all of which are responsible for its increased stiffness and reduced permeability. Chondrocytes within ankle cartilage have a decreased response to catabolic factors such as interleukin-1 and fibronectin fragments, compared to the chondrocytes of knee cartilage. Moreover, in response to damage, ankle chondrocytes synthesize proteoglycans at a higher rate than that found in knee cartilage chondrocytes, which suggests a greater capacity for repair. In addition to the cartilages of the two joints, the underlying bones also respond differently to degenerative changes. Taken together, these metabolic, biochemical and biomechanical differences may provide protection to the ankle.

Objective To delineate the role of endogenous osteogenic protein 1 (OP-1) in human articular cartilage homeostasis via the inhibition of OP-1 gene expression by antisense oligonucleotides. Methods Human adult normal articular cartilage was obtained from the knee and ankle joints of 34 organ donors. Chondrocytes were cultured as tissue explants or isolated cells in alginate or high-density monolayers for 48 hours in the presence of OP-1 antisense or sense oligonucleotides. The effect of OP-1 antisense inhibition was evaluated by reverse transcription–polymerase chain reaction, 35S incorporation, dimethylmethylene blue assay, histology with Safranin O staining, and immunohistochemistry with anti–proOP-1, anti–mature OP-1, and anti-aggrecan antibodies. Results Antisense treatment inhibited OP-1 gene expression by a mean ± SD of 34 ± 12% (P < 0.01) in chondrocytes cultured in monolayers and by 77 ± 27% (P < 0.03) in alginate beads. The inhibition of autocrine OP-1 caused a striking decrease in aggrecan gene expression, in total proteoglycan content accumulated in cartilage matrix, and in the ability of chondrocytes to newly synthesize proteoglycans. OP-1 antisense reduced aggrecan messenger RNA expression by 42 ± 17% (P < 0.05) and proteoglycan synthesis by 48 ± 23% (P < 0.01). Histology and immunohistochemistry revealed a dramatic decrease in Safranin O staining and reduced anti-aggrecan staining (primarily in the superficial and middle cartilage layers) with OP-1 antisense treatment. Conclusion Our results suggest that OP-1 is an important endogenous cartilage factor that regulates matrix integrity and possibly needs to be induced or up-regulated to maintain normal cartilage homeostasis. These findings confirm our hypothesis that a lack of autocrine OP-1 may lead to an elevated susceptibility of chondrocytes to the catabolic processes, thus contributing/promoting cartilage degeneration.

Objective Although cartilage lesions occur in the ankles, osteoarthritis rarely develops in the ankles, suggesting that ankle cartilage can up-regulate mechanisms to repair the damaged matrix. To define these processes, we compared cartilage samples obtained from normal tali and from lesional sites of damaged tali. Methods Cartilage samples were obtained from the tali of normal ankles and from 3 sites on tali with lesions (the lesion, adjacent to the lesion, and far removed from the lesion). Cartilage was analyzed for type II collagen (CII) messenger RNA, C-terminal type II procollagen propeptide (CPII), the collagenase cleavage neoepitope (Col2-3/4Cshort), and the denaturation epitope (Col2-3/4m). For the assessment of type IX collagen, the COL2 and NC4 domains were evaluated. The cartilage samples were also assayed for glycosaminoglycans, epitope 846 of aggrecan, and DNA. Results The DNA content, epitope 846, COL2(IX), and the denaturation epitope were significantly increased in lesional cartilage. Although there was a tendency toward an increase in CII content and CPII, the increase did not reach significance. Neither the NC4(IX) domain nor Col2-3/4C was elevated. Surprisingly, changes in cartilage both adjacent to and remote from the lesion were similar to those in the lesion. Conclusion The changes observed in cartilage obtained from the lesion and from sites adjacent to the lesion were not surprising; however, the changes in cartilage obtained from sites remote from the lesion were unexpected. This up-regulation of matrix turnover in ankles with degenerative lesions may indicate a physiologic response of the entire articular surface to repair the damaged matrix, which is not restricted to the lesion site. This suggests that there may be some mechanism of communication across the cartilage. The response by ankle cartilage obtained from a site remote from the lesion has not been observed in the knee.

