Search

Pain-related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P-induced inflammatory processes are mediated via signaling through a G-protein-coupled receptor, that is, neurokinin-1 tachykinin receptor (NK(1)-R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF-2) or IL-1beta accelerate matrix degradation via a neural pathway upregulation of substance P and NK(1)-R. We show here that substance P stimulates the production of cartilage-degrading enzymes, such as matrix metalloproteinase-13 (MMP-13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK(1)-R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein-7, suggesting the dual role of substance P as both a pro-catabolic and anti-anabolic mediator of cartilage homeostasis. We report that bFGF-mediated stimulation of substance P and its receptor NK(1)-R is, in part, through an IL-1beta-dependent pathway.

To report an unusual phenotype of macular corneal dystrophy (MCDC1) associated with a novel CHST6 mutation transmitted via maternal isodisomy.Slit lamp examination of the patient and his parents was performed. DNA was collected from each individual for amplification and sequencing of the CHST6 coding region, as well as exons 4 and 12 of TGFBI. Serum antigenic keratan sulfate (AgKS) levels were measured for confirmation of the diagnosis and subtyping of MCDC1. Quantitative real-time PCR (qPCR) was performed to differentiate between homozygous and hemizygous sequence variants. Genotyping at 12 single nucleotide polymorphisms (SNPs) within and surrounding CHST6 was performed to determine the pattern of inheritance of mutations identified in CHST6.Examination of the proband revealed bilateral, discrete, axially distributed, gray-white deposits at the level of Bowman's layer, with diffuse fine corneal stromal haze. Screening of TGFBI exons 4 and 12 in the proband did not reveal any allelic variants. However, screening of CHST6 in the proband demonstrated a novel homozygous missense mutation involving a highly conserved amino acid (c.518TC; Leu173Pro) and undetectable serum AgKS levels in the proband confirmed the diagnosis of type I MCDC1. Quantitative PCR confirmed that both copies of CHST6 were present in the patient, excluding the possibility that the mutation was present in the hemizygous state. The results of genotyping were consistent with maternal isodisomy, as the patient was homozygous for an allele possessed by his mother at each SNP, two of which were informative and demonstrated nonpaternal inheritance.A phenotypically unusual variant of MCDC1 was found to be associated with the novel Leu173Pro mutation in CHST6, transmitted via uniparental isodisomy, a previously unreported pattern of inheritance in the corneal dystrophies.

Lipopolysaccharide (LPS) induces matrix degradation and markedly stimulates the production of several cytokines, i.e., interleukin-1beta, -6, and -10, by disc cells and chondrocytes. We performed a series of experiments to compare cellular responses of cells from the bovine intervertebral disc (nucleus pulposus and annulus fibrosus) and from bovine articular cartilage to LPS. Alginate beads containing cells isolated from bovine intervertebral discs and articular cartilage were cultured with or without LPS in the presence of 10% fetal bovine serum. The DNA content and the rate of proteoglycan synthesis and degradation were determined. In articular chondrocytes, LPS strongly suppressed cell proliferation and proteoglycan synthesis in a dose-dependent manner and stimulated proteoglycan degradation. Compared with articular chondrocytes, nucleus pulposus cells responded in a similar, although less pronounced manner. However, treatment of annulus fibrosus cells with LPS showed no significant effects on proteoglycan synthesis or degradation. A slight, but statistically significant, inhibition of cell proliferation was observed at high concentrations of LPS in annulus fibrosus cells. Thus, LPS suppressed proteoglycan synthesis and stimulated proteoglycan degradation by articular chondrocytes and nucleus pulposus cells. The effects of LPS on annulus fibrosus cells were minor compared with those on the other two cell types. The dissimilar effects of LPS on the various cell types suggest metabolic differences between these cells and may further indicate a divergence in pathways of LPS signaling and a differential sensitivity to exogenous stimuli such as LPS.

Summary Objective This study examined the effects of a growth factor, recombinant human osteogenic protein-1 (rhOP-1), on the formation of tissue-engineered cartilaginous tissue by adult bovine articular chondrocytes using the alginate-recovered-chondrocyte (ARC) method. Design To ascertain if rhOP-1 enhances the formation of the cell-associated matrix (CM) and the characteristics of CM formation, bovine articular chondrocytes were first cultured for up to 14 days in alginate beads in medium supplemented with serum, with or without rhOP-1. Then, the recovered chondrocytes and their associated CM were resuspended in medium, with or without OP-1, seeded onto culture inserts, and incubated for an additional 14 days. The fabricated ARC tissues were subjected to biochemical and histological analyses. Results The addition of rhOP-1 to the medium in the alginate bead culture step resulted in an increased accumulation of both proteoglycan (PG) and collagen, with a ratio of PG to collagen that was higher than that found in native adult cartilage. The addition of rhOP-1 in the second step had a similar stimulatory effect during 14 days of culture. Histological examination of the tissue formed under all conditions revealed a cartilage-like matrix, stained strongly by toluidine blue. The thickness of the tissues obtained from culture conditions that included the addition of rhOP-1 was four times greater than that of the tissues cultured without rhOP-1. Conclusions Using the ARC method, rhOP-1 enhanced the formation of matrix and generated a voluminous tissue-engineered cartilaginous construct. These characteristics may be beneficial in generating constructs that can cover large defects.

