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Purpose: To analyze chondral flaps debrided during hip arthroscopy to determine their biochemical and cellular composition., Methods: Thirty-one full-thickness acetabular chondral flaps were collected during hip arthroscopy. Biochemical analysis was undertaken in 21 flaps from 20 patients, and cellular viability was determined in 10 flaps from 10 patients. Biochemical analysis included concentrations of (1) DNA (an indicator of chondrocyte content), (2) hydroxyproline (an indicator of collagen content), and (3) glycosaminoglycan (an indicator of chondrocyte biosynthesis). Higher values for these parameters indicated more healthy tissue. The flaps were examined to determine the percentage of viable chondrocytes., Results: The percentage of acetabular chondral flap specimens that had concentrations within 1 SD of the mean values reported in previous normal cartilage studies was 38% for DNA, 0% for glycosaminoglycan, and 43% for hydroxyproline. The average cellular viability of our acetabular chondral flap specimens was 39% (SD, 14%). Only 2 of the 10 specimens had more than half the cells still viable. There was no correlation between (1) the gross examination of the joint or knowledge of the patient's demographic characteristics and symptoms and (2) biochemical properties and cell viability of the flap, with one exception: a degenerative appearance of the surrounding cartilage correlated with a higher hydroxyproline concentration., Conclusions: Although full-thickness acetabular chondral flaps can appear normal grossly, the biochemical properties and percentage of live chondrocytes in full-thickness chondral flaps encountered in hip arthroscopy show that this tissue is not normal., Clinical Relevance: There has been recent interest in repairing chondral flaps encountered during hip arthroscopy. These data suggest that acetabular chondral flaps are not biochemically and cellularly normal. Although these flaps may still be valuable mechanically and/or as a scaffold in some conductive or inductive capacity, further study is required to assess the clinical benefit of repair., (Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Regeneration of human articular cartilage is inherently limited and extensive efforts have focused on engineering the cartilage tissue. Various cellular sources have been studied for cartilage tissue engineering including adult chondrocytes, and embryonic or adult stem cells. Juvenile chondrocytes (from donors below 13 years of age) have recently been reported to be a promising cell source for cartilage regeneration. Previous studies have compared the potential of adult and juvenile chondrocytes or adult and osteoarthritic (OA) chondrocytes. To comprehensively characterize the comparative potential of young, old, and diseased chondrocytes, here we examined cartilage formation by juvenile, adult, and OA chondrocytes in three-dimensional (3D) biomimetic hydrogels composed of poly(ethylene glycol) and chondroitin sulfate. All three human articular chondrocytes were encapsulated in the 3D biomimetic hydrogels and cultured for 3 or 6 weeks to allow maturation and extracellular matrix formation. Outcomes were analyzed using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. After 3 and 6 weeks, juvenile chondrocytes showed a greater upregulation of chondrogenic gene expression than adult chondrocytes, while OA chondrocytes showed a downregulation. Aggrecan and type II collagen deposition and glycosaminoglycan accumulation were high for juvenile and adult chondrocytes but not for OA chondrocytes. Similar trend was observed in the compressive moduli of the cartilage constructs generated by the three different chondrocytes. In conclusion, the juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogels. The 3D culture model described here may also provide a useful tool to further study the molecular differences among chondrocytes from different stages, which can help elucidate the mechanisms for age-related decline in the intrinsic capacity for cartilage repair.

Cartilage loss is a leading cause of disability among adults and effective therapy remains elusive. Neonatal chondrocytes (NChons) are an attractive allogeneic cell source for cartilage repair, but their clinical translation has been hindered by scarce donor availability. Here we examine the potential for catalyzing cartilage tissue formation using a minimal number of NChons by co-culturing them with adipose-derived stem cells (ADSCs) in 3D hydrogels. Using three different co-culture models, we demonstrated that the effects of co-culture on cartilage tissue formation are dependent on the intercellular distance and cell distribution in 3D. Unexpectedly, increasing ADSC ratio in mixed co-culture led to increased synergy between NChons and ADSCs, and resulted in the formation of large neocartilage nodules. This work raises the potential of utilizing stem cells to catalyze tissue formation by neonatal chondrocytes via paracrine signaling, and highlights the importance of controlling cell distribution in 3D matrices to achieve optimal synergy.

This study tested the hypothesis that heme oxygenase-1 (HO-1) expression counteracts bacterial antigen-induced catabolic metabolism in human articular chondrocytes. HO-1 expression was induced in chondrocytes by the iron-containing porphoryin, hemin. Anti-catabolic and anti-apoptotic effects of HO-1 expression were evaluated following bacterial antigen (lipopolysaccharides, LPS) activation of chondrocytes by quantification of cytokine and cartilage matrix protein expression. Effects of HO-1 over-expression on chondrocyte matrix metabolism were evaluated using plasmid-driven protein synthesis. Hemin increased HO-1 expression and LPS increased interleukin-1beta and interleukin-6 gene and protein expression in chondrocytes. Hemin-induced HO-1 decreased LPS-induced interleukin-1beta and interleukin-6 gene and protein expression. Increased HO-1 expression partially reversed LPS-suppression of aggrecan and type II collagen gene expression and suppressed LPS-induced gene expression of IL-6, inducible nitric oxide synthase (iNOS), matrix metalloproteinases (MMPs), and IL-1beta. HO-1 induction was inversely correlated with LPS-induced chondrocyte apoptosis. HO-1 over-expression in chondrocytes decreased matrix protein gene expression. With LPS activation, increased HO-1 expression decreased chondrocyte catabolism, partially reversed LPS-dependent inhibition of cartilage matrix protein expression and protected against apoptosis. Without LPS, hemin-induced HO-1 and plasmid-based over-expression of HO-1 inhibited cartilage matrix gene expression. The results suggest that elevated HO-1 expression in chondrocytes is protective of cartilage in inflamed joints but may otherwise suppress matrix turn over., (© 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Objective: To evaluate the hypothesis that a mechanical stimulus (30-min walk) will produce a change in serum concentrations of cartilage oligomeric matrix protein (COMP) that is associated with cartilage thickness changes on magnetic resonance imaging (MRI)., Methods: Serum COMP concentrations were measured by enzyme-linked immunosorbent assay in 17 patients (11 females, age: 59.0±9.2 years) with medial compartment knee osteoarthritis (OA) at study entry immediately before, immediately after, 3.5 h, and 5.5 h after a 30-min walking activity. Cartilage thickness changes in the medial femur and medial tibia were determined from MR images taken at study entry and at 5-year follow-up. Relationships between changes in cartilage thickness and COMP levels, with post-activity concentrations expressed as a percentage of pre-activity levels, were assessed by the calculation of Pearson correlation coefficients and by multiple linear regression analysis, with adjustments for age, sex, and body mass index (BMI)., Results: Changes in COMP levels 3.5 h and 5.5 h post-activity were correlated with changes in cartilage thickness in the medial femur and tibia at the 5-year follow-up. The results were strengthened after analyses were adjusted for age, sex, and BMI. Neither baseline pre-activity COMP levels nor changes in COMP levels immediately post-activity were correlated with cartilage thickness changes., Conclusions: The results of this study support the hypothesis that a change in COMP concentration induced by a mechanical stimulus is associated with cartilage thinning at 5 years. Mechanically-induced changes in mechano-sensitive biomarkers should be further explored in the context of stimulus-response models to improve the ability to assess OA progression., (Published by Elsevier Ltd.)