The purpose of this review is to summarize the current scientific knowledge of bone morphogenetic proteins (BMPs) in adult articular cartilage. We specifically focus on adult cartilage, since one of the major potential applications of the members of the BMP family may be a repair of adult tissue after trauma and/or disease. After reviewing cartilage physiology and BMPs, we analyze the data on the role of recombinant BMPs as anabolic agents in tissue formation and restoration in different in vitro and in vivo models following with the endogenous expression of BMPs and factors that regulate their expression. We also discuss recent transgenic modifications of BMP genes and subsequent effect on cartilage matrix synthesis. We found that the most studied BMPs in adult articular cartilage are BMP-7 and BMP-2 as well as transforming growth factor-beta (TGF-beta). There are a number of contradicting reports for some of these growth factors, since different models, animals, doses, time points, culture conditions and devices were used. However, regardless of the experimental conditions, only BMP-7 or osteogenic protein-1 (OP-1) exhibits the most convincing effects. It is the only BMP studied thus far in adult cartilage that demonstrates strong anabolic activity in vitro and in vivo with and without serum. OP-1 stimulates the synthesis of the majority of cartilage extracellular matrix proteins in adult articular chondrocytes derived from different species and of different age. OP-1 counteracts the degenerative effect of numerous catabolic mediators; it is also expressed in adult human, bovine, rabbit and goat articular cartilage. This review reveals the importance of the exploration of the BMPs in the cartilage field and highlights their significance for clinical applications in the treatment of cartilage-related diseases.

A synchronized balance between synthesis and breakdown of extracellular matrix (ECM) molecules in normal articular cartilage is disturbed in osteoarthritis (OA). The focus of our study is the anabolic factor, osteogenic protein-1 (OP-1) that is expressed in articular cartilage and is able to induce the synthesis of ECM components. The major aim was to investigate both qualitatively and quantitatively endogenous OP-1 in normal, degenerative, and OA cartilage. Normal and degenerative cartilage was obtained at autopsies from femoral condyles of human organ donors with no documented history of joint disease; OA cartilage was obtained from patients undergoing joint arthroplasty. Appearance of donor cartilage was evaluated by Collins scale, where normal cartilage is assigned grades 0–1, and degenerated cartilage is assigned grades 2–4. OP-1 mRNA expression was assessed by RT-PCR; OP-1 protein (pro- and active forms) was qualitatively analyzed by Western blotting and quantified by OP-1 ELISA. The highest levels of OP-1 expression (mRNA and protein) were detected in normal cartilage of grade 0. The concentration of OP-1 protein was about 50 ng per gram cartilage dry weight. With the progression of cartilage degeneration (increased Collins grades and OA) OP-1 protein was down-regulated up to 9-fold. These changes affected primarily the active form of OP-1. OP-1 message also declined in cartilages with the increase of degenerative changes. In conclusion, an overall decrease in endogenous OP-1 in degenerated and OA tissue suggests that OP-1 could be one of the factors responsible for normal homeostasis and matrix integrity in cartilage.

Degenerative changes in the tall and femoral distal cartilages of more than 2,000 tissue donors were graded based on the appearance of articular cartilage and osteophytes. In the ankle and the knee the degenerative changes increased with age; however, the rate of degeneration in the ankle was slower than in the knee. The degenerative changes in the ankle were more severe in men than in women, were predominantly bilateral, and seemed to be correlated with weight. The slower rate of change in the ankle may be caused, in part, by the biochemical and biomechanical tissue properties that distinguish ankle cartilage from that of the knee.

Articular cartilage of synovial joints is not visible with conventional X-ray imaging. Hence, the gradual degeneration and destruction of articular cartilage, which is characteristic of degenerative joint diseases, is only detected at a late stage when the cartilage is lost and the joint space that it once occupied narrows. The development of an X-ray imaging technique that could detect both the degenerative cartilage and bone features of joint diseases is of special interest. Here we show, for the first time, that a high-contrast imaging technique, diffraction-enhanced X-ray imaging (DEI), allows the visualization of articular cartilage of both disarticulated and articulated rabbit knee joints. Furthermore, a single cartilage lesion can be visualized within an intact joint. The results suggest that DEI has the potential to be of use in the study of cartilage degeneration.

Articular cartilage has a poor reparative capacity. This feature is exacerbated with aging and during degenerative joint conditions, contributing to loss of motion and impairment of quality of life. This study focused on osteogenic protein-1 (OP-1) and its ability to serve as a repair-stimulating factor in articular cartilage. The purpose of this work was to develop a quantitative method for the assessment of the content of OP-1 protein in extracts from human articular cartilage and to investigate the changes in OP-1 mRNA expression and protein levels with aging of normal adult cartilage. Full thickness cartilage was dissected from femoral condyles of donors with no history of joint disease within 24 h of death. Levels of OP-1 mRNA expression were measured by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method; concentration of OP-1 protein was detected by new sandwich enzyme-linked immunosorbent assay (ELISA); qualitative changes in OP-1 forms were evaluated by Western blots with various anti-OP-1 antibodies. The sensitivity of the ELISA method allowed the detection of picogram quantities of OP-1 in cartilage extracts. We found that (1) concentration of OP-1 in normal cartilage is within the range of biological activity of OP-1 in vitro; and (2) during aging of human adult, articular cartilage, levels of OP-1 protein and message are dramatically reduced (more than 4-fold; p

