Deficiency of hyaluronan synthase 1 (Has1) results in chronic joint inflammation and widespread intra-articular fibrosis in a murine model of knee joint cartilage damage.

OBJECTIVE: Articular cartilage defects commonly result from traumatic injury and predispose to degenerative joint diseases. To test the hypothesis that aberrant healing responses and chronic inflammation lead to osteoarthritis (OA), we examined spatiotemporal changes in joint tissues after cartilage injury in murine knees. Since intra-articular injection of hyaluronan (HA) can attenuate injury-induced osteoarthritis in wild-type (WT) mice, we investigated a role for HA in the response to cartilage injury in mice lacking HA synthase 1 (Has1(-/-)). DESIGN: Femoral groove cartilage of WT and Has1(-/-) mice was debrided to generate a non-bleeding wound. Macroscopic imaging, histology, and gene expression were used to evaluate naïve, sham-operated, and injured joints. RESULTS: Acute responses (1-2 weeks) in injured joints from WT mice included synovial hyperplasia with HA deposition and joint-wide increases in expression of genes associated with inflammation, fibrosis, and extracellular matrix (ECM) production. By 4 weeks, some resurfacing of damaged cartilage occurred, and early cell responses were normalized. Cartilage damage in Has1(-/-) mice also induced early responses; however, at 4 weeks, inflammation and fibrosis genes remained elevated with widespread cartilage degeneration and fibrotic scarring in the synovium and joint capsule. CONCLUSIONS: We conclude that the ineffective repair of injured cartilage in Has1(-/-) joints can be at least partly explained by the markedly enhanced expression of particular genes in pathways linked to ECM turnover, IL-17/IL-6 cytokine signaling, and apoptosis. Notably, Has1 ablation does not alter gross HA content in the ECM, suggesting that HAS1 has a unique function in the metabolism of inflammatory HA matrices.

Human genome-wide expression analysis reorients the study of inflammatory mediators and biomechanics in osteoarthritis.

A major objective of this article is to examine the research implications of recently available genome-wide expression profiles of cartilage from human osteoarthritis (OA) joints. We propose that, when viewed in the light of extensive earlier work, this novel data provides a unique opportunity to reorient the design of experimental systems toward clinical relevance. Specifically, in the area of cartilage explant biology, this will require a fresh evaluation of existing paradigms, so as to optimize the choices of tissue source, cytokine/growth factor/nutrient addition, and biomechanical environment for discovery. Within this context, we firstly discuss the literature on the nature and role of potential catabolic mediators in OA pathology, including data from human OA cartilage, animal models of OA, and ex vivo studies. Secondly, due to the number and breadth of studies on IL-1ß in this area, a major focus of the article is a critical analysis of the design and interpretation of cartilage studies where IL-1ß has been used as a model cytokine. Thirdly, the article provides a data-driven perspective (including genome-wide analysis of clinical samples, studies on mutant mice, and clinical trials), which concludes that IL-1ß should be replaced by soluble mediators such as IL-17 or TGF-ß1, which are much more likely to mimic the disease in OA model systems. We also discuss the evidence that changes in early OA can be attributed to the activity of such soluble mediators, whereas late-stage disease results more from a chronic biomechanical effect on the matrix and cells of the remaining cartilage and on other local mediator-secreting cells. Lastly, an updated protocol for in vitro studies with cartilage explants and chondrocytes (including the use of specific gene expression arrays) is provided to motivate more disease-relevant studies on the interplay of cytokines, growth factors, and biomechanics on cellular behavior.

Initial application of EPIC-µCT to assess mouse articular cartilage morphology and composition: effects of aging and treadmill running.