One approach to repairing articular defects is to regenerate cartilage by recapitulating the changes that occur during fetal and postnatal growth into adulthood, and to thereby restore functional biomechanical properties, especially those of the normally strong superficial region. The objectives of this study were (1) to characterize and compare tensile biomechanical properties of the superficial region of articular cartilage of the patellofemoral groove (PFG) and femoral condyle (FC) from bovine animals over a range of growth stages (third-trimester fetal, 1-3 week-old calf, and adult), and (2) to determine if these properties were correlated with collagen network components. With growth from the fetus to the adult, the equilibrium and dynamic tensile moduli and strength of cartilage samples increased by an average of 391-1060%, while the strain at the failure decreased by 43%. The collagen concentration (per wet weight) increased by 98%, and the pyridinoline cross-link concentration increased by 730%, while the glycosaminoglycan concentration remained unchanged or decreased slightly. Some growth-associated changes were location-specific, with tensile moduli and strength attaining higher values in the PFG than the FC. The growth-associated variation in tensile moduli and strength were associated strongly with variation in the contents of collagen and pyridinoline cross-link, but not sulfated glycosaminoglycan. The marked changes in the tensile properties and collagen network components of articular cartilage with growth suggest that such parameters may be used to evaluate the degrees to which regenerated cartilage recapitulates normal development and growth.

Articular cartilage biochemical composition and mechanical properties evolve during in utero and in vivo growth, with marked differences between fetus, newborn, and young adult. The objectives of this study were to test whether in vitro growth of bovine fetal and newborn calf articular cartilage explants resulted in changes in biochemical and tensile properties during up to 6 weeks of free-swelling culture in serum-supplemented medium. During this culture period, both fetal and calf cartilage grew markedly in size, increasing in dry and wet mass by 150-270%. This was due in part to increases in sulfated glycosaminoglycan (+248%), collagen (+96%), and pyridinoline cross-link (+133%). This was accompanied by an increase in water content so that the concentration of matrix components decreased, despite the overall net increase in mass. The ratio of pyridinoline cross-link to collagen remained low and characteristic of immature tissue. The equilibrium and dynamic tensile moduli and strength of both fetal and calf cartilage decreased during the culture period. The biochemical and biomechanical properties of the cartilage explants were correlated, such that the low values of modulus and strength were associated with low concentrations of collagen and pyridinoline. Thus, the tested culture conditions supported growth and maintenance cartilage in an immature state, but did not induce biomechanical or collagen network maturation.

Most attempts to tissue-engineer cartilage have involved seeding of cultured cells into a biological or synthetic scaffold. We have developed a novel two-step culture approach that makes possible the in vitro formation of cartilaginous-like tissue by mature adult bovine chondrocytes without the aid of a synthetic matrix. The first step consists of culturing chondrocytes under conditions that maintain their rounded shape and their molecular phenotype as assessed by type II collagen and aggrecan production. This step was accomplished by culturing the isolated chondrocytes in alginate beads until the cells have reestablished a proteoglycan-rich cell-associated matrix (CM). The second step consists of culturing the cells with their CM, after recovery from the beads, on a tissue culture insert with a porous membrane. In this study, young adult bovine articular chondrocytes were cultured in alginate beads in the presence of 10% or 20% fetal bovine serum (FBS). After 7 days of culture, the alginate beads were dissolved by incubating the beads for 20 min in sodium citrate buffer, a calcium chelator. Following a brief centrifugation, the cells with their CM were recovered, resuspended in medium containing 10% or 20% FBS and seeded onto a tissue culture insert. After 1 week of culture on the insert, the individual cells with their CM progressively became incorporated into a mass of cartilaginous tissue. Culture with 20% FBS resulted in the best formation of tissues. These tissues, easily recovered from the insert, were then subjected to biochemical and histological analyses. The biochemical results showed that the chondrocytes remain phenotypically stable in the tissues. The de novo tissue has a relatively high ratio of PG/collagen. Histological examination of the tissue revealed it contained a cartilage-like matrix strongly stained with toluidine blue. This scaffold-free system appears ideal to study, in vitro, the development of transplantable cartilaginous tissue.

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

Objective To determine the effects of interleukin-1α (IL-1α) on the expression of hyaluronan synthase (HAS), CD44, and aggrecan in human articular chondrocytes, and to assess the net result of these metabolic changes on the accumulation of hyaluronan within articular cartilage. Methods Normal human articular cartilage slices, as well as isolated chondrocytes, were treated with IL-1α. Changes in the relative expression of messenger RNA (mRNA) for HAS-2, CD44, and aggrecan were determined by competitive, quantitative reverse transcriptase–polymerase chain reaction. Hyaluronan accumulation was characterized by staining with a hyaluronan-specific binding protein and by fluorophore-assisted carbohydrate electrophoresis, while proteoglycan content was determined by alcian blue and Safranin O staining, CD44 protein expression by immunohistochemistry, and aggrecan biosynthesis by 35S-sulfate incorporation. Changes in cell-associated matrix sizes were visualized by a particle exclusion assay. Results IL-1α stimulated the expression of HAS-2 and CD44 mRNA (3.5-fold and 3-fold, respectively), but inhibited the expression of aggrecan mRNA. In IL-1–treated chondrocytes, extracellular hyaluronan decreased, while intracellular accumulation of hyaluronan was enhanced. Together with the decrease in expression of aggrecan, a dramatic reduction in cell-associated matrix was observed. IL-1–treated cartilage slices displayed a prominent depletion of aggrecan as well as hyaluronan within the upper layers of the tissue. The regional loss of hyaluronan coincided with a regional up-regulation of CD44. Conclusion These data demonstrate that IL-1α stimulates HAS-2 at the same time as it inhibits the expression of aggrecan. Although hyaluronan biosynthesis is up-regulated, so too is the expression of CD44 and the internalization/catabolism of hyaluronan. The net result is a loss of hyaluronan in areas of the articular cartilage where increases in CD44 expression are most prominent. This depletion of hyaluronan in the upper layers of the tissue likely facilitates the prominent loss of aggrecan from the tissue.