The prevalence of osteoarthritis (OA) is lower in some joints, i.e., the ankle, than in the knee. We have compared the cartilages from these two joints of the same limb in adult donors (matched pairs). Our data to date suggest that there are metabolic, biochemical and biomechanical differences between the cartilages of the two joints. The current study has focused on extending the metabolic studies comparing the response of chondrocytes to Interleukin-1beta (IL-1beta) and osteogenic protein 1 (OP-1) by analyzing changes in sulfate incorporation into glycosaminoglycans (GAGs) as a measure of proteoglycan (PG) synthesis. Human adult chondrocytes from normal knees (tibiofemoral) and ankles (talocrural) joints cultured as explants both responded to IL-1beta after 72 h by decreasing PG synthesis; however, the IC50 for the knee chondrocytes was 6.2 pg/ml, while that for the ankle was 35 pg/ml. When the explants were incubated for 72 h with IL-1beta and allowed to rebound without IL-1beta, synthesis of PG was significantly elevated by ankle chondrocytes within five days; knee chondrocytes were unable to significantly increase synthesis even after eight days. However, in both knee and ankle, application of OP-I enhanced PG synthesis in the rebound phase. In response to IL-1, an upregulation of proteinase activity was detectable by an increase in the neoepitopes proteolytically-generated by both aggrecanase and matrix metalloproteinases (MMPs), in the deep zone of the knee cartilage. Stromelysin and collagenase were upregulated as well. The data emerging from these studies confirm that the ankle is less responsive to catabolic stimulation and more responsive to anabolic stimulation following IL-1 removal. These differences in metabolic activity between the cartilages of the two joints could in part help to explain their differences in susceptibility to OA.

Objective The purpose of this work was to test the effect of inhibition of bone remodeling, through the use of the bisphosphonate, zoledronic acid, on cartilage matrix damage in an animal model of cartilage matrix damage. Design New Zealand white rabbits were divided into four groups for treatment purposes: (1) untreated controls; (2) injected into one knee joint with the cartilage matrix degradation enzyme, chymopapain; (3) injected into one knee joint with chymopapain and also given subcutaneous injections of the bisphosphonate, zoledronic acid, three times per week until sacrifice at either day 28 or 56 post-chymopapain-injection; (4) received only the zoledronic acid injections. At sacrifice, the knee joints were examined grossly and histologically, and biochemically for proteoglycan content. Urine samples were analysed, at intervals, for levels of collagen cross-links which are biochemical markers of cartilage and bone. Results Animals receiving both intraarticular chymopapain injections and subcutaneous zoledronic acid injections displayed a significantly lower degree of grossly and histologically detectable cartilage degeneration on the tibial articular surfaces (the articular surface displaying the greatest degree of degeneration) than did animals only receiving the chymopapain injections. In addition, urinary levels of collagen cross-links for bone and cartilage were significantly higher in those animals only receiving chymopapain injections. Conclusion The bone resorption observed after chymopapain injection into the rabbit knee joint can be inhibited through the use of the bisphosphonate, zoledronic acid. Furthermore, zoledronic acid does not increase the level of cartilage degeneration and appears to provide some level of chondroprotection in this model.

Osteoarthritis (OA) is a common, disabling condition of synovial joints that can eventually lead to reduced, or lost, mobility. It is characterized by both articular cartilage degeneration and subchondral bone changes. However, a cause-and-effect relationship between the two tissues remains controversial. Increased subchondral bone density has been associated with early degenerative changes in the cartilage of knee, hip, and finger joints-joints in which progressive changes to OA are common. In contrast, the ankle joint is known to exhibit early cartilage changes, but is not prone to the development of OA. In the present study, it was found that cartilage degeneration on the talus is not associated with an increase in bone density, as assessed through peripheral quantitative computed tomography (pQCT).

Articular hyaline cartilage takes on the contours of the subchondral bone on which it lies, but its thickness varies between joints, within a single joint and within a single articular surface. Previous studies have correlated articular cartilage thickness distribution with the degree of stress and weight bearing on joint surfaces, but few studies have considered the thickness of the calcified cartilage in relation to these parameters. Here we report a correlation between the cartilage thickness distribution and weight bearing distribution on the head of the 1st metatarsal bone, a component of one of the major weight bearing joints in the lower extremity during the gait cycle. The greatest total and uncalcified articular cartilage thickness was found on the central and lateral distal aspects of the metatarsal head, a region that receives maximal ground reactive force during the propulsive phase of the normal gait cycle. Although the thickness of the calcified cartilage was correlated with the thickness of the uncalcified cartilage, it varied to a lesser extent across the articular surface than did that of the uncalcified cartilage.