OBJECTIVE: The current study was undertaken to adapt Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-µCT) to mouse articular cartilage (AC), which presents a particular challenge because it is thin (30 µm) and has a small volume (0.2-0.4 mm(3)), meaning there is only approximately 2-4 µg of chondroitin sulfate (CS) glycosaminoglycan per joint surface cartilage. DESIGN: Using 6 µm isotropic voxels and the negatively charged contrast agent ioxaglate (Hexabrix), we optimized contrast agent concentration and incubation time, assessed two methods of tissue preservation (formalin fixation and freezing), examined the effect of ex vivo chondroitinase ABC digestion on X-ray attenuation, assessed accuracy and precision, compared young and skeletally mature cartilage, and determined patterns of degradation in a murine cartilage damage model induced by treadmill running. RESULTS: The optimal concentration of the contrast agent was 15%, formalin fixation was preferred to freezing, and 2 h of incubation was needed to reach contrast agent equilibrium with formalin-fixed specimens. There was good agreement with histologic measurements of cartilage thickness, although µCT over-estimated thickness by 13% (5 µm) in 6-week-old mice. Enzymatic release of 0.8 µg of chondrotin sulfate (about 40% of the total) increased X-ray attenuation by 17%. There was a 15% increase in X-ray attenuation in 14-week-old mice compared to 6-week-old mice (P < 0.001) and this corresponded to 65% decrease in CS content at 14 weeks. The older mice also had reductions of 33% in cartilage thickness and 44% in cartilage volume (P < 0.001). Treadmill running induced a 16% decrease in cartilage thickness (P = 0.012) and a 12% increase in X-ray attenuation (P = 0.006) in 14-week-old mice. CONCLUSION: This technique enables non-destructive visualization and quantification of murine femoral AC in three dimensions with anatomic specificity and should prove to be a useful new tool in studying degeneration of cartilage in mouse models.

The relationship between fibrogenic TGFß1 signaling in the joint and cartilage degradation in post-injury osteoarthritis.

OBJECTIVE: To review the literature on modulation of chondrocyte activities in the osteoarthritic joint, and to discuss these changes in relation to established hard and soft tissue repair paradigms, with an emphasis on transforming growth factor beta (TGFß1)-mediated signaling which can promote either a chondrogenic or fibrogenic phenotype. METHODS: Papers addressing the close relationship between repair in general, and the specific post-injury response of joint tissues are summarized. Different interpretations of the role of TGFß1 in the emergence of an "osteoarthritic" chondrocyte are compared and the phenotypic plasticity of "reparative" progenitor cells is examined. Lastly, emerging data on a central role for A-Disintegrin-And-Metalloproteinase-with-Thrombospondin-like-Sequences-5 (ADAMTS5) activity in modulating TGFß1 signaling through activin receptor-like kinase 1 (ALK1) and activin receptor-like kinase 5 (ALK5) pathways is discussed. RESULTS: The review illustrates how a transition from ALK5-mediated fibrogenic signaling to ALK1-mediated chondrogenic signaling in joint cells represents the critical transition from a non-reparative to a reparative cell phenotype. Data from cell and in vivo studies illustrates the mechanism by which ablation of ADAMTS5 activity allows the transition to reparative chondrogenesis. Multiple large gene expression studies of normal and osteoarthritis (OA) human cartilages (CAs) also support an important role for TGFß1-mediated pro-fibrogenic activities during disease progression. CONCLUSIONS: We conclude that progressive articular CA damage in post-injury OA results primarily from biomechanical, cell biologic and mediator changes that promote a fibroblastic phenotype in joint cells. Since ADAMTS5 and TGFß1 appear to control this process, agents which interfere with their activities may not only enhance endogenous CA repair in vivo, but also improve the properties of tissue-engineered CA for implantation.

The effects of oral glucosamine on joint health: is a change in research approach needed?