In this article, cross-relaxation imaging is applied to human ex vivo knee cartilage, and correlations of the cross-relaxation imaging parameters with macromolecular content in articular cartilage are reported. We show that, unlike the more commonly used magnetization transfer ratio, the bound pool fraction, the cross-relaxation rate (k) and the longitudinal relaxation time (T(1)) vary with depth and can therefore provide insight into the differences between the top and bottom layers of articular cartilage. Our cross-relaxation imaging model is more sensitive to macromolecular content in the top layers of cartilage, with bound pool fraction showing moderate correlations with proteoglycan content, and k and T(1) exhibiting moderate correlations with collagen., (Copyright © 2011 Wiley-Liss, Inc.)

Objective: A relationship between T1ρ relaxation time and glycosaminoglycan (GAG) content has been demonstrated in chemically degraded bovine cartilage, but has not been demonstrated with quantitative biochemistry in human cartilage. A relationship has also been established between T2 relaxation time in cartilage and osteoarthritis (OA) severity. We hypothesized that T1ρ relaxation time would be associated with GAG content in human cartilage with normal T2 relaxation times., Methods: T2 relaxation time, T1ρ relaxation time, and glycosaminoglycan as a percentage of wet weight (sGAG) were measured for top and bottom regions at 7 anatomical locations in 21 human cadaver patellae. For our analysis, T2 relaxation time was classified as normal or elevated based on a threshold defined by the mean plus one standard deviation of the T2 relaxation time for all samples., Results: In the normal T2 relaxation time subset, T1ρ relaxation time correlated with sGAG content in the full-thickness and bottom regions, but only marginally in the top region alone. sGAG content decreased significantly with age in all regions., Conclusion: In the subset of cartilage specimens with normal T2 relaxation time, T1ρ relaxation time was inversely associated with sGAG content, as hypothesized. A predictive model, which accounts for T2 relaxation time and the effects of age, might be able to determine longitudinal trends in GAG content in the same person based on T1ρ relaxation time maps., (Copyright © 2010 Osteoarthritis Research Society International. All rights reserved.)

Homeostasis of articular cartilage depends in part on mechanical loads generated during daily activity whereas inappropriate joint loads result in focal degeneration of cartilage, as occurs in osteoarthritis. We will review results of a series of questions regarding the effects of two types of mechanical loads-intermittent hydrostatic pressure and shear stress-on adult human articular chondrocytes in high-density monolayer culture. Intermittent hydrostatic pressure increased aggrecan and Type II collagen gene expression in normal chondrocytes and induced changes in the cell-associated proteins of normal and osteoarthritic chondrocytes. Hydrostatic pressure also counteracted inhibitory effects of bacterial lipopolysaccharide on matrix protein expression by cultured chondrocytes. Application of shear stress to osteoarthritic chondrocytes increased the release of the proinflammatory mediator, nitric oxide, decreased aggrecan and Type II collagen expression, and induced molecular changes associated with apoptosis whereas hydrostatic pressure increased matrix macromolecule expression. The findings show that the types of load comprising the mechanical loading environment of articular cartilage considerably alter chondrocyte metabolism and suggest that mechanical stimulation may be used for in vitro or in vivo approaches for cartilage engineering.

The development, maintenance, and destruction of cartilage are regulated by mechanical factors throughout life. Mechanical cues in the cartilage fetal endoskeleton influence the expression of genes that guide the processes of growth, vascular invasion, and ossification. Intermittent fluid pressure maintains the cartilage phenotype whereas mild tension (or shear) promotes growth and ossification. The articular cartilage thickness is determined by the position at which the subchondral growth front stabilizes. In mature joints, cartilage is thickest and healthiest where the contact pressure and cartilage fluid pressure are greatest. The depth-dependent histomorphology reflects the local fluid pressure, tensile strain, and fluid exudation. Osteoarthritis represents the final demise and loss of cartilage in the skeletal elements. The initiation and progression of osteoarthritis can follow many pathways and can be promoted by mechanical factors including: (1) reduced loading, which activates the subchondral growth front by reducing fluid pressure; (2) blunt impact, causing microdamage and activation of the subchondral growth front by local shear stress; (3) mechanical abnormalities that increase wear at the articulating surface; and (4) other mechanically related factors. Research should be directed at integrating our mechanical understanding of osteoarthritis pathogenesis and progression within the framework of cellular and molecular events throughout ontogeny.

Objective: This study tested the hypothesis that intermittent hydrostatic pressure applied to human osteoarthritic chondrocytes modulates matrix metalloproteinase and pro-inflammatory mediator release in vitro., Design: Human osteoarthritic articular chondrocytes were isolated and cultured as primary high-density monolayers. For testing, chondrocyte cultures were transferred to serum-free medium and maintained without loading or with exposure to intermittent hydrostatic pressure (IHP) at 10 MPa at a frequency of 1 Hz for periods of 6, 12 and 24 h. Levels of matrix metalloproteinase-2, -9 (MMP-2, -9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and the pro-inflammatory mediators, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), released into the culture medium were assessed by ELISA. Matrix metalloproteinase activity was confirmed by zymographic analysis., Results: In the absence of IHP, levels of MMP-2, TIMP-1, IL-6, and MCP-1 in the chondrocyte culture medium increased in a time-dependent manner. Application of IHP decreased MMP-2 levels at all time periods tested, relative to unloaded control cultures maintained for the same time periods. Although 84/82 kDa bands were faintly detectable by zymography, MMP-9 levels were not quantifiable in medium from loaded or unloaded cultures by ELISA. TIMP-1 levels were not altered in response to IHP at any time period tested. IL-6 and MCP-1 levels decreased in cultures exposed to IHP at 12 and 24 h, relative to unloaded control cultures maintained for the same time periods., Conclusion: IHP decreased release of MMP-2, IL-6 and MCP-1 by osteoarthritic chondrocytes in vitro suggesting that pressure influences cartilage stability by modulating chondrocyte expression of these degradative and pro-inflammatory proteins in vivo.