OBJECTIVE: To relate the rate of bone resorption to serum levels of both hyaluronan (HA) and antigenic keratan sulfate (KS) in canine experimental osteoarthritis (OA) and to evaluate the effects of calcitonin on these parameters and the OA lesions of the unstable knee. METHODS: Twenty-two dogs underwent anterior cruciate ligament transection (ACLT) and 6 dogs underwent sham operation. Urinary pyridinium crosslinks were quantified by high-performance liquid chromatography. Immunoassays quantified hyaluronan (HA) and antigenic KS. Macroscopic and histologic OA lesions were scored. Calcitonin treatment was started on day 14 postsurgery and stopped on either day 49 or day 104 postsurgery. Control dogs and all treated dogs were killed on day 105. RESULTS: All ACLT joints developed OA. In contrast to sham-operated animals, all operated dogs exhibited an early and sustained rise in the levels of their urinary and serum markers. Calcitonin markedly reduced the levels of these markers and the severity of OA lesions. Furthermore, the longer the period of calcitonin therapy, the lower the score of the OA lesions. CONCLUSION: Bone, synovium, and articular cartilage all appear to be involved in the state of hypermetabolism that develops in unstable joints. Furthermore, the rate of bone resorption increases markedly in the early stages of this OA model and is likely to contribute to cartilage breakdown. Since calcitonin reduced the severity of OA changes, this form of therapy may have benefits for humans who have recently experienced a traumatic knee injury.

STUDY DESIGN: This study determined whether entrapment of a rabbit intervertebral disc in alginate gel helped to promote the retention of normal metabolic activities by the nucleus pulposus and anulus fibrosus in tissue culture. OBJECTIVES: To establish an in vitro culture system to study the metabolism of the intervertebral disc as a whole integral organ. SUMMARY OF BACKGROUND DATA: In vitro studies of the metabolism of intervertebral discs have been scarce because of the difficulties involved in maintaining the integrity of the tissues, especially that of the nucleus pulposus, in culture medium. METHODS: Rabbit intervertebral discs were embedded in alginate gel and maintained in culture for as long as 1 month. At weekly intervals, experiments were performed to measure the rate of proteoglycan synthesis and to characterize proteoglycans newly synthesized by cells in the anulus fibrosus and nucleus pulposus. In addition, at these same time intervals, the contents of sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, and collagen in these two intervertebral disc tissues were measured to evaluate tissue integrity. Intervertebral discs cultured in medium alone were used as controls and analyzed in parallel. RESULTS: The anulus fibrosus and nucleus pulposus of intervertebral discs cultured in alginate gel sustained a higher rate of proteoglycan synthesis and maintained a higher content of extracellular matrix components than the respective controls at all times. CONCLUSIONS: This new alginate tissue culture system should prove useful for studying the metabolism of whole intervertebral discs.

Summary The injection of 2.0 mg chymopapain into the adolescent rabbit knee causes loss of articular cartilage proteoglycans (PG). Although chondrocytes attempt to restore lost PG, failure to repair ensues. Pure chondroitin 4,6-sulfate (Condrosulf ® , IBSA Lugano, Switzerland) has been used in clinical studies of human osteoarthritis (OA) as a slow-acting drug for OA (SYSADOA). Using our model of articular cartilage injury, we examined the effects of oral and intramuscular administration of Condrosulf ® after chymopapain-induced cartilage injury. In this study, animals received an injection of 2.0 mg chymopapain (Chymodiactin ® , Boots Pharmaceuticals) into the left knee and were sacrificed after 84 days. The contralateral right knee served as a noninjected control. Some animals received oral Condrosulf ® while others received intramuscular injections of Condrosulf ® . Serum keratan sulfate (KS) levels were monitored to ensure degradation of the cartilage PG. Those animals not exhibiting at least a 100% increase of serum KS following chymopapain injection were excluded from the study. At sacrifice, cartilage PG contents were markedly reduced in animals receiving an injection of 2.0 mg chymopapain with no further treatment. In contrast, oral administration of Condrosulf ® beginning 11 days prior to chymopapain injury resulted in significantly higher ( P =0.0036) cartilage PG contents. Intramuscular administration of Condrosulf ® resulted in higher, but less significantly so ( P =0.0457), cartilage PG contents. These results suggest that daily Condrosulf ® treatment prior to and continuing after chymopapain injury may have a protective effect on the damaged cartilage, allowing it to continue to re-synthesize matrix PG after the treatment is discontinued.