OBJECTIVE: Oral glucosamine (GlcN) has been widely studied for its potential therapeutic benefits in alleviating the pain and disability of osteoarthritis (OA). Its popularity has grown despite ongoing controversy regarding its effectiveness vs placebo in clinical trials, and lack of information regarding possible mechanisms of action. Here, we review the state of knowledge concerning the biology of GlcN as it relates to OA, and discuss a framework for future research directions. METHODS: An editorial "narrative" review of peer-reviewed publications is organized into four topics (1) Chemistry and pharmacokinetics of GlcN salts (2) Biological effects of GlcN salts in vitro (3) Therapeutic effects of GlcN salts in animal models of OA and (4) GlcN salts in the treatment of clinical OA. RESULTS: Data reporting potent pleiotropic activities of GlcN in in vitro cell and explant cultures are discussed in the context of the established pharmacokinetic data in humans and animals. The available clinical trial data are discussed to place the patient in the context of controlled research on disease management. CONCLUSIONS: Future research to determine therapeutic mechanisms of GlcN salt preparations will require use of standardized and clinically relevant in vitro assay systems and in vivo animal models for testing, as well as development of new outcome measures for inflammation and pain pathways in human OA.

Adult bone marrow stromal cell-based tissue-engineered aggrecan exhibits ultrastructure and nanomechanical properties superior to native cartilage.

OBJECTIVE: To quantify the structural characteristics and nanomechanical properties of aggrecan produced by adult bone marrow stromal cells (BMSCs) in peptide hydrogel scaffolds and compare to aggrecan from adult articular cartilage. DESIGN: Adult equine BMSCs were encapsulated in 3D-peptide hydrogels and cultured for 21 days with TGF-ß1 to induce chondrogenic differentiation. BMSC-aggrecan was extracted and compared with aggrecan from age-matched adult equine articular cartilage. Single molecules of aggrecan were visualized by atomic force microscopy-based imaging and aggrecan nanomechanical stiffness was quantified by high resolution force microscopy. Population-averaged measures of aggrecan hydrodynamic size, core protein structures and CS sulfation compositions were determined by size-exclusion chromatography, Western analysis, and fluorescence-assisted carbohydrate electrophoresis (FACE). RESULTS: BMSC-aggrecan was primarily full-length while cartilage-aggrecan had many fragments. Single molecule measurements showed that core protein and GAG chains of BMSC-aggrecan were markedly longer than those of cartilage-aggrecan. Comparing full-length aggrecan of both species, BMSC-aggrecan had longer GAG chains, while the core protein trace lengths were similar. FACE analysis detected a ∼ 1:1 ratio of chondroitin-4-sulfate to chondroitin-6-sulfate in BMSC-GAG, a phenotype consistent with aggrecan from skeletally-immature cartilage. The nanomechanical stiffness of BMSC-aggrecan was demonstrably greater than that of cartilage-aggrecan at the same total sGAG (fixed charge) density. CONCLUSIONS: The higher proportion of full-length monomers, longer GAG chains and greater stiffness of the BMSC-aggrecan makes it biomechanically superior to adult cartilage-aggrecan. Aggrecan stiffness was not solely dependent on fixed charge density, but also on GAG molecular ultrastructure. These results support the use of adult BMSCs for cell-based cartilage repair.

The human pharmacokinetics of oral ingestion of glucosamine and chondroitin sulfate taken separately or in combination.

OBJECTIVE: As part of the National Institutes of Health (NIH)-sponsored Glucosamine/Chondroitin sulfate Arthritis Intervention Trial (GAIT) our objective here was to examine (1) the pharmacokinetics (PK) of glucosamine (GlcN) and chondroitin sulfate (CS) when taken separately or in combination as a single dose in normal individuals (n=29) and (2) the PK of GlcN and CS when taken as a single dose after 3 months daily dosing with GlcN, CS or GlcN+CS, in patients with symptomatic knee pain (n=28). METHODS: The concentration of GlcN in the circulation was determined by established fluorophore-assisted carbohydrate electrophoresis (FACE) methods. The hydrodynamic size and disaccharide composition of CS chains in the circulation and dosage samples was determined by Superose 6 chromatography and FACE. RESULTS: We show that circulating levels of CS in human plasma are about 20 microg/ml. Most significantly, the endogenous concentration and CS disaccharide composition were not detectably altered by ingestion of CS, when the CS was taken alone or in combination with GlcN. On the other hand, the Cmax (single-dose study) and AUC values (multiple-dose study) for ingested GlcN were significantly reduced by combination dosing with CS, relative to GlcN dosing alone. CONCLUSIONS: We conclude that pain relief perceived following ingestion of CS probably does not depend on simultaneous or prior intake of GlcN. Further, such effects on joint pain, if present, probably do not result from ingested CS reaching the joint space but may result from changes in cellular activities in the gut lining or in the liver, where concentrations of ingested CS, or its breakdown products, could be substantially elevated following oral ingestion. Moreover, since combined dosing of GlcN with CS was found to reduce the plasma levels seen with GlcN dosing alone, any improved pain relief by combination dosing cannot be explained by higher circulating concentrations of GlcN.