The in vivo pathomechanics of osteoarthritis (OA) at the knee is described in a framework that is based on an analysis of studies describing assays of biomarkers, cartilage morphology, and human function (gait analysis). The framework is divided into an Initiation Phase and a Progression Phase. The Initiation Phase is associated with kinematic changes that shift load bearing to infrequently loaded regions of the cartilage that cannot accommodate the loads. The Progression Phase is defined following cartilage breakdown. During the Progression Phase, the disease progresses more rapidly with increased load. While this framework was developed from an analysis of in vivo pathomechanics, it also explains how the convergence of biological, morphological, and neuromuscular changes to the musculoskeletal system during aging or during menopause lead to the increased rate of idiopathic OA with aging. Understanding the in vivo response of articular cartilage to its physical environment requires an integrated view of the problem that considers functional, anatomical, and biological interactions. The integrated in vivo framework presented here will be helpful for the interpretation of laboratory experiments as well as for the development of new methods for the evaluation of OA at the knee.

This study examined the relationship between acetabular cartilage properties after hemiarthroplasty surgery and surgical variables including femoral head size and position. Nineteen sheep received unilateral hip arthroplasties and were euthanized one year post-operatively to harvest the femora and acetabula. Cartilage histology, biochemistry and material properties were determined from samples located in the superior load-bearing region. Femoral head size mismatch, leg length difference, anterior-posterior and medial lateral offset and anteversion were measured. In the acetabulum. substantial cartilage degradation occurred with widespread librillation and significant changes in the biochemical and material properties compared to the intact contralateral joint. Regression analyses on the surgical variables explained 75-80% of the changes in tissue biochemistry but did not explain the material changes. Head size mismatch and leg length difference were the most significant contributors of the five variables examined and therefore may be critical to successful outcome in hemiarthroplasty.

The normal loading of joints during daily activities causes the articular cartilage to be exposed to high levels of intermittent hydrostatic pressure. This study quantified effects of intermittent hydrostatic pressure on expression of mRNA for important extracellular matrix constituents. Normal adult bovine articular chondrocytes were isolated and tested in primary culture, either as high-density monolayers or formed aggregates. Loaded cells were exposed to 10 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for periods of 2, 4, 8, 12, and 24 hrs. Other cells were intermittently loaded for a period of 4 hrs per day for 4 days. Semiquantitative reverse transcription polymerase chain reaction assays were used to assess mRNA signal levels for collagen types II and I and aggrecan. The results showed that type II collagen mRNA signal levels exhibited a biphasic pattern, with an initial increase of approximately five-fold at 4 and 8 hrs that subsequently decreased by 24 hrs. In contrast, aggrecan mRNA signal increased progressively up to three-fold throughout the loading period. Changing the loading profile to 4 hrs per day for 4 days increased the mRNA signal levels for type II collagen nine-fold and for aggrecan twenty-fold when compared to unloaded cultures. These data suggest that specific mechanical loading protocols may be required to optimally promote repair and regeneration of diseased joints.

Mechanical loading alters the metabolism of articular cartilage, possibly due to effects of shear stress on chondrocytes. In cultured chondrocytes, glycosaminoglycan synthesis increases in response to fluid-induced shear. This study tested the hypothesis that shear stress increases nitric oxide production in chondrocytes, and nitric oxide then influences glycosaminoglycan metabolism. Inhibitors of nitric oxide synthase, G proteins, phospholipase C, potassium channels, and calcium channels were also analyzed for effects on nitric oxide release and glycosaminoglycan synthesis. Fluid-induced shear was applied to primary high-density monolayer cultures of adult bovine articular chondrocytes using a cone viscometer. Nitric oxide release in chondrocytes increased in response to the duration and the magnitude of the fluid-induced shear. Shear-induced nitric oxide production was reduced in the presence of nitric oxide synthase inhibitors-but was unaffected by pertussis toxin, neomycin, tetraethyl ammonium chloride, or verapamil. The increase in glycosaminoglycan synthesis in response to shear stress was blocked by nitric oxide synthase inhibitors, pertussis toxin, and neomycin but not by tetraethyl ammonium chloride or verapamil. The phospholipase C inhibitor, neomycin, also decreased glycosaminoglycan synthesis in the absence of flow-induced shear. As studied here, shear stress increased nitric oxide production by chondrocytes, and the shear-induced change in matrix macromolecule metabolism was influenced by nitric oxide synthesis, G protein activation, and phospholipase C activation.

Expression of matrix metalloproteinase-9 mRNA in osteoarthritic and normal cartilage was analyzed using reverse transcription-polymerase chain reaction and in situ hybridization. Fifty-four osteoarthritic cartilage samples were obtained from 24 patients undergoing total knee arthroplasty. Sixteen normal cartilage samples were obtained from non-osteoarthritic knees of four autopsy cases. With normal cartilage, reverse transcription-polymerase chain reaction analysis for matrix metalloproteinase-9 mRNA showed that chondrocytes exhibited only a trace signal. In analysis of osteoarthritic cartilage, chondrocytes of moderately and severely fibrillated cartilage exhibited a 73-fold and 110-fold increase in matrix metalloproteinase-9 mRNA signal, respectively, relative to normal cartilage. Chondrocytes of nonfibrillated osteoarthritic cartilage exhibited a 6-fold increase (p < 0.02) in matrix metalloproteinase-9 mRNA signal relative to normal cartilage. Analysis of matrix metalloproteinase-9 mRNA expression in fresh-frozen sections of normal and osteoarthritic cartilage by in situ hybridization confirmed these results. This study showed that reverse transcription-polymerase chain reaction provides a sensitive index of mRNA levels in normal and osteoarthritic cartilage samples and suggests that increased expression of matrix metalloproteinase-9 precedes fibrillation of cartilage in the development of osteoarthritis.