Summary The aim of this study was to assess the clinical, radiological and biological efficacy and tolerability of the SYSADOA, chondroitin 4- and 6-sulfate (CS, CondrosulP ~, IBSA, Lugano, Switzerland), in patients suffering from knee osteoarthritis. This was a 1-year, randomized, double-blind, controlled pilot study which included 42 patients of both sexes, aged 35--78 years with symptomatic knee OA. Patients were treated orally with 800 mg chondroitin sulfate (CS) per day or with a placebo (PBO) administred in identical sachets. The main outcome criteria were the degree of spontaneous joint pain and the overall mobility capacity. Secondary outcome criteria included the actual joint space measurement and the levels of biochemical markers of bone and joint metabolism. This limited study confirmed that chondroitin sulfate was well-tolerated and both significantly reduced pain and increased overall mobility capacity. Treatment with CS was also associated in a limited group of patients with a stabilization of the medial femoro-tibial joint width, measured with a digitized automatic image analyzer, whereas joint space narrowing did occur in placebo-treated patients. In addition, the metabolism of bone and joint assessed by various biochemical markers also stabilized in the CS patients whereas it was still abnormal in the PBO patients. These results confirm that oral chondroitin 4- and 6-sulfate is an effective and safe symptomatic slow-acting drug for the treatment of knee OA. In addition, CS might be able to stabilize the joint space width and to modulate bone and joint metabolism. This is the first preliminary demonstration that a SYSADOA might influence the natural course of OA in humans.

OBJECTIVE Recombinant human osteogenic protein 1 (OP-1) is an effective stimulator of human cartilage 35S-proteoglycan synthesis. The present study was conducted to determine whether stimulation of human articular chondrocytes with OP-1 can help overcome interleukin-1beta (IL-1beta)-induced suppression of 35S-proteoglycan synthesis. METHODS Human articular chondrocytes in alginate beads were maintained for 3 days in the absence (control) or presence of IL-1beta at 0.1-100 pg/ml with or without OP-1 at 50 ng/ml, in medium containing 10% fetal bovine serum (FBS). Incorporation of 35S-sulfate into proteoglycans was quantified during the last 4 hours of culture and reported as counts per minute per microg DNA. Release of interleukin-1 receptor antagonist (IL-1Ra) and prostaglandin E2 into the medium was monitored by immunoassay. RESULTS IL-1beta at 10 pg/ml caused a 60% decrease in 35S-proteoglycan synthesis. This could be blocked by including 500 ng/ml IL-1Ra in the medium. The presence of 50 ng/ml OP-1 in the IL-1beta-containing medium was effective in restoring 35S-proteoglycan synthesis to the level of that found in cultures not treated with IL-1beta. The restorative effects of OP-1 and IL-1Ra were cumulative. The rate of release of prostaglandin E2 and IL-1Ra into the medium was not affected by the presence of OP-1. CONCLUSION Treatment of human articular chondrocytes with OP-1 cultured in the presence of FBS is effective in overcoming the down-regulation of proteoglycan synthesis induced by low doses of IL-1beta.

Study design Cells from normal rabbit nucleus pulposus (NP) and anulus fibrosus (AF) were cultured in alginate beads for as long as 14 days to allow them to reform a matrix made up of two compartments: the cell-associated matrix (CM) and further removed matrix (FRM). At different time points, the CM and FRM made by each cell population were analyzed using histologic, biochemical, and immunologic assays. Objectives To study the metabolism of normal rabbit NP and AF cells in alginate by characterizing the CM and FRM formed by each cell population, and to identify metabolic properties that may shed light on mechanisms at play in disc degeneration. Summary of background data Little is known about the metabolism of intervertebral disc cells, in part because of the lack of microculture systems appropriate for the study of these cells in vitro. In recent studies from our laboratories, it was suggested that articular chondrocytes cultured in alginate beads remain phenotypically stable and reform a matrix similar to the one they populate in vivo. This culture system appears ideally suited for the study of intervertebral cells available only in limited numbers. Methods Rabbit NP and AF cells released from the matrix by sequential enzyme digestion were encapsulated in alginate beads (20,000 cells/bead) and cultured for as long as 14 days. At selected time points, beads were solubilized with calcium chelating agents, and the CM and FRM were isolated. The rate of 35S-sulfate incorporation into proteoglycans, and the contents of various extracellular matrix molecules (total sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, collagen, and pyridinium crosslinks) were measured. Results Both NP and AF cells remained phenotypically stable in the alginate gel throughout the culture period and reestablished a matrix composed of CM and FRM compartments. The two cell populations exhibited numerous differences in their metabolic activities in vitro. Nucleus pulposus cells synthesized fewer proteoglycan and collagen molecules and were less effective in incorporating these into the CM than AF cells. Conclusions Intervertebral disc cells, especially NP cells, are extremely sluggish in reforming a CM, a protective shell rich in proteoglycans and collagen molecules. This may help explain why damage to the NP often is accompanied by progressive degeneration of the disc in vivo.

To determine the immunophenotype or immunophenotypes of macular corneal dystrophy in Saudi Arabia.We studied 56 cases of macular corneal dystrophy. Tissue from 60 corneal transplant buttons was stained by the avidin-biotin complex method using an anti-keratan sulfate monoclonal antibody. The serum antigenic keratan sulfate was measured in 23 of the 56 patients, four unaffected relatives, and 13 individuals with chronic actinic keratopathy. Serum and corneal tissue were studied in 17 of the 50 affected individuals with corneal transplant material.Thirty-five corneas (58.3%) of 29 of 50 patients did not react with anti-keratan sulfate monoclonal antibody. The stroma and abnormal intracellular and extracellular corneal accumulations reacted with anti-keratan sulfate monoclonal antibody in seven corneas (11.7%). The stroma in the other 18 corneas (30.0%) from 15 patients did not react with the anti-keratan sulfate monoclonal antibody, but corneal fibroblasts did. Twenty-one of the 23 patients with macular corneal dystrophy had no detectable serum antigenic keratan sulfate (9 ng/ml); two had values of 12 and 51 ng/ml, respectively, and their corneal stroma and abnormal accumulations reacted with anti-keratan sulfate monoclonal antibody.We detected macular corneal dystrophy type IA, a new immunophenotype characterized by the lack of detectable antigenic keratan sulfate in the serum (9 ng/ml), and a corneal stroma that did not react with the keratan sulfate monoclonal antibody but in which corneal fibroblasts did react with keratan sulfate monoclonal antibody (in 15 of 50 patients).