Keratan sulphate in the interglobular domain has a microstructure that is distinct from keratan sulphate elsewhere on pig aggrecan.

The microstructure of keratan sulphate purified from the interglobular domain, the keratan sulphate-rich region and total aggrecan was compared using fluorophore-assisted-carbohydrate-electrophoresis. Keratan sulphate in the interglobular domain was substantially less sulphated than keratan sulphate elsewhere on aggrecan, based on the ratio of unsulphated: monosulphated disaccharides generated by endo-beta-galactosidase digestion, and the ratio of monosulphated: disulphated disaccharides generated by keratanase II digestion. The ratio of unsulphated: monosulphated: disulphated disaccharides was 1:4:5 for keratan sulphate from total aggrecan and the keratan sulphate-rich region, but only 1:0.9:0.8 for the interglobular domain. These results show that keratan sulphate in the interglobular domain of pig aggrecan has a microstructure that is distinct from keratan sulphate in the keratan sulphate-rich region.

Co-culture of mechanically injured cartilage with joint capsule tissue alters chondrocyte expression patterns and increases ADAMTS5 production.

We studied changes in chondrocyte gene expression, aggrecan degradation, and aggrecanase production and activity in normal and mechanically injured cartilage co-cultured with joint capsule tissue. Chondrocyte expression of 21 genes was measured at 1, 2, 4, 6, 12, and 24h after treatment; clustering analysis enabled identification of co-expression profiles. Aggrecan fragments retained in cartilage and released to medium and loss of cartilage sGAG were quantified. Increased expression of MMP-13 and ADAMTS4 clustered with effects of co-culture, while increased expression of ADAMTS5, MMP-3, TGF-beta, c-fos, c-jun clustered with cartilage injury. ADAMTS5 protein within cartilage (immunohistochemistry) increased following injury and with co-culture. Cartilage sGAG decreased over 16-days, most severely following injury plus co-culture. Cartilage aggrecan was cleaved at aggrecanase sites in the interglobular and C-terminal domains, resulting in loss of the G3 domain, especially after injury plus co-culture. Together, these results support the hypothesis that interactions between injured cartilage and other joint tissues are important in matrix catabolism after joint injury.

Superficial zone chondrocytes in normal and osteoarthritic human articular cartilages synthesize novel truncated forms of inter-alpha-trypsin inhibitor heavy chains which are attached to a chondroitin sulfate proteoglycan other than bikunin.

OBJECTIVE: We have examined the occurrence of the inflammation-associated inter-alpha-trypsin inhibitor (IalphaI) components, bikunin, heavy chain (HC)1 and HC2 in normal cartilage and osteoarthritis (OA) cartilage and synovial fluids. DESIGN/METHODS: Cartilage extracts from normal donors and late-stage OA patients, and synovial fluids from OA patients were studied by Western blot with multiple antibodies to bikunin, HC1 and HC2. Cell and matrix localization was determined by immunohistochemistry and mRNA by RT-PCR. RESULTS: Bikunin.chondroitin sulfate (CS) and IalphaI were abundant in OA cartilages, but virtually undetectable in normal. In both OA and normal cartilages, HCs were largely present in a novel C-terminally truncated 50-kDa form, with most, if not all of these being attached to CS on a proteoglycan other than bikunin. Synovial fluids from OA patients contained bikunin.CS and full-length (approximately 90 kDa) HCs linked to hyaluronan (HA) as HC.HA (SHAP.HA). Immunohistochemistry showed intracellular and cell-associated staining for bikunin and HCs, consistent with their synthesis by superficial zone chondrocytes. PCR on multiple human normal and OA cartilage samples detected transcripts for HC1 and HC2 but not for bikunin. In OA cartilages, immunostaining was predominantly matrix-associated, being most intense in regions with a pannus-like fibrotic overgrowth. CONCLUSION: The truncated structure of HCs, their attachment to a proteoglycan other than bikunin, PCR data and intracellular staining are all consistent with synthesis of HC1 and HC2 by human articular chondrocytes. The presence of bikunin.CS and IalphaI in OA cartilage, but not in normal, appears to be due to diffusional uptake and retention through fibrillated (but not deeply fissured) cartilage surfaces.