Cartilage was obtained from eight matched knee (tibiofemoral and femoropatellar) and ankle (talocrural) joints of five different donors (both left and right from donors 14, 22, and 38 years of age, and left only from donors 31 and 45 years of age) within 24 hours of death. All cartilage was graded as normal by the macroscopic visual Collins' scale and the histological Mankin scale. Cylindrical disks of cartilage were harvested from 10 sites within the tibiofemoral and femoropatellar joint surfaces and four sites within the talocrural joint, and uniaxial confined compression measurements were performed to quantify a spectrum of physical properties including the equilibrium modulus, hydraulic permeability, dynamic stiffness, streaming potential, electrokinetic coupling coefficient, and electrical conductivity. Matched specimens from the same 14 sites were used for complementary measurements of biochemical composition and molecular interaction, including water content, hypotonic swelling behavior, and sulfated glycosaminoglycan and collagen contents. In comparison of the top 1-mm slices of talar cartilage with the top 1-mm of tibiofemoral cartilage, the talar cartilage appeared denser with a higher sulfated glycosaminoglycan content, lower water content, higher equilibrium modulus and dynamic stiffness, and lower hydraulic permeability. The equilibrium modulus increased with increasing sulfated glycosaminoglycans per wet weight and decreased with increasing water content for all joint surfaces. Multiple linear regression showed that greater than 80% of the variation in the equilibrium modulus could be accounted for by variations in the biochemical parameters (water content, sulfated glycosaminoglycans/wet weight, and hydroxyproline content/wet weight) for each joint surface. Nonhomogeneous depth-dependent changes in the physical properties and biochemical composition of full-thickness distal femoral cartilage were consistent with previous reports. Since the compressive deformation of cartilage during cyclic loading is confined to the more superficial regions, the differences in properties of the upper regions of the talar compared with tibiofemoral or femoropatellar cartilage may be important in the etiology of osteoarthritis.

Objective We have used recombinant osteogenic protein-1 to investigate our hypothesis that proper repair and maintenance of cartilage requires not only enhanced biosynthesis and replenishment of the extracellular matrix but also the enhancement of components necessary for matrix retention. Design The effects of osteogenic protein-1 were examined on bovine articular cartilage slices as well as isolated chondrocytes grown in alginate beads. Cartilage slices were examined for accumulation of proteoglycan by incorporation of 35 S-sulfate and staining using Safranin O or, a biotinylated probe specific for hyaluronan. Bovine chondrocytes were characterized by use of a particle exclusion assay, in-situ hybridization, quantitative-competitive RT-PCR and a hyaluronan-binding assay. Results Osteogenic protein-1 treatment substantially enhanced the accumulation of hyaluronan and proteoglycan within cartilage tissue slices. As with the tissue, osteogenic protein-1 enhanced the size of cell-associated matrices assembled and retained by chondrocytes in vitro . This enhanced matrix assembly was paralleled by an increased expression of mRNA for aggrecan, hyaluronan synthase-2 and CD44. Of the two hyaluronan synthase genes expressed by chondrocytes, only hyaluronan synthase-2 was upregulated by osteogenic protein-1. Coupled with the increase in the CD44 mRNA was an increase in functional hyaluronan binding activity present at the chondrocyte cell surface. Conclusions These results demonstrate that osteogenic protein-1 stimulates not only the synthesis of the major cartilage extracellular matrix component aggrecan, but also two associated molecules necessary for the retention of aggrecan, namely hyaluronan and CD44.

Successful and cartilage-specific cultivation of chondrocytes requires a stable phenotype during the in vitro culture period. This is based on a differentiated extracellular matrix synthesis. The alginate system as a three-dimensional support is a useful system to culture chondrocytes and to analyze the biochemical processes in this system. Talar cartilage from the talocrural joints of 40 different donors were obtained through the Regional Organ Bank of Illinois within 24 hours of death. In 65% of the tissues the cartilage was classified as being undamaged. In these studies we were interested in the results of short-term culture over 14 days. Cell proliferation, total collagen content and total proteoglycan content were measured in the different matrix compartments and were visualized by histology and immunohistochemistry. Already after 7 days in culture the adult human chondroctes looked intact and formed a stable and cell-associated cartilage-specific extracellular matrix in the presence of 10% calf serum. This could be also demonstrated in the presence of IGF-I. With regards to the collagen content IGF-I at a concentration of 50 ng/ml seemed to induce an equal effect to 10% serum; with regards to the proteoglycan content IGF-I at a concentration of 20 ng/ml was equivalent. These encouraging preliminary results may lead to a new approach in tissue engineering for chondrocyte transplantation in combination with their extracellular matrix.