This report presents a method for analyzing matrix metalloproteinase-9 (92 kD gelatinase) MMP-9 mRNA expression using reverse transcription polymerase chain reaction (RT-PCR). With this method, chondrocytes isolated from small samples of osteoarthritic cartilage showed significantly elevated signal for MMP-9 mRNA compared to normal cartilage. In addition, cells of synovial fluid samples aspirated from osteoarthritic joints also exhibited MMP-9 expression using this technique. RT-PCR proved to be a sensitive method for assessing MMP-9 regulation in osteoarthritic and normal cartilage, and may provide a useful index of arthritic cartilage and synovial fluid cell metabolism when limited tissue is available.

Osteoarthritis is a disease in which articular cartilage metabolism is altered, leading to cartilage destruction. As insulin-like growth factor-I (IGF-I) is the major anabolic mediator for articular cartilage, and the IGF-binding proteins (IGFBPs) are an integral part of the IGF axis, they may play a role in the pathophysiology of osteoarthritis. Chondrocytes isolated from fibrillated and normal appearing areas of osteoarthritic human cartilage and from normal cartilage were studied for IGF and IGFBP expression. IGF and IGFBP messenger ribonucleic acids were analyzed by a RT-quantitative PCR technique and Northern blotting. In osteoarthritic chondrocytes, IGF-I message was increased 3.5-fold, IGFBP-3 was increased 24-fold, and IGFBP-5 was increased 16-fold over normal chondrocytes. Chondrocytes from normal appearing areas of cartilage from osteoarthritic joints had intermediate levels. Message levels for beta-actin, IGF-II, and IGFBP-4 were unchanged between the cartilage types. IGF and IGFBP production were analyzed by Western ligand blots and RIAs of conditioned medium from cartilage cultured in serum-free conditions. IGF-I was undetectable in conditioned medium from normal cartilage and increased in that from osteoarthritic cartilage. Osteoarthritic cartilage samples produced IGFBP-2, -3, and -4; glycosylated IGFBP-4; and IGFBP-5. IGFBP-2, -3, and -5 production was increased in osteoarthritic cartilage. Proteases with activity against IGFBP-3 and -5 were also produced by osteoarthritic cartilage. The observation that IGFBP-3 and -5 expression and production are elevated in osteoarthritic cartilage suggests that they may be acting as a competitor for IGF-I in osteoarthritic cartilage, thus reducing the anabolic stimulation of this tissue and contributing to the net loss of cartilage in this disease.

Comparison of perilesional cartilage, lesional repair tissue, and subchondral bone activity 6 months after application of holmium-yttrium-aluminum-garnet (Ho:YAG) laser energy to chronic (10 week) induced 10-mm full-thickness (FT) circular articular cartilage craters followed by 6 months' intermittent active motion (IAM) in a free exercise environment was investigated. The 2.1-microns wavelength was delivered in hand-controlled near-contact mode by arthroscopic surgery in a saline medium. Bilateral arthroscopy was performed on normal antebrachiocarpal, intercarpal, and metacarpophalangeal joints of six adult horses. Full-thickness craters were created in nine sites per limb on weight-bearing articular surfaces with a motorized bur. Right limb craters served as sham operated controls. Animals were killed at 10 weeks after FT crater creation (n = 2), and at 24 weeks (6 months) after laser energy application (n = 4). Histological analysis using hematoxylineosin (HE) and Safranin-O staining consisted of a modified Mankin grading of perilesional cartilage and lesional repair tissue scoring. Biochemical analysis was performed for cellularity and glycosaminoglycan (GAG) synthesis. Histological analysis showed clustering of chondrocytes or perilesional zonal cloning (PZC) in 83% of laser-treated lesions and in no control lesions. No differences were observed between treated and control lesional repair activity. Laser-treated perilesional cartilage showed a significant (P < .02) decrease in GAG synthesis. No adverse effects to distant cartilage were observed after application of laser energy regarding cell proliferation or GAG synthesis. Significance of decreased GAG synthesis in treated perilesional cartilage and perilesional zonal cloning of chondrocytes in treated cartilage is unknown. Additional study of Ho:YAG laser energy application to cartilage and subchondral bone is needed before its application in the surgical management and repair of cartilage damage.

This study tested the effect of fluid-induced shear on interleukin-6 expression in normal human articular chondrocytes in vitro. As determined by Northern blot analysis, interleukin-6 mRNA expression occurs in chondrocytes from osteoarthritic cartilage but not in normal chondrocytes. Applying fluid-induced shear stress to primary high density cultures of chondrocytes increased interleukin-6 mRNA signal 4-fold at 1 hour and 10 to 15-fold at 48 hours compared with unsheared control cultures. At 48 hours, fluid-induced shear stress increased interleukin-6 protein levels in the culture medium 9 to 10-fold compared with unsheared controls. mRNA signals for interleukin-1alpha, interleukin-1beta, and tumor necrosis factor-alpha in RNA from sheared or control chondrocytes were not detected by Northern blotting. Transforming growth factor-beta mRNA signal was detectable but was not affected by shear. In contrast, human lung fibroblasts (WI-38) responded to fluid-induced shear with increased signal for transforming growth factor-beta, but not interleukin-6, mRNA. Both cell types did respond to interleukin-1alpha with increased interleukin-6 mRNA signal. These data demonstrated that distortional forces, such as fluid-induced shear stress, alter interleukin-6 levels in normal chondrocytes in vitro and suggest that increased interleukin-6 expression in osteoarthritic cartilage may result, in part, from alterations in the mechanical loading of the tissue.

This study tested the effects of hydrostatic pressure (10 MPa) on adult articular chondrocyte mRNA and extracellular matrix synthesis in vitro. High density primary cultures of bovine chondrocytes were exposed to hydrostatic pressure applied intermittently at 1 Hz or constantly for 4 hours in serum-free medium or in medium containing 1% fetal bovine serum. mRNAs for aggrecan, types I and II collagen, and beta-actin were analyzed by Northern blots and quantified by slot blots. Proteoglycan synthesis was quantified by 35SO4 uptake into cetylpyridinium chloride-precipitable glycosaminoglycans, and cell-associated aggrecan and type-II collagen were detected by immunohistochemical techniques. In serum-free medium, intermittent pressure increased aggrecan mRNA signal by 14% and constant pressure decreased type-II collagen mRNA signal by 16% (p < 0.05). In the presence of 1% fetal bovine serum, intermittent pressure increased aggrecan and type-II collagen mRNA signals by 31% (p < 0.01) and 36% (p < 0.001), respectively, whereas constant pressure had no effect on either mRNA. Intermittent and constant pressure stimulated glycosaminoglycan synthesis 65% (p < 0.001) and 32% (p < 0.05), respectively. Immunohistochemical detection of cell-associated aggrecan and type-II collagen was increased in response to both intermittent and constant pressure. These data support the hypothesis that physiologic hydrostatic pressure directly influences the extracellular matrix metabolism of articular chondrocytes.