We investigated an individual macular corneal dystrophy (MCD) type II cornea from a 42-year-old woman with markedly reduced antigenic keratan sulphate levels. A characteristic 4.6 A X-ray reflection was evident, and the mid-stroma contained 30% less sulphur than normal. Close packing of collagen was restricted to the superficial stroma. Abnormally large proteoglycan filaments were noted throughout the extracellular matrix and Descemet's membrane's posterior non-banded zone, but not its anterior banded zone. Small, collagen-associated stromal proteoglycans were susceptible to digestion with chondroitinase ABC, but not keratanase I or N-glycanase. On occasion, collagen fibrils ranged in size from 20 nm to 58 nm, with preferential diameters of 34 nm and 42 nm. Corneal guttae were evident, as were numerous endothelial inclusions, most probably due to intracellular fibrillogranular vacuoles similar to those found in the stroma. The endothelium expressed reduced anti-keratan sulphate labelling.

The matrix formed by adult human chondrocytes in alginate beads is composed of two compartments: a thin rim of cell-associated matrix that corresponds to the pericellular and territorial matrix of articular cartilage and a more abundant further-removed matrix, the equivalent of the interterritorial matrix in the tissue. On day 30 of culture, the relative and absolute volumes occupied by the cells and each of the two matrix compartments in the beads were nearly identical to those in native articular cartilage. Furthermore, the concentration of aggrecan in the cell-associated matrix was similar to that in adult human articular cartilage and was approximately 40-fold higher than in the further removed matrix compartment. Fluorescence-activated cell sorting revealed that the cell-associated matrix was built on the cell membrane in part via interactions between hyaluronic acid and CD44-like receptors. Approximately 25% of the aggrecan molecules synthesized by the chondrocytes during a 4-h pulse in the presence of [35S]sulfate on day 9 of culture were retained in the cell-associated matrix where they turned over with a half-life (t1/2) = 29 days. Most [35S]aggrecan molecules reached the further removed matrix compartment where they turned over much more slowly (t1/2 > 100 days). These results add support to the contention that aggrecan molecules residing in the pericellular and territorial areas of the adult human articular cartilage matrix are more susceptible to degradation by proteolytic enzymes synthesized by the chondrocytes than those that inhabit the interterritorial areas further removed from the cells.

Background: The frequency of different types of macular corneal dystrophy (MCD) was determined in Iceland where MCD accounts for one third of every penetrating keratoplasty. Methods: The authors determined the serum levels of antigenic keratan sulfate (aKS) in 27 patients with MCD and 53 unaffected family members by an enzyme-linked immunosorbent assay that uses an anti-KS monoclonal antibody (5-D-4). The authors also stained sections from 37 corneal buttons (including 2 regrafts) from 23 patients with MCD by the avidin-biotin complex method using the same anti-KS monoclonal antibody. Results: Based on the serum analyses, 22 patients had MCD type I and 5 had MCD type II. The corneas from patients without detectable KS in the serum lacked immunohistochemical reactivity to the anti-KS antibody. Every MCD cornea examined from individuals with normal serum KS levels showed KS reactivity. All 53 unaffected siblings and parents carrying the recessive gene had normal serum KS levels. Conclusions: Macular corneal dystrophy types I (78.6%)and II (21.4%)both occur in Iceland. Members of affected sibships had only one of these types, not both. Nine patients with MCD type I and four persons with MCD type II belonged to a large pedigree in which individuals have been traced as far back as the beginning of the 16th century. The linking of patients with MCD types I and II in an inbred pedigree suggests that both types may be manifestations of the same abnormal gene rather than independent entities. The serum KS levels were not helpful in detecting heterozygous MCD carriers.

We have used polarized light (POL) to monitor changes in the organization of the articular cartilage collagen network and matrix proteoglycans (PGs) after intra-articular injection of chymopapain (CP). POL viewing of sirius red stained sections revealed a loss of normal birefringence suggesting an apparent collapse of the collagen network following intra-articular CP. After 21 days, knees injected with 2.0 mg CP showed no return of normal birefringence, however, normal birefringence was noted in knees injected with only 0.2 mg CP. POL viewing of toluidine blue stained sections revealed a severe loss of matrix PGs followed by PG restoration in animals injected with 0.2 mg CP. The most important inference from the data is that articular cartilage can recover from enzyme-induced alterations in the spatial collapse of its fibrillar network. This is an important finding since it has often been inferred that damage to the collagen network leads invariably to progressive articular cartilage destruction.

OBJECTIVE. To measure serum levels of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of MMP-1 (TIMP-1) in patients with rheumatoid arthritis (RA) and in age-matched control subjects, and to determine how these correlate with serum levels of antigenic keratan sulfate (KS) and other biochemical and clinical indicators of disease activity. METHODS. Immunoassays were used to measure levels of MMP-1, MMP-3, TIMP-1, and antigenic KS. Radiologic and functional joint scores were based upon Steinbrocker's criteria. Erythrocyte sedimentation rates (ESR) and levels of C-reactive proteins (CRP) were measured. RESULTS. In RA patients, levels of MMP-3 and TIMP-1 were significantly increased, and strongly correlated with the ESR and CRP levels but not with radiologic or functional joint scores. Levels of antigenic KS were significantly lower in RA patients and correlated negatively with systemic parameters of inflammation and serum levels of TIMP-1. CONCLUSIONS. The increase in serum levels of MMP-3 and TIMP-1 appears to reflect systemic inflammation in RA. The inverse correlation between serum levels of TIMP-1 and antigenic KS suggests that an upregulation of TIMP-1 synthesis might be responsible for the apparent suppression of cartilage aggrecan catabolism in patients with severe inflammatory changes.