The prevalence of osteoarthritis (OA) is higher in some joints than in others. Fibrillation and full-thickness cartilage defects in the knee have been considered to be evidence of developing OA (pre-OA). While similar changes have been reported in the ankle (talocrural joint), the frequency of these changes is much higher than expected if the degeneration represents pre-OA. These observations suggest that in the ankle degenerative changes do not proceed to OA. The current study was to determine the prevalence of articular cartilage degeneration in ankles in a population of 470 bone donors with no history of joint disease. Knees from 50 donors were also available. Our data suggest that degeneration in the ankle cartilage does not appear to be a normal part of aging, was more frequent in men than women, increased with age, and occurred most often in both limbs with the same severity. In those donors with degeneration in the ankle, the knee also showed degenerative changes with an equal or higher grade. These data suggest that factors (such as altered mechanics) responsible for degeneration in one limb also cause changes in the contralateral limb and that factors affecting the ankle joints also appear to influence the knee joints.

Objective and Design: The abilities of osteogenic protein-1 (OP-1) and TGF-β1 to affect cartilage damage caused by fibronectin fragments (Fn-fs) that are known to greatly enhance cartilage proteoglycan (PG) degradation were compared.¶Material: Articular cartilage was obtained from 18 month old bovines.¶Treatment: To test blocking of damage, cartilage was cultured with or without OP-1 or TGF-β in the presence of 100 nM Fn-fs. To test restoration of PG, cartilage was first cultured with Fn-fs and the cartilage then treated with factors.¶Methods: Cartilage PG content was measured in papain digests using the dimethylmethylene blue assay. PG synthesis was measured by incorporation of 35S labeled sulfate.¶Results: OP-1 blocked damage and restored PG in damaged cartilage, apparently due to enhanced PG synthesis. However, TGF-β1 alone decreased PG content.¶Conclusions: These results clearly demonstrate differences between OP-1 and TGF-β1, both members of the TGF-β superfamily and illustrate the efficacy of OP-1 in blocking Fn-f mediated damage.

We have previously described a large proteoglycan named superficial zone protein that was isolated and purified from culture medium of superficial slices of bovine articular cartilage. Monoclonal antibodies were raised against superficial zone protein and used as probes in Western blot analyses for immunohistochemical studies both to determine precisely which cells within the joint synthesize the proteoglycan and to isolate a cDNA fragment from a bovine chondrocyte lambdagt11 library that encodes part of the proteoglycan. The cDNA fragment that was obtained with use of monoclonal antibody 6-A-1 encodes the 3' end of the sequence for superficial zone protein. On Western blots, monoclonal antibody 3-A-4 recognized an epitope on native, but not reduced, superficial zone protein, whereas monoclonal antibody 6-A-1 reacted with both native and denatured antigen. The proteoglycan was immunolocalized with monoclonal antibody 3-A-4 in chondrocytes predominantly within the superficial zone of fetal and adult articular cartilage and in some cells of the synovial lining. However, the proteoglycan was not detected in chondrocytes deep in articular cartilage, in nasal septal cartilage, or in synovial stromal cells. The only matrix staining positively for superficial zone protein was at the articular surface bordering the synovial cavity in adult, but not fetal, joints. Isolated chondrocytes and synovial cells showed intracellular binding of monoclonal antibody 3-A-4, and flow-cytometric analysis with the antibody gave the following percentages of immunopositive cells: 37.4, 52.5, 3.4, and 7.5 from chondrocytes from the full-thickness, superficial, and deep zones and from synovial cells, respectively. Thus, both chondrocytes and synovial cells bordering the joint cavity synthesize superficial zone protein and substantiate its usefulness as a phenotypic marker of particular cellular species lining the articular cavity.

According to numerous cadaveric, radiographic, and clinical studies, ankle and knee joints differ in susceptibility to osteoarthritis. To test for biochemical differences in susceptibility to damage, a chondrocytic chondrolysis system has been utilized. In this system, fibronectin fragments are added to cultured cartilage explants, resulting in enhanced release of catabolic cytokines, induction of matrix metalloproteinases, temporary suppression of proteoglycan synthesis, and consequently, severe loss of cartilage proteoglycan. We found that the addition of an amino-terminal thrombin-generated 29-kDa fibronectin fragment to cultured knee cartilage from 14 donors (average age: 53 years) usually caused a 30-50% decrease in proteoglycan content by day 7. However, of the ankle cartilage specimens examined from 21 donors (average age: 50 years), only three showed damage by day 7, one by day 14, and six by day 21, and 11 were not damaged until day 28. For eight of the donors (average age: 44 years), both knee and ankle cartilages were obtained: this allowed comparison between tissues from the same donor. The analysis showed that the ankle cartilage was much more refractory to damage than was the knee cartilage from the same donor. These data clearly show differences between ankle and knee cartilage in susceptibility to the fibronectin fragments and suggest the feasibility of use of these fragments for discerning differences in homeostasis of the ankle and knee cartilage.