This study tested the effects of fluid-induced shear on high density monolayer cultures of adult articular chondrocytes. Fluid-induced shear (1.6 Pa) was applied by cone viscometer to normal human and bovine articular chondrocytes for periods of 24, 48, and 72 hours. At 48 and 72 hours, fluid-induced shear caused individual chondrocytes to elongate and align tangential to the direction of cone rotation. Fluid-induced shear stimulated glycosaminoglycan synthesis by 2-fold (p < 0.05) and increased the length of newly synthesized chains in human and bovine chondrocytes. In human chondrocytes, the hydrodynamic size of newly synthesized proteoglycans also was increased. After 48 hours of fluid-induced shear, the release of prostaglandin E2 from the chondrocytes was increased 10 to 20-fold. In human chondrocytes, mRNA signal levels for tissue inhibitor of metalloproteinase increased 9-fold in response to shear compared with the controls. In contrast, mRNA signal levels for the neutral metalloproteinases, collagenase, stromelysin, and 72 kD gelatinase, did not show such major changes. This study demonstrated that articular chondrocyte metabolism responds directly to physical stimulation in vitro and suggests that mechanical loading may directly influence cartilage homeostasis in vivo.

IGF-I is the major anabolic factor for cartilage matrix production. Chondrocytes and cartilage treated with interleukin-1 alpha (IL-1 alpha), and chondrocytes from several models of inflammatory joint disease, exhibit reduced responsiveness to IGF-I. Since the IGF-binding proteins (IGFBPs) modulate the effects of IGF-I, we examined the effect of IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) on IGFBP production by normal human articular chondrocytes in primary culture. Western ligand blots and immunoprecipitation of conditioned medium samples showed that articular chondrocytes produced IGFBPs-2, -3 and -4 and glycosylated IGFBP-4. Both IL-1 alpha and TNF-alpha increased chondrocyte production of IGFBP-3, but did not alter IGFBP-4 production. The activity of a neutral metalloprotease with the ability to cleave IGFBP-3 was also increased by IL-1 alpha. These data suggest that the cytokines IL-1 alpha and TNF-alpha may act to reduce IGF-I access to chondrocytes by increasing production of IGFBP-3. This may be a factor in the decreased matrix production in the inflammatory arthritides.

Objective: To test the effects of nonsteroidal antiinflammatory drugs (NSAID), naproxen, ibuprofen and diclofenac, and salicylates on normal and interleukin 1 (IL-1) treated human articular chondrocyte metabolism in vitro., Methods: Normal adult human articular chondrocytes were isolated and cultured as primary monolayers; the cells were treated with NSAID and salicylates at low and high plating density for assessing effects on proliferation and matrix synthesis and IL-1 modulated cell metabolism, respectively., Results: Cell proliferation was inhibited by ibuprofen and high doses of salicylates. Glycosaminoglycan (GAG) synthesis was stimulated by ibuprofen at 10 micrograms/ml but was not changed by any other drugs at similarly low concentrations; at medium to high concentrations, only the salicylates inhibited GAG synthesis. Collagen synthesis was unaffected by any drug at the concentrations tested. IL-1 induced prostaglandin E2 release was completely inhibited by the NSAID and partially inhibited by the salicylates. IL-1 induced IL-6 release was inhibited by ibuprofen and the salicylates where as IL-1 induced APMA-activated collagenase was only inhibited by the salicylates., Conclusion: Normal human chondrocytes respond differentially to naproxen, ibuprofen, dicolfenac, and the salicylates; these observations suggest that each drug may exhibit unique attributes with respect to longterm efficacy on cartilage metabolism.

Biochemical analysis was performed on hyaline cartilage from four distinct quadrants in surgically harvested osteoarthritic thumb trapeziometacarpal joints and both normal and osteoarthritic cadaver specimens. Chondromalacic surfaces in the palmar contact areas of arthritic specimens were characterized by preferential loss of glycosaminoglycan, retention of collagen, and a shift in glycosaminoglycan subspecies as indicated by an increasing chondroitin sulfate/keratan sulfate ratio. In contrast, articular cartilage from the dorsal portion of arthritic joints biochemically resembled that from both the adjacent normal surfaces of the scaphoid and trapezoid facets and normal postmortem specimens. No significant difference was noted between the biochemical profiles of osteoarthritic surgical specimens and arthritic cadaver specimens. Such selective glycosaminoglycan depletion localized to the contact areas of the joint suggests an interdependent relationship between mechanical factors and biochemical matrix degradation in production of the cartilage lesion of trapeziometacarpal osteoarthritis.

Regional variation in the surface ultrastructure of hyaline cartilage in the osteoarthritic trapeziometacarpal joint was studied by means of scanning electron microscopy. Articular surface degeneration on intact osteochondral specimens was analyzed and correlated with known patterns of gross, histologic, and biochemical disease. The earliest changes were observed in the tangential surface lamina and occurred in areas of chondromalacia in the palmar contact area of the joint. Progressive disruption of this superficial fibrillar layer of the articular cartilage was followed by disorganization of the underlying chondrocytes and matrix. In palmar regions with more advanced chondromalacia and loss of eosinophilic staining, free cellular elements and craters 20-30 microns in diameter, resembling empty chondrocyte lacunae, appeared in conjunction with the absence of any tangential surface meshwork. Eburnated areas demonstrated no fibrillar or cellular elements. Disruption of the protective surface lamina of trapeziometacarpal articular cartilage occurs in a geographic pattern corresponding to joint contact areas and regions of selective biochemical decomposition of proteoglycan matrix. These observations support an interdependent relationship between mechanically induced abrasive surface wear and biochemical matrix degradation in the production of the cartilage lesion of trapeziometacarpal osteoarthritis.