Objective. To investigate the prognostic value of serum hyaluronic acid (HA) and keratan sulfate (KS) levels in relation to tibiofemoral osteoarthritis (OA) of the knee. Methods. Clinical and demographic data were collected on 94 patients. Radiographs were obtained at study entry and at 5-year followup. Disease progression was defined as 2 mm of joint space narrowing of any tibiofemoral compartment, and/or knee joint surgery during the study period. Serum HA and KS were measured and levels were correlated with entry data and disease progression. Results. At entry, HA levels were significantly related to disease duration (P = 0.036), minimum joint space (P = 0.049), and previous surgery (P = 0.001). After these variables were taken into account, patients whose disease had progressed were shown to have had significantly higher levels of HA at baseline compared with those whose disease had not progressed (P = 0.019). However, there were no significant differences in levels of serum KS between those with and those without disease progression, at entry (P = 0.779) or at subsequent visits. Conclusion. These results suggest that serum HA levels predict disease outcome in OA of the knee and confirm that a single measurement of the serum level of KS is not useful as a prognostic marker in OA.

Keratan sulfate (KS) is a highly-negatively charged glycosaminoglycan which is found principally in aggrecan, the most abundant proteoglycan in the extracellular matrix of hyaline, fibrous and elastic cartilage. In 1985, we discovered that small peptidoglycans bearing antigenic KS (agKS) were present in small amounts in human serum and postulated that the great majority of these were derived from the degradation of cartilage aggrecan (Thonar et al. 1985). During the past decade, studies of antigenic KS in joint fluid and serum have provided strong evidence in support of this hypothesis. By asking questions which could not have been addressed previously, these studies have shed new light upon the metabolism of aggrecan in vivo. In this manuscript, we describe some of these findings and put in perspective their contribution to our understanding of the metabolism of aggrecan in joint diseases.

Proteoglycans synthesized by chondrocytes in alginate beads are found in two compartments: the cell-associated matrix and the further removed matrix (Hauselmann, H. J., Aydelotte M. B., Schumacher B. L., Kuettner K. E., Gitelis, S. H., and Thonar, E. J.-M. A. (1992) Matrix 12, 116-129). To study the metabolism of aggrecan in these two compartments, mature bovine articular chondrocytes in alginate beads were pulsed with [35S]sulfate for 30 min or 16 h on day 7 of culture and then chased in isotope-free medium for up to 21 days. At different times, the two matrix pools were separately isolated, and the 35S-proteoglycans quantified, purified, and characterized. Radiolabeled aggrecan molecules exhibited a very long average half-life in the beads (t1/2 = 95 days). In contrast, small non-aggregating proteoglycans, which made up approximately 4% of the 35S-proteoglycans synthesized, were rapidly lost from the beads (t1/2 = 95 days versus 15 days). Radiolabeled aggrecan subunits in the two matrix compartments had a similar average hydrodynamic size and polydispersity; importantly, the size of these molecules did not change during the chase period. Catabolism of 35S-aggrecan in the cell-associated matrix was the only significant contributor to the appearance in the medium of partially degraded 35S-aggrecan which had lost the ability to bind to hyaluronan. These results strongly suggest aggrecan molecules which reside in the pericellular and territorial matrix compartments in close proximity to the chondrocytes have a much faster rate of turnover than their counterpart in the interterritorial areas further removed from the cells.

OBJECTIVE. To measure serum levels of collagenase (MMP-1), stromelysin-1 (MMP-3), and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in normal subjects and in patients with osteoarthritis (OA), and to assess how these correlate with biochemical and clinical indicators of disease activity in OA. METHODS. Specific immunoassays were used to measure MMPs, TIMP-1, and antigenic keratan sulfate (KS). The total area of cartilage affected by the disease was measured (expressed as an articular index). RESULTS. In the normal population (n = 118), the serum concentration of MMP-3, but not of MMP-1 or TIMP-1, increased with age and was approximately 2 times higher in males than in females. In the OA patients (n = 33), the serum levels of MMP-3, but not of MMP-1 or TIMP-1, were significantly elevated and correlated strongly with the articular index but poorly with objective and subjective functional capacity scores as well as with serum levels of antigenic KS and systemic parameters of inflammation. CONCLUSION. These findings illustrate the importance of matching patients and normal controls for age and sex in further studies of MMP-3 and are consistent with the hypothesis that MMP-3 might play an important role in the degradation of joint cartilage in OA. Further, serum levels of MMP-3 may prove useful for monitoring therapy for OA.