Although there is sparse information concerning the properties of foot-joint cartilages, knowledge of the morphology and biochemistry of these cartilages is important in the study of changes that occur in the development of osteoarthritis. Normal first and fifth metatarsophalangeal joints were chosen for comparison because of the difference between these two joints in the prevalence of osteoarthritis, particularly with advancing age. The authors' study shows that there is no age-related decrease in articular-cartilage thickness; however, there is an age-related decrease in the chondrocyte density in the superficial zone in both joints. There is, however, a difference between the two joints in the level of expression of matrix-degrading enzymes. This difference may indicate differences in specific chondrocyte activity that precedes or accompanies the development of osteoarthritis or other degenerative morphological changes.

SummaryIntra-articular sodium hyaluronic acid (HA) has been used as a treatment intervention in the management of osteoarthritis. It has been observed that HA can coat the articular surface, and thus, has been suggested to provide a possible prophylactic barrier for the articular cartilage. In an accompanying manuscript (Homandberg et al.), we report that a commercially available high-molecular-weight HA (approximately 800-kDa, ARTZ, Seikagaku Corp.) can partially block fibronectin fragment (Fn-f)-mediated cartilage injury in vitro. Herein we report a study of the effects of intra-articular HA on an in vivo animal model of Fn-f-mediated cartilage injury. Rabbit knees were injected with Fn-f, and after 1 week, the cartilage proteoglycan (PG) content had decreased to 59 ± 8% of control. In sharp contrast, PG content in knees receiving pre-treatment with HA followed by Fn-f injection had only decreased to 85 ± 27% of control (P < 0.01). Similarly, the PG content in knees receiving an injection of Fn-f, followed by an injection of HA were significantly higher (74 ± 18% of control) than Fn-f injured knees with no treatment (P < 0.02). Intra-articular HA alone had no effect on cartilage PG content. The results in this study suggest that HA is effective in partially preventing Fn-f mediated cartilage injury, most likely by coating the articular surface. Further, HA treatment after Fn-f injury may facilitate restoration of matrix components.

Objective. To detect the message for membrane type 1 (MT1) matrix metalloproteinase (MMP) in articular cartilage and chondrosarcoma cells, to study its expression in osteoarthritis (OA), and to determine whether interleukin-1β (IL-1β) influences its expression. Methods. Reverse transcription–polymerase chain reaction methods were used to detect message. Cloning and sequencing were applied to confirm the sequence. Northern blotting was used to quantify the message for MT1-MMP and to compare its expression in OA cartilage with or without IL-1β treatment. In situ hybridization was utilized to show MT1-MMP transcripts in cartilage and to study the influence of IL-1β. Results. The results show that MT1-MMP messenger RNA (mRNA) is expressed in chondrosarcoma cells and OA chondrocytes. Results of the in situ hybridization confirmed the expression in OA cartilage as well as in normal cartilage. The level of mRNA was not modulated following IL-1β stimulation. Conclusion. This study shows that MT1-MMP is expressed by chondrocytes. The similarities in mRNA levels in OA and normal chondrocytes suggest that regulation of MT1-MMP mRNA may not be a primary factor in OA.

Ankle and knee joints differ in their susceptibility to osteoarthritis (OA). This article reviews literature on differences between these joints. A Medline search and search of bibliographies of review articles was conducted. Knee cartilage degeneration leads to the development of OA with clinical symptoms, whereas the ankle cartilage develops fissures that do not appear to progress to later stages of OA. Epidemiological studies support these findings. Factors that might explain this phenomena include differences in joint motion, cartilage thickness, congruency, mechanical forces, and even evolutionary changes. Data suggest that chondrocytes from the two joints may respond differently to stimuli. Comparisons of cartilage from the knee and ankle joint of the same donor may provide a better understanding of the biochemical and molecular processes that induce the pathogenesis of OA and may provide new approaches to early detection and treatment.