Structural studies on proteoglycan catabolism in rabbit articular cartilage explant cultures.

Mature rabbit articular cartilage cultures have been used to study the catabolism of aggregating proteoglycan monomers in normal cartilage. During the first 4 days of culture, about 40% of monomers are degraded and lose the ability to bind to hyaluronate. The non-aggregating products (NAgg-PG) have been isolated and compared structurally and immunologically to aggregating monomers (Agg-PG) purified from fresh tissue. The results show that: (1) NAgg-PG are smaller, more heterogeneous in size and have a lower protein/glycosaminoglycan ratio than Agg-PG. (2) NAgg-PG and Agg-PG have a very similar chondroitin sulfate/keratan sulfate ratio. (3) NAgg-PG have 25-50% lower disulfide content than Agg-PG. (4) NAgg-PG have only about 20% of the reactivity of Agg-PG towards a monoclonal antibody (12-20/1-C-6) specific for the hyaluronate binding region of the core protein. These results provide further evidence that proteoglycan catabolism in cartilage explants involves proteolysis of core protein resulting in separation of the hyaluronate binding region from the glycosaminoglycan-rich regions.

Age-related changes in the kinetics of release of proteoglycans from normal rabbit cartilage explants.

The release of proteoglycans from explant cultures of articular cartilage from immature and mature rabbits has been studied with the following results. At both ages the tissue proteoglycan was released in two phases: an initial extensive release (day 0 to 3) and a period of slow release (day 4 to 15). The percentage released in the initial phase was, however, significantly greater for mature (55%) than immature (38%) explants. At both ages the newly synthesized proteoglycans (in vivo labeled) were also released in two kinetic pools. Thus, graphical analysis of release data readily resolved the disappearance curves into two linear components with in vitro half-lives of 1 day and 22 days. Again, the percentage in the short half-life pool was much greater for mature (70%) than immature (40%) explants. At both ages the initial release was largely chondrocyte-mediated since freeze-thawing the tissue before culture markedly reduced proteoglycan release. At both ages the released proteoglycans were smaller than equivalent preparations of extracted proteoglycans and they were much less capable of forming aggregates with hyaluronate. The results show that there are age-dependent changes in rabbit articular cartilage that increase the proportion of proteoglycans, both total and newly synthesized, that are susceptible to rapid chondrocyte-mediated catabolism in explant cultures.

Structure of chondroitin sulfate on aggrecan isolated from bovine tibial and costochondral growth plates.