We examined the relationship between serum and synovial fluid (SF) levels of antigenic keratan sulfate (KS) and the clinical, laboratory, and radiologic features of disease in 125 well-characterized patients with knee osteoarthritis (OA). KS was quantified by enzyme-linked immunosorbent assay, using an antibody specific for a highly sulfated epitope on KS chains; the results were calculated as equivalents of an international standard of KS from human costal cartilage. The mean level of serum KS (393 ng/ml) was significantly higher than those previously reported for populations of adults without OA. There was a wide scatter of serum KS values (range 156–912 ng/ml), with little correlation with clinical or radiologic features. Men had significantly higher levels than women (456 ± 135 ng/ml versus 368 ± 110 ng/ml, mean ± SD), and there was a statistically significant but weak association with indicators of polyarticular involvement (number of symptomatic joints, Heberden's nodes, hip symptoms) in women. Despite the wide scatter of results in the population as a whole, individual levels of KS were stable for up to 4 consecutive years in the 9 patients studied. Levels of KS were much higher in SF (n = 25) than in serum, but the two were not correlated. There was an inverse correlation between radiographic evidence of cartilage loss and the level of KS in SF. The large variations in serum KS values suggest that this measure may not be of diagnostic significance among populations of patients. However, the stability of serum levels with time and the relationship between cartilage mass and SF levels may be useful for monitoring the individual patient's response to therapy, both systemically (serum KS) and in single joints (SF KS).

The serum of most patients with type 1 macular corneal dystrophy (MCD), the most prevalent subtype, lacks detectable antigenic keratan sulfate (KS), and it has been postulated that such individuals may lack antigenic KS in their cartilage as well. To test this hypothesis, we studied the cornea, serum, and nasal cartilage from an MCD patient using light and electron microscopy, immunohistochemistry, and a quantitative enzyme-linked immunosorbent assay (ELISA) which uses a monoclonal antibody against a sulfated epitope on the KS chain to measure KS content. Histologically, corneal deposits seen were characteristic of MCD. No abnormal deposits were noted in the cartilage. The lack of immunoreactivity in corneal sections with antibodies against sulfated epitope on KS and the absence of this epitope in serum showed that the patient had type 1 MCD. The cartilage specimen showed no immunoreactivity in the chondrocytes or extracellular matrix. Quantitative analysis by ELISA demonstrated that the antigenic KS content of the cornea and cartilage was at least 800 times lower than that in normal controls. This provided direct evidence that the abnormality in the sulfation of keratan in type 1 MCD involves the cornea and cartilage.

Keratan sulfate is a glycosaminoglycan; in man, most KS is present in cartilage proteoglycans, 1 but small amounts have been identified in blood.l, 2 Recent evidence indicates that this KS is almost exclusively derived from catabolism of cartilage proteoglycans.l-3 As a result, it has been postulated that the concentration of KS in the blood is directly proportional to the rate of degradation of cartilage proteoglycans.I, 2 Linear growth results from bone formation on a cartilage scaffold (endochondral ossification) and depends on adequate function of the hypothalamic-pituitary-somatomedin-insulin-like growth factor I axis and the genetic ability of cartilage to respond to these secretions. 4, 5 In the growing child, serum levels of KS are elevated, which is not unexpected because growing cartilages are constantly undergoing degradation and replacement by new bone formation. We previously reported that serum levels of keratan sulfate rise from low levels in infancy and reach a plateau by the age of 4 to 5 years. Serum concentrations of KS appear to remain high until age 12 years, when they rapidly decline toward the levels found in normal adult sera. Serum levels of KS in a growing child correlate with the individual child's age-adjusted percentile height. 6 In our investigation, we measured the serum concentrations of KS in two groups of children with short stature: one group with Constitutional delay of maturity and the other with growth hormone deficiency. In the latter group, we compared KS levels before and after treatment with growth

Tendon rupture is a common sports injury in adults. However, the mechanical properties of repair tissue are inferior to those of normal tissue. To accelerate tendon healing, an in vivo approach using growth factors has been applied and has shown evidence for the efficacy of biological stimulation of the repair process. Recombinant human osteogenic protein-1 (rhOP-1) has been shown to be effective in stimulating matrix production by various connective tissues. To test the effect of rhOP-1 on the matrix metabolism of tendon cells in vitro, bovine tendon cells were cultured in monolayer with various doses of rhOP-1 for 7 days. The addition of rhOP-1 to cell culture media resulted in significant increases in cell proliferation, DNA content, and the synthesis of proteoglycans (PGs) and collagen, compared to control cultures. The relative percentage of large PGs in the OP-1 culture was higher than that in the control culture. In conclusion, we show for the first time that rhOP-1 stimulates the proliferation of tendon cells and their ability to synthesize and accumulate PGs and collagen in their extracellular matrix. These biological properties may be used in the tissue-engineering of tendon tissues. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:42–48, 2008

Although growth plate response to mechanical stress has been increasingly studied, our understanding of mechanical modulation of neonatal growth plate is incomplete, especially concerning biochemical changes. This study was designed to explore the cellular and biochemical responses of the cranial base growth plate (CBGP) explant upon cyclic loading. The growth plate with subchondral bone was aseptically isolated from each of 24 neonatal rabbits and fixated in an organ culture system. Cyclic loading was applied to growth plate explants at 200 mN and 1 Hz for 60 min (N=12), whereas control explants were immersed in organ culture for 60 min without mechanical loading (N=12). Computerized image analysis revealed that cyclic loading induced significantly more proliferating chondrocytes than unloaded controls (p

The aim of this study was to design in vitro growth protocols that can comprehensively quantify articular cartilage structure-function relations via measurement of mechanical and biochemical properties. Newborn bovine patellofemoral groove articular cartilage explants were tested sequentially in confined compression (CC), unconfined compression (UCC), and torsional shear before (D0, i.e. day zero) and after (D14, i.e. day 14) unstimulated in vitro growth. The contents of collagen (COL), collagen-specific pyridinoline (PYR) crosslinks, glycosaminoglycan, and DNA significantly decreased during in vitro growth; consequently, a wide range of biochemical properties existed for investigating structure-function relations when pooling the D0 and D14 groups. All D0 mechanical properties were independent of compression strain while only Poisson's ratios were dependent on direction (i.e. anisotropic). Select D0 and D14 group mechanical properties were correlated with biochemical measures; including (but not limited to) results that CC/UCC moduli and UCC Poisson's ratios were correlated with COL and PYR. COL network weakening during in vitro growth due to reduced COL and PYR was accompanied by reduced CC/UCC moduli and increased UCC Poisson's ratios.