Objective. To compare the responses of chondrocytes from superficial and deep layers of normal human articular cartilage to interleukin-1 (IL-1) and IL-1 receptor antagonist protein (IRAP), and to evaluate the binding sites for IL-1 on these cells. Methods. Cartilage and chondrocytes from superficial and deeper layers of human femoral condyles were cultured with and without IL-1 in the presence and absence of IRAP. The effect of these agents on 35S-proteoglycan synthesis and catabolism and production of stromelysin and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by biochemical and immunologic assays. Receptor binding was evaluated using 125I-labeled IL-1. Results. IL-1 induced more severe inhibition of proteoglycan synthesis and a lower ratio of secreted TIMP-1:stromelysin in chondrocytes from superficial cartilage than those from deeper cartilage. IRAP blocked responses to IL-1 more effectively in chondrocytes from deep cartilage than those from superficial cartilage. Chondrocytes from the articular surface showed approximately twice the number of high-affinity binding sites for IL-1 as did cells from deep cartilage. Conclusion. Chondrocytes from the surface of articular cartilage show a greater vulnerability to the harmful effects of IL-1 and are less responsive to the potential therapeutic effects of IRAP than cells in the deeper layers of the tissue.

Osteoarthrosis is characterized by a progressive destruction of articular cartilage resulting from an imbalance of synthesis and degradation of aggrecan and collagen, the major matrix components. The process is governed by inflammatory episodes, thus the disease is called Osteoarthritis (OA). Aggrecan is a large proteoglycan (PG) which is responsible for the biomechanical function of the cartilage to withstand compressive loading. Aggrecan consists of a core protein to which chains of chondroitin and keratan sulfate are attached (Hascall 1988; Heinegard 1992). Cleavage of the core protein between the two globular domains at the animo terminal end results in loss of aggrecan from the cartilage and impaired function. A number of potential cleavage sites have been identified within the interglobular domain for different proteases including members of the family of matrix metalloproteinases (MMPs), lysosomal enzymes, plasmin, elastase and urokinase (Fosang et al. 1991; 1992; 1993). The predominant proteolytic...

Objective. The effects of doxycycline were tested in an in vitro system in which the cartilages of embryonic avian tibias are completely degraded. Methods. Tibias were cultured with 5, 20, or 40 μg/ml doxycycline. Control tibias were cultured without doxycycline. Conditioned media and tissue sections were examined for enzyme activity and matrix loss. Results. Cartilages were not resorbed in the presence of doxycycline, whereas control cartilages were completely degraded. Collagen degradation was reduced in association with treatment with doxycycline at all doses studied. Higher concentrations of doxycycline reduced collagenase and gelatinase activity and prevented proteoglycan loss, cell death, and deposition of type X collagen in the cartilage matrix; in addition, treatment with doxycycline at higher concentrations caused increases in the length of the hypertrophic region. Conclusion. The effects of doxycycline extend beyond inhibition of the proteolytic enzymes by stimulating cartilage growth and disrupting the terminal differentiation of chondrocytes.

SummaryMolecules derived from cartilage are present in body fluids where they act as markers of the degradation or synthesis of specific components of the extracellular matrix of this shock-absorbing tissue. In joint fluid, the level of a marker molecule can be used to detect changes taking place in

Objective Human superficial chondrocytes show distinct spatial organizations, and they commonly aggregate near osteoarthritic (OA) fissures. The aim of this study was to determine whether remodeling or destruction of the spatial chondrocyte organization might occur at a distance from focal (early) lesions in patients with OA. Methods Samples of intact cartilage (condyles, patellofemoral groove, and proximal tibia) lying distant from focal lesions of OA in grade 2 joints were compared with location-matched nondegenerative (grade 0–1) cartilage samples. Chondrocyte nuclei were stained with propidium iodide, examined by fluorescence microscopy, and the findings were recorded in a top-down view. Chondrocyte arrangements were tested for randomness or significant grouping via point pattern analyses (Clark and Evans Aggregation Index) and were correlated with the OA grade and the surface cell densities. Results In grade 2 cartilage samples, superficial chondrocytes were situated in horizontal patterns, such as strings, clusters, pairs, and singles, comparable to the patterns in nondegenerative cartilage. In intact cartilage samples from grade 2 joints, the spatial organization included a novel pattern, consisting of chondrocytes that were aligned in 2 parallel lines, building double strings. These double strings correlated significantly with an increased number of chondrocytes per group and an increased corresponding superficial zone cell density. They were observed in all grade 2 condyles and some grade 2 tibiae, but never in grade 0–1 cartilage. Conclusion This study is the first to identify a distinct spatial reorganization of human superficial chondrocytes in response to distant early OA lesions, suggesting that proliferation had occurred distant from focal early OA lesions. This spatial reorganization may serve to recruit metabolically active units as an attempt to repair focal damage., National Institutes of Health (U.S.) (grant P5O-AR39239), National Institutes of Health (U.S.) (grant R01-AR33236), Deutsche Forschungsgemeinschaft (DFG) (grant RO 2511/1-1), Deutsche Forschungsgemeinschaft (DFG) (grant RO 2511/2-1)