The structure of chondroitin sulfate on aggrecan isolated from the rib and proximal tibial growth plates of bovine fetuses was investigated, and the previously reported increase in the hydrodynamic size of chondroitin sulfate chains between the reserve and hypertrophic zones of the rib was confirmed in the tibial growth plate. Superose 6 gel chromatography, calibrated for chondroitin sulfate chain length by monosaccharide analysis, showed that the average molecular mass of chondroitin sulfate in the reserve and maturing zones of both growth plates was 21,600 and 30,400, respectively. Determination by capillary zone electrophoresis of the disaccharide composition of chains following chondroitinase digestion showed that delta Di-0S, delta Di-4S, and delta Di-6S together accounted for more than 98% of the disaccharides in the digests from all zones of both growth plates; delta disulfated and delta trisulfated disaccharides were not detected. Furthermore, this analysis revealed a gradient in chondroitin sulfate composition from the reserve to the hypertrophic zone, characterized by a marked increase in the content of delta Di-6S (from about 32% to about 52%) and a marked decrease in the content of delta Di-4S (from about 53% to about 35%). Moreover, this altered pattern of sulfation was detected on chains of all sizes in the hypertrophic zone, suggesting that a proportion of the reserve zone aggrecan might be removed and replaced with aggrecan rich in chondroitin-6-sulfate synthesized during the proliferative and maturation stages of the resident chondrocytes. These data are discussed in relation to the biosynthetic mechanisms that control chondroitin sulfate chain length and sulfation on aggrecan and their modification during chondrocyte proliferation, maturation, and hypertrophy in the growth plate.

Proteoglycans and glycosaminoglycan fine structure in the mouse tail tendon fascicle.

The isolated mouse tail tendon fascicle, a functional and homogenous volume of tendon extracellular matrix, was utilized as an experimental system to examine the structure function relationships in tendon. Our previous work using this model system demonstrated relationships between mean collagen fibril diameter and fascicle mechanical properties in isolated tail tendon fascicles from three different groups of mice (3-week and 8-week control and 8-week Mov13 transgenic) K.A. Derwin, L.J. Soslowsky, J. Biomech. Eng. 121 (1999) 598-604. These groups of mice were chosen to obtain tendon tissues with varying collagen fibril structure and/or biochemistry, such that relationships with material properties could be investigated. To further investigate the molecular details of matrix composition and organization underlying tendon function, we report now on the preparation, characterization, and quantitation of fascicle PGs (proteoglycans) from these three groups. The chondroitin sulfate/dermatan sulfate (CS/DS)-substituted PGs, biglycan and decorin, which are the abundant proteoglycans of whole tendons, were also shown to be the predominant PGs in isolated fascicles. Furthermore, similar to the postnatal maturation changes in matrix composition previously reported for whole tendons, isolated fascicles from 8-week mice had lower CS/DS PG contents (both decorin and biglycan) and a higher collagen content than 3-week mice. In addition, CS/DS chains substituted on PGs from 8-week fascicles were shorter (based on a number average) and richer in disulfated disaccharide residues than chains from 3-week mice. Fascicles from 8-week Mov13 transgenic mice were found to contain similar amounts of total collagen and total CS/DS PG as age-matched controls, and CS/DS chain lengths and sulfation also appeared normal. However, both decorin and biglycan in Mov13 tissue migrated slightly faster on sodium dodecyl sulfate polyacrylamide gel electorphoresis (SDS-PAGE) than the corresponding species from 8-week control, and biglycan from the 8-week Mov 13 fascicles appeared to migrate as a more polydisperse band, suggesting the presence of a unique PG population in the transgenic tissue. These observations, together with our biomechanical data [Derwin and Soslowsky, 1999] suggest that compensatory pathways of extracellular matrix assembly and maturation may exist, and that tissue mechanical properties may not be simply determined by the contents of individual matrix components or collagen fibril size.

Proteoglycans of human rotator cuff tendons.

Rotator cuff and biceps tendons that appeared grossly normal were procured from adult cadavers without a history of shoulder problems. These tendons were analyzed for the amount and type of glycosaminoglycan, type of proteoglycan, and histology. When compared with the distal/tensional region of biceps tendon, the glycosaminoglycan content of supraspinatus, infraspinatus, and subscapularis tendons was 2.5-fold higher and the glycosaminoglycan content of the proximal/compressed region of biceps tendon was 3-fold higher. The ratio of hyaluronic acid to chondroitin sulfate/dermatan sulfate in all three cuff tendons was approximately 1. Rotator cuff tendons contained large proteoglycan similar to aggrecan, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel migration elution from Sepharose CL-4B, and content of both chondroitin sulfate and keratan sulfate chains. Both decorin and biglycan were also present, as demonstrated by migration in sodium dodecyl sulfate-polyacrylamide gels and core protein immunoreactivity. In contrast decorin was the only proteoglycan prominent in distal/tensional regions of biceps tendon. Histological analysis showed layers of loosely organized alcian blue-stained material running between the longitudinal collagen fiber bundles. The proteoglycan content of rotator cuff tendons was similar to fibrocartilage in tendons that have been subjected to compressive loads in situ. This suggests that cells of normal adult rotator cuff tendons have adapted to loads distinct from pure tension. However, the histological organization did not resemble mature fibrocartilage. The increased amount of proteoglycan in rotator cuff tendons may serve to separate and lubricate collagen bundles as they move relative to each other during normal shoulder motion.