We report here that a 92-kD gelatinolytic metalloproteinase is expressed as protein and mRNA in human osteoarthritic cartilage, but not in normal adult articular cartilage. Western immunoblotting demonstrated that the 92-kD gelatinolytic activity corresponded to 92-kD type IV collagenase/gelatinase (gelatinase B); mRNA for gelatinase B was identified by Northern blotting. Chondrocytes from normal cartilage also exhibited mRNA for 72-kD type IV collagenase/gelatinase (gelatinase A), tissue collagenase, and stromelysin-1, and these mRNAs were increased in osteoarthritic cartilage. Regional analysis of osteoarthritic cartilage samples from four individuals revealed that gelatinase B mRNA was expressed in grossly fibrillated areas; two of four nonfibrillated cartilage samples failed to exhibit the mRNA, but did have increased levels of mRNA for other neutral metalloproteinases. IL-1 alpha treatment of normal human cartilage explants or isolated chondrocytes induced increased levels of gelatinase B and increased mRNA for tissue collagenase and stromelysin-1. Under identical conditions, mRNA levels for gelatinase A were not increased indicating that regulation of this enzyme in human articular chondrocytes is distinct from that of other metalloproteinases. Our data showing expression of gelatinase B in fibrillated cartilage suggest that it is a marker of progressive articular cartilage degradation in osteoarthritis.

This study analyzed processes underlying osteoporosis and osteoarthrosis after short-term immobilization of the right hind limb of postadolescent (2.8 kg) and mature (4.0 kg) rabbits. After 3 weeks, the lateral posterior aspect of the lateral tibial plateau and the lateral femoral condyle of the immobilized limb exhibited prominent subchondral vascular eruptions. Femoral metaphyseal bone density decreased 27 and 18% in the immobilized limbs of postadolescent and mature rabbits, respectively. Calcein green fluorescence increased 1.9-fold (p less than 0.001) in the metaphyseal trabeculae of immobilized femurs. With immobilization, sulfate incorporation into femoral cartilage glycosaminoglycan increased, although total cartilage glycosaminoglycan and hydroxyproline levels were unchanged. Thymidine incorporation into DNA increased four- to fivefold in tibial and femoral cartilage of the immobilized limb. In this study, bone loss and remodeling preceded erosive cartilage degradation.

We report that administration of the corticosteroid, methylprednisolone (PRED) inhibited interleukin 1 (IL-1) induction of chondrocyte caseinolytic activity (25-55%) and collagenolytic activity (15-24%). The nonsteroidal antiinflammatory drug (NSAID), naproxen (NAP) had no effect on either enzyme activity over a therapeutic range (7-30 micrograms/ml) but at 120 micrograms/ml inhibited IL-1 induced caseinolytic and collagenolytic activity by 17 and 19%, respectively. However, PRED (2 micrograms/ml) in combination with NAP (30 micrograms/ml) significantly increased the inhibition of caseinolytic activity (p less than 0.001) compared to that observed with PRED (2 micrograms/ml) alone. The suppression of IL-1 induced collagenolytic activity noted with PRED in combination with NAP did not exceed that observed with PRED alone.

Human articular cartilage released significantly increased levels of metal-dependent enzymes capable of degrading collagen, casein, and gelatin at a neutral pH following exposure to a sterile, purified fraction of Staphylococcus aureus culture medium. Neutral metalloprotease activity was determined by radiolabeled substrate assays and substrate gel analysis. The enzymes were activated with 4-aminophenylmercuric acetate and were inhibited by 1,10-phenanthroline and ethylenediamine tetraacetic acid. Protein immunoblots demonstrated that type I collagenase and stromelysin (matrix metalloproteinase III) secretion was increased following staphylococcal medium challenge. The profile of enzymatic activity induced by staphylococcal medium was directly comparable to that observed with interleukin-1, which was used as a positive control. The staphylococcal medium had no inherent proteolytic activity. Increased production of the neutral metalloproteases collagenase and stromelysin may significantly contribute to the extensive cartilage destruction noted in staphylococcal septic arthritis.

Juvenile rheumatoid arthritis (JRA) involves a wide range of joint tissues. Tissue changes include proliferation of synovial cells, alterations in synovial fluid, and degradation of articular cartilage. Synovial cell proliferation results in increased numbers of fibroblasts and lymphocytes. Changes in the synovial fluid include increased content of antibodies, altered complement ratios, increased levels of factors stimulating cartilage-mediated proteoglycan degradation, and decreased levels of insulinlike growth factor I. Levels of cytokine such as interleukin-1 and interleukin-2 vary with cell culture and assay technique. Cartilage degradation is apparent from increased quantities of proteoglycan and glycosaminoglycan in serum and synovial fluid. Type II collagen peptide antibodies are also prevalent in JRA patients. Cartilage degradation appears linked to factors in JRA synovial fluid. Conditioned medium of peripheral blood mononuclear cells from patients with JRA also stimulates increased release of cartilage proteoglycan. Thus, the outcome in JRA likely reflects activities of interacting soluble factors that directly influence cartilage homeostasis.

We report herein that cartilage proteolytic activity increased in bovine and rabbit articular cartilage after treatment with a purified staphylococcal culture medium or intraarticular infection with Staphylococcus aureus. Staphylococcal culture medium increased the release of gelatinolytic, collagenolytic, and caseinolytic activity into the medium of isolated chondrocytes or cartilage organ culture. The proteolytic activities were determined in assays using radiolabeled substrate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staphylococcal culture medium was proteolytically inactive by both assay techniques. RNA synthesis of isolated chondrocytes was unaffected by staphylococcal culture medium, whereas overall protein synthesis was inhibited by 84%. An analysis of extracts of Staphylococcus aureus-infected rabbit knee cartilage by substrate gels showed increased gelatinolytic and caseinolytic activity compared with extracts of uninfected knee cartilage. Our data suggest that the rapid loss of proteoglycan and persistent degradation of cartilage in staphylococcal septic arthritis is due to the production and activation of chondrocyte proteases.