Bovine articular chondrocytes cultured in agarose gel in the presence of serum elaborated a highly organized extracellular matrix rich in proteoglycans and collagens. The cultures were evaluated quantitatively by radiosulfate labeling of proteoglycans, and by densitometry following staining with alcian blue. In addition, immunohistochemical methods were used to demonstrate the presence of several components of cartilage proteoglycan molecules. Treatment with Interleukin-1 (Il-1) or retinol resulted in diminished synthesis and enhanced catabolism of matrix proteoglycans, but the chondrocytes were more sensitive to human recombinant Il-1 alpha than to Il-1 beta. Treatment with Il-1 alpha or retinol resulted in a profound disorganization of the residual matrix around the majority of the chondrocytes, while Il-1 beta caused much less severe changes. Some variation in cellular response to Il-1 alpha may result from the heterogeneity previously reported among articular chondrocytes.

Neovascularization of developing, repairing or neoplastic tissues is regulated, at least partially, by a family of proteins of low molecular mass (1000 less than mol mass less than 50 000 Da) which can be extracted from avascular tissues, such as hyaline cartilage, aorta or bladder epithelium, by mild salt solutions. These extractable proteins, functionally defined as anti-invasion factor (AIF), act as local regulators for some of the major mechanistic pathways by which endothelial cells are thought to invade tissues during neovascularization, mainly by matrix-degrading enzymes and by increased rates of migration and proliferation. AIF contains a spectrum of proteinase (collagenase) inhibitory activities, as well as an endothelial cell growth inhibitor. The endothelial cell growth inhibitor is directed against actively dividing endothelial cells in culture but has no effect on endothelial cell monolayers or any other cell lines tested. In tumours, the AIF-derived endothelial cell growth inhibitor may limit tumour growth to less than 2 mm in diameter by inhibiting tumour neovascularization.

Difficulty in maintaining phenotypic stability of the Swarm rat chondrosarcoma in long-term monolayer cultures has prompted investigation of alternative conditions that would enable extended maintenance of these cells, permitting use of the tumor as a model system for the long-term study of proteoglycan metabolism. Morphological analysis of the growth of the chondrosarcoma chondrocytes in agarose has shown stability of the culture over a 20 day period with respect to the ability of the cells to proliferate and synthesize an Alcian blue-positive extracellular matrix. The present study confirms these findings through analysis of the growth characteristics of the culture and the pattern of proteoglycan and collagen synthesis. The chondrocytes actively synthesize a proteoglycan-rich matrix at a rate dependent on the initial plating density and concentration of serum in the culture medium. These factors similarly affect the proliferative capabilities of the culture as demonstrated by the growth curves obtained at different culture conditions. During 20 days in culture, the cells synthesize an aggregating chondroitin sulfate proteoglycan and collagen type II, typical of cartilage and this chondrosarcoma. In addition, small molecular weight proteoglycans were found to be present at concentrations of up to 10% of the total proteoglycan population. Degradative rates are slow, the proteoglycan half-life is about 30 days, but can be enhanced with retinol, reducing the half-life to 2 days.

While traumatic joint injuries are known to increase the risk of osteoarthritis (OA), the mechanism is not known. Models for injurious compression of cartilage may identify predictors of injury that suggest a clinical mechanism. We investigated the relationship between peak stress during compression and glycosaminoglycan (GAG) loss after injury for knee and ankle cartilages. Human cartilage explant disks were harvested post-mortem from the knee and ankle of three organ donors with no history of OA and subjected to injurious compression to 65% strain in uniaxial unconfined compression at 2 mm/s (400%/s). The GAG content of the conditioned medium was measured 3 days after injury. After injury of knee cartilage disks, damage was visible in 18 of 39 disks (36%). Three days after injury, the increase in GAG loss to the medium (GAG loss from injured disks minus GAG loss from location-matched uncompressed controls) was 1.5+/-0.3 microg/disk (mean +/- SEM). With final strain and compression velocity held constant, we observed that increasing peak stress during injury was associated with less GAG loss after injury (P0.001). In contrast, ankle cartilage appeared damaged after injury in only 1 of 16 disks (6%), there was no increase in GAG loss (0.0+/-0.3 microg/disk), and no relationship between peak stress and increase in GAG loss was detected (P=0.51). By itself, increasing peak stress did not appear to be an important cause of GAG loss from human cartilage in our injurious compression model. However, we observed further evidence for differences in the response of knee and ankle cartilages to injury.