Biosynthetic response of cartilage explants to dynamic compression.

The biosynthetic response of calf articular cartilage explants to dynamic compression was examined over a wide range of amplitudes, waveforms, and frequencies. Glycosaminoglycan synthesis was assessed by 35S-sulfate incorporation, and amino acid uptake and protein synthesis were assessed by 3H-proline incorporation. Two culture chambers were designed to allow uniaxial radially unconfined compression and mechanical testing of cartilage disks: one chamber was used inside a standard incubator; the other was used with a mechanical spectrometer and allowed load and displacement to be monitored during compression. Dynamic stiffness measurements of 3-mm diameter disks identified a characteristic frequency [0.001 Hz (cycles/sec)] that separated low- and high-frequency regimes in which different flow and deformation phenomena predominated; e.g., at 0.0001-0.0001 Hz, significant fluid was exuded from cartilage disks, whereas at 0.01-1 Hz, hydrostratic pressure increased within disks. At the higher frequencies, oscillatory strains of only approximately 1-5% stimulated 3H-proline and 35S-sulfate incorporation by approximately 20-40%. In contrast, at the lower frequencies (a) compressions of less than 5% had no effect, consistent with the dosimetry of biosynthetic inhibition by static compression (approximately 25% compression caused a approximately 20% inhibition of radiolabel incorporation), and (b) higher amplitudes (cycling between disk thicknesses of 1.25 and 0.88-1.00 mm) stimulated 3S-sulfate incorporation by approximately 20-40%, consistent with the kinetics of response to a single 2-h compression and release. None of the compression protocols was associated with detectable alterations in (e.g., compression-induced depletion of) total glycosaminoglycan content. This study provides a framework for identifying both the physical and biological mechanisms by which dynamic compression can modulate chondrocyte biosynthesis. In addition, the culture and compression methodology potentially allows in vitro evaluation of clinical strategies of continuous passive motion therapy to stimulate cartilage remodeling.

Immunology of chondroitin/dermatan sulfate.

Variable substitutions and locations of the sulfate esters along the backbone of chondroitin/dermatan sulfate chains, combined with their carbohydrate structures, present topographies to immune systems which can be recognized as antigenic. This has led to the development of a number of monoclonal antibodies which recognize distinct epitopes in the native structures of these glycosaminoglycan chains. In some studies, the original chondroitin/dermatan sulfate proteoglycan was digested with chondroitinase enzymes before being used as an immunogen. in this case, the linkage oligosaccharides remaining bound to the core protein contain a modified (4,5-unsaturated) hexuronic acid derivative at their non-reducing ends as a result of the eliminase mechanism of the enzyme. This 'haptenic' structure is highly antigenic and has led to the development of a number of monoclonal antibodies which recognize this structure as part of their epitopes. Examples of the use of some of these monoclonal antibodies for localization of proteoglycan structures in tissue sections and on transblots are described. The precise structures are known for only a few of the native epitopes recognized by these monoclonal antibodies. Recent analytical methods have been developed for determining structures of chondroitin sulfate oligosaccharides. An example of the use of these methods to analyze the structures of the non-reducing termini of chondroitin/dermatan sulfate chains is discussed. The results show their potential value for quantifying the native epitope recognized by a monoclonal antibody, designated 3B3, which recognizes chains terminated by glucuronic acid-N-acetylgalactosamine-6-sulfate. Such methods should be useful for determining the epitope structures for other monoclonal antibodies in this class.