This paper reports the effects of adding partially purified bovine thyroid calcitonin, thyrocalcitonin, to adult bovine articular cartilage cells. Thyrocalcitonin stimulated chondrocyte proliferation fourfold under low serum (0.5%) culture conditions. In serum-free medium, thyrocalcitonin stimulated cell proliferation more than twofold. With high-density cultures in serum-free medium, chondrocyte glycosaminoglycan (GAG) synthesis was stimulated 60% by thyrocalcitonin. Cell-associated radioactivity was increased twofold. In contrast to thyrocalcitonin, addition of human and salmon calcitonin peptides as well as the thyroid hormones T3 and T4 had no effect on adult cartilage cell proliferation or GAG synthesis. The data reported here suggest the existence of a thyroid-derived factor, independent of calcitonin peptides or thyroid hormones, which stimulates adult articular chondrocyte metabolism.

Inflammatory and noninflammatory juvenile synovial fluid (SF) samples were examined in vitro for their effect on cartilage. SF from 3 of 6 patients with pauciarticular juvenile arthritis (JA), 7 of 7 patients with polyarticular JA and 2 of 2 patients with ankylosing spondylitis had significantly increased proteoglycan (PG) releasing activity in 4 day cultures of living but not of freeze-killed cartilage. Noninflammatory SF did not cause increased PG release. Cartilage collagen content was not significantly altered by either inflammatory or noninflammatory SF. Our in vitro results showed PG releasing activity was present in inflammatory but not in normal juvenile SF.

The purpose of this study was to determine the effect of orientation of pulsed electromagnetic fields (PEMFs) on cellular proliferation and extracellular matrix synthesis. Bovine articular chondrocytes were cultured in PEMFs (repetitive pulse at 72 Hz) generated using Helmholtz coils oriented either parallel (horizontal) or perpendicular (vertical) to the plane of cell adhesion. Dissipation of signal energy in the form of heat increased the temperature of the PEMF coils by 2 degrees C and the tissue culture medium by 1 degree C. Therefore, control coils, which emitted no PEMFs, were heated to the temperature of PEMF coils by circulating water. Chondrocytes were cultured in 16-mm-well culture plates, and the data for individual wells were pooled as triplicates. Although not observed by microscopic examination of individual wells, positionally dependent electric field effects may be minimized by this approach. PEMFs generated by coils oriented vertically significantly decreased chondrocyte proliferation. The effect was dependent on the concentration of serum in the culture media. At 3% serum concentration, the total cell number attained after 10 days of culture was reduced by 50% in stimulated cultures when compared with controls. At 5% serum concentration, there was no effect. PEMFs applied by coils oriented horizontally did not alter proliferation of articular chondrocytes. PEMFs had no effect on synthesis of extracellular matrix by chondrocytes plated at high density, irrespective of orientation.

Unlabelled: In joints with bacterial arthritis, continuing prolonged destruction of cartilage may occur in spite of prompt, effective antibiotic therapy. We measured the extent to which early antibiotic therapy with ceforanide altered the degradation of the cartilage after arthritis due to Staphylococcus aureus had been produced in the knee joint in rabbits. Degradation of the cartilage was quantified by analyses for glycosaminoglycan and collagen. Three weeks after the infection was produced, the cartilage had lost more than half of its glycosaminoglycan whether the antibiotic therapy had been started at one, two, or seven days after infection. Beginning the antibiotic treatment one day after infection reduced over-all loss of collagen by 37 per cent and decreased the area of erosion of the infected articular surfaces. When antibiotic treatment was begun at four, eight, or twelve hours after infection, the loss of glycosaminoglycan averaged 18 per cent. Prophylaxis with antibiotics completely prevented any degradation of the cartilage., Clinical Relevance: The findings reported here show how rapidly cartilage loses glycosaminoglycan when it is involved by arthritis caused by staphylococci and how early treatment of the infection reduces the loss of collagen. There is less protection against loss of glycosaminoglycan. The results emphasize the need for early diagnosis and treatment of infectious synovitis and support the rationale for early administration of antibiotics without waiting for identification of the responsible bacteria.

The purpose of this study was to test whether cartilage serves as the source or repository of antigenic components active in the stimulation of inflammation in rheumatoid arthritis through an analysis of peripheral blood lymphocyte proliferation. Articular cartilage samples were obtained from patients with osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis undergoing joint replacement surgery. Each sample was homogenized and characterized biochemically with respect to the content of proteoglycan, collagen, and immunoglobulin. Proteoglycan content of rheumatoid cartilage was reduced by 71% when compared to osteoarthritic cartilage; the proteoglycan content of ankylosing spondylitis cartilage was reduced by 40% when compared to osteoarthritic cartilage. Immunoglobulins were detectable in all cartilage samples when analyzed by ELISA or end-plate titration. Lymphocyte proliferation, quantified by uptake of 3H-thymidine, was unaltered by addition of cartilage fragments, low (saline) and high salt extracts (2.0 M CaCl2), or cartilage residues. Both autologous and heterologous lymphocytes were tested against the cartilage samples with no difference in reactivity. Purified bovine articular proteoglycans and Type II collagen were also inactive. Although tetanus toxoid and phytohemagglutinin were effective stimulants of proliferation, lymphocytes from arthritis patients were suppressed relative to those of normal individuals. Analysis of arthritic articular cartilage by these techniques failed to demonstrate the presence of antigen(s) stimulating proliferation of peripheral blood lymphocytes.

Observations made during treatment of juvenile pseudarthrosis by pulsing electromagnetic fields (PEMF) suggested that bone growth might be altered. PEMF applied to immature rabbits under conditions of continuous stimulation (24 hours/day for 8 weeks) produced no major changes in bone growth. Continuous stimulation by PEMF induced a statistically significant increase (22%) in femoral articular cartilage glycosaminoglycan. Intermittent PEMF stimulation (12 hours with stimulation/12 hours without stimulation) for 18 weeks produced no significant change in bone growth or time of epiphyseal plate closure. No significant changes in the physical characteristics of growing bone were observed with any treatment.