Changes in sulfation patterns of chondroitin sulfate in equine articular cartilage and synovial fluid in response to aging and osteoarthritis.

OBJECTIVES: To determine effects of aging on sulfation of chondroitin sulfate (CS) in articular cartilage and synovial fluid from normal equine middle carpal joints, and to determine whether CS compositional analysis can be used to assess alterations in proteoglycan turnover in degenerative cartilage obtained from horses with carpal osteochondral fractures. SAMPLE POPULATION: Carpal articular cartilage and synovial fluid from 44 cadavers with normal joints and from 16 Thoroughbred racehorses during routine carpal arthroscopic surgery. PROCEDURE: After papain/chondroitinase digestion of cartilage, CS disaccharides (unsulfated disaccharide delta Di0S, and monosulfated disaccharides delta Di4S and delta Di6S) were quantified by capillary zone electrophoresis. The CS was purified from synovial fluid chondroitinase digested, and analyzed. The CS nonreducing terminal residues, N-acetylgalctosamine (galNAc) or glucuronic acid adjacent to a 4-sulfated or 6-sulfated galNAc, were quantified. RESULTS: In cartilage, the delta Di6S-to-delta Di4S ratio increased with age; in degenerative cartilage, this ratio was not significantly different from the normal value. Percentage of delta Di0S decreased with age and was significantly higher in degenerative than in normal cartilage. The galNAc4S and galNAc4,6S represented > or = 96% of the terminal residues. There was a significant decrease in 6-sulfation of the terminal residues in degenerative cartilage. CONCLUSIONS: 6-Sulfation of internal and terminal CS residues increased with age. Cartilage degeneration in racehorses was accompanied by deposition of CS chains with altered sulfation patterns, in normal and diseased joints of horses > 2 years old, synovial fluid CS was not indicative of cartilage CS and may represent turnover products of a subpopulation of proteoglycan within the matrix.

Adult equine bone marrow stromal cells produce a cartilage-like ECM mechanically superior to animal-matched adult chondrocytes.

Our objective was to evaluate the age-dependent mechanical phenotype of bone marrow stromal cell- (BMSC-) and chondrocyte-produced cartilage-like neo-tissue and to elucidate the matrix-associated mechanisms which generate this phenotype. Cells from both immature (2-4 month-old foals) and skeletally-mature (2-5 year-old adults) mixed-breed horses were isolated from animal-matched bone marrow and cartilage tissue, encapsulated in self-assembling-peptide hydrogels, and cultured with and without TGF-beta1 supplementation. BMSCs and chondrocytes from both donor ages were encapsulated with high viability. BMSCs from both ages produced neo-tissue with higher mechanical stiffness than that produced by either young or adult chondrocytes. Young, but not adult, chondrocytes proliferated in response to TGF-beta1 while BMSCs from both age groups proliferated with TGF-beta1. Young chondrocytes stimulated by TGF-beta1 accumulated ECM with 10-fold higher sulfated-glycosaminoglycan content than adult chondrocytes and 2-3-fold higher than BMSCs of either age. The opposite trend was observed for hydroxyproline content, with BMSCs accumulating 2-3-fold more than chondrocytes, independent of age. Size-exclusion chromatography of extracted proteoglycans showed that an aggrecan-like peak was the predominant sulfated proteoglycan for all cell types. Direct measurement of aggrecan core protein length and chondroitin sulfate chain length by single molecule atomic force microscopy imaging revealed that, independent of age, BMSCs produced longer core protein and longer chondroitin sulfate chains, and fewer short core protein molecules than chondrocytes, suggesting that the BMSC-produced aggrecan has a phenotype more characteristic of young tissue than chondrocyte-produced aggrecan. Aggrecan ultrastructure, ECM composition, and cellular proliferation combine to suggest a mechanism by which BMSCs produce a superior cartilage-like neo-tissue than either young or adult chondrocytes.