In order to obtain a phenotypically stable cell population of chondrocytes, high density primary monolayer cultures of bovine articular chondrocytes were established. Using these cultures, a specific insulin-like growth factor-I/somatomedin-C (IGF-I/SM-C) receptor was demonstrated and characterized. At 15 degrees C steady-state binding was attained by 5 h, and averaged 25% per 2.2 X 10(6) cells. Fifty per cent displacement of 125I-labelled IGF-I/SM-C by unlabelled IGF-I/SM-C occurred at concentrations of only 2.3 ng/ml, whereas IGF-II and porcine insulin were approximately 15- and 1000-fold less potent respectively. Scatchard analysis gave a linear plot, with a calculated association constant of 2.26 X 10(9) l/mol and a receptor number of 15 400 sites per cell. Preincubation of chondrocyte monolayers with either IGF-I SM-C or porcine insulin at 37 degrees C for 20 h resulted in reduction of 125I-labelled IGF-I/SM-C binding in a dose-dependent manner, although higher concentrations were required with insulin. More than 40% down-regulation of the receptor occurred with IGF-I/SM-C at concentrations of 10 nmol/l and nearly 70% reduction at 50 nmol/l. Interestingly, after preincubation with either human (h) or bovine (b) GH, 40% down-regulation of 125I-labelled IGF-I/SM-C binding was observed at concentrations of 10 mumol/l. Local production of IGF-I/SM-C by chondrocytes in response to GH stimulation may have occurred, but, because only 120 pmol IGF-I/SM-C and less than 30 pmol IGF-I/SM-C per litre were recovered from serum-free conditioned media preincubated with bGH and hGH respectively, this was not established.(ABSTRACT TRUNCATED AT 250 WORDS)

We report effects of adding insulin-like growth factor I (IGF-I) and methionyl human growth hormone (GH), alone or in combination, to adult bovine articular chondrocytes plated at high density. Purified human and synthetic IGF-I stimulated chondrocyte DNA and proteoglycan synthesis. GH had no effect on either process. However, GH added in combination with IGF-I increased proteoglycan, cell-associated proteoglycan, and keratan sulfate synthesis over levels observed with IGF-I alone. IGF-I and GH did not alter the hydrodynamic size of proteoglycans or synthesis of collagen. Our results show that GH and IGF-I act together to stimulate adult chondrocyte extracellular matrix synthesis.

The purpose of this study was to compare the effects of human recombinant interleukin 1, alpha and beta, on articular cartilage. The effects of rIL-1 alpha and rIL-1 beta on proteoglycan degradation and synthesis following treatment of bovine articular cartilage in serum-free organ culture were quantified. Purified human IL-1 and both rIL-1 alpha and rIL-1 beta induced a two-fold or greater increase in glycosaminoglycan (GAG) release from cultured articular cartilage. Levels or rIL-1 alpha as low as 15 pM induced increased proteoglycan degradation whereas identical levels of rIL-1 beta did not. Killing of the cartilage cells abolished induced GAG release by all forms of IL-1. Analysis of proteoglycan size following IL-1 treatment showed limited degradation of material released into the culture medium or remaining within cartilage. Both forms of recombinant IL-1 inhibited GAG synthesis when continually present in the culture medium. Actinomycin D and cycloheximide inhibited IL-1 dependent cartilage destruction whereas indomethacin did not.

The timing and molecular profile of cartilage destruction in Escherichia coli and Staphylococcus aureus infectious arthritis and killed Mycobacterium butyricum adjuvant arthritis are presented. Infectious arthritis was studied for 3 weeks; cartilage samples were analyzed at 2, 10, and 21 days. At 48 h postinfection, glycosaminoglycan content was reduced by 20% (p less than 0.05) in E. coli infected knees and by 42% (p less than 0.05) in tibial plateau cartilage of S. aureus infected knees. By the 3rd week of infection, glycosaminoglycan losses amounted to as much as 73% (p less than 0.005). In comparison, collagen losses were not significant prior to the 3rd week of infection, at which time 42% (p less than 0.05) was lost. Adjuvant arthritic tibial plateau cartilage was examined at 1, 3 and 12 weeks. Glycosaminoglycans decreased by 42% the 1st week, plateauing at 62% by the 3rd and 12th weeks. Collagen degradation began at 3 weeks (28% loss, p less than 0.10) and by the 12th week was reduced by 49% (p less than 0.005). Analysis of the individual species of glycosaminoglycan showed a parallel loss of chondroitin sulfate and keratan sulfate. Fractionation of glycosaminoglycans with respect to size produced no evidence of shortened chains in cartilage from infected joints. Hyaluronic acid losses were greatest when collagen was significantly decreased. The pattern by which chondroitin and keratan sulfates are lost demonstrates that a prominent feature of infectious and noninfectious inflammatory arthritis is a rapid loss of proteoglycan subunits that precedes collagen loss.

Patients with juvenile arthritis (JA) spontaneously produced a substance which accelerated proteoglycan loss from cultured articular cartilage. Peripheral blood mononuclear cells (PBMC) from 15 patients with JA were cultured for varying days, and cell-free PBMC conditioned media were added to articular cartilage cultures. Release of proteoglycan and collagen from cartilage was quantified by analysis of chondroitin sulfate and hydroxyproline content, respectively, after 4 days of culture. Conditioned media from PBMC of patients with systemic onset JA (3/3) and 5/7 patients with polyarticular JA increased release of proteoglycan when added to cartilage cultures. Mitogen stimulation of the PBMC was unnecessary for activity and addition of mitogen did not alter proteoglycan release. The PBMC conditioned media from the other patients (2/7) with polyarticular JA, from patients (3/3) with systemic onset JA which had progressed to polyarticular JA, and from patients with pauciarticular JA, did not enhance proteoglycan release without mitogen stimulation. PBMC of normal children produced media which enhanced proteoglycan release after mitogen stimulation. No conditioned medium accelerated proteoglycan release if cartilage was freeze killed before culture and none tested reduced cartilage collagen content.

This article describes the macromolecular changes in keratan sulfate and proteoglycan that occur in rabbit articular cartilage during postnatal development. Articular cartilage glycosaminoglycans from femoral condyles and the tibial plateaus of rabbits at 8, 12, 18, and 26 weeks and 2 years of age were extracted, fractionated, and quantified. The predominant glycosaminoglycan present in articular cartilage at 8 weeks was chondroitin sulfate. During subsequent maturation the relative proportions of keratan sulfate and chondroitin sulfate varied inversely. The greatest increase in the amount of keratan sulfate present in cartilage was observed between 12 and 26 weeks of age. Hyaluronic acid content was measurable at 12 weeks; afterward the amount remained relatively constant with age. Proteoglycans, extracted from 6-, 12-, and 22-week-old rabbit femoral and tibial cartilage in the presence of protease inhibitors, were analyzed on columns of Sepharose CL-2B. Cartilage proteoglycans decreased in hydrodynamic size between 12 and 22 weeks, corresponding to the period of maximal change in content of keratan and chondroitin sulfate.