Objective To examine the cartilage growth–associated effects of a disruption in the balance between the swelling pressure of glycosaminoglycans (GAGs) and the restraining function of the collagen network, by diminishing GAG content prior to culture using enzymatic treatment with chondroitinase ABC. Methods Immature bovine articular cartilage explants from the superficial and middle layers were analyzed immediately or after incubation in serum-supplemented medium for 13 days. Other explants were treated with chondroitinase ABC to deplete tissue GAG and also either analyzed immediately or after incubation in serum-supplemented medium for 13 days. Treatment- and incubation-associated variations in tissue volume, contents of proteoglycan and collagen network components, and tensile mechanical properties were assessed. Results Incubation in serum-supplemented medium resulted in expansive growth with a marked increase in tissue volume that was associated with a diminution of tensile integrity. In contrast, chondroitinase ABC treatment on day 0 led to a marked reduction of GAG content and enhancement of tensile integrity, and subsequent incubation led to maturational growth with minimal changes in tissue volume and maintenance of tensile integrity at the enhanced levels. Conclusion The data demonstrate that a manipulation of GAG content in articular cartilage explants can distinctly alter the growth phenotype of cartilage. This may have practical utility for tissue engineering and cartilage repair. For example, the expansive growth phenotype may be useful to fill cartilage defects, while the maturational growth phenotype may be useful to induce matrix stabilization after filling defect spaces.

Objective—To examine longitudinal changes in serum and synovial fluid concentrations of keratan sulfate (KS) and hyaluronan (HA) after cranial cruciate ligament (CCL) transection in dogs. Animals—12 clinically normal adult mixed-breed dogs. Procedure—Following CCL transection in the right stifle joint, KS and HA concentrations were determined in serum and neat (undiluted) synovial fluid prior to and 1, 2, 3, and 12 months after surgery. Postsurgical dilution of synovial fluid was corrected by use of urea as a passive marker. Results—Synovial fluid KS and HA concentrations decreased at 1, 2, and 3 months after surgery in operated stifle joints, compared with baseline values. Synovial fluid KS concentration decreased in unoperated stifle joints at 1 month. A decrease in synovial fluid KS concentration was found in operated stifle joints, compared with unoperated stifle joints, at 2 and 3 months, and a decrease in synovial fluid HA concentrations was also found in operated stifle joints, compared with unoperated stifle joints, at 1, 2, and 3 months. Serum KS concentrations increased from baseline values at 3 months after surgery. Hyaluronan concentrations in operated stifle joints were lower than baseline values at 1, 2, and 3 months. Urea-adjusted synovial fluid concentrations revealed that dilution did not account for the decline in biomarker concentrations. Conclusions and Clinical Relevance—The initial decrease and subsequent increase in synovial fluid concentrations of HA and KS may be caused by an acute inflammatory response to surgical intervention that negatively affects cartilage metabolism or an increase in production of immature proteoglycans.

Background context Chondroitinase ABC (C-ABC) is used in chemonucleolysis to degrade, with great specificity, the chondroitin sulfate and dermatan sulfate chains of proteoglycans (PGs). A recent study showed that osteogenic protein-1 (OP-1) is very effective in stimulating the production and formation of the extracellular matrix by rabbit intervertebral disc cells. Purpose To test the hypothesis that the repair of the extracellular matrix of the intervertebral disc after chemonucleolysis by C-ABC can be stimulated by exposure to a low dose of a growth factor, OP-1. Study design An alginate bead cell culture system was used to monitor the effects of OP-1 on the repair of damaged matrices after in vitro chemonucleolysis with C-ABC. Methods Rabbit nucleus pulposus (NP) or annulus fibrosus (AF) cells cultured for 2 weeks in alginate gel were briefly exposed to low concentrations of C-ABC and then cultured in the presence or absence of OP-1. The control group was cultured without enzyme treatment for the same period in the absence of OP-1. At each time point, the contents of DNA and proteoglycan accumulation and proteoglycan synthesis were measured. Results NP or AF cells cultured in alginate beads, which were digested with C-ABC and then treated with OP-1, recover PG content more rapidly than those cultured in the absence of OP-1. The major contributor to the superior matrix repair in the cells treated with OP-1 was an up-regulation of proteoglycan synthesis. Conclusions OP-1 was effective in stimulating matrix repair by NP and AF cells after their matrices were nearly totally depleted of sulfated glycosaminoglycans. The use of OP-1 after chemonucleolysis might help the disc to regain biomechanical strength, weakened by enzyme digestion, by stimulating matrix metabolism.

Objectives: Tissue engineering of nasal septal cartilage has numerous potential applications in craniofacial reconstruction. Chondrocytes suspended in alginate gel have been shown to produce a substantial cell-associated matrix. The objective of this study was to determine whether cartilage tissue could be generated using the alginate-recovered-chondrocyte (ARC) method, in which chondrocytes are cultured in alginate as an intermediate step in tissue fabrication. Methods: Nasal septal chondrocytes from five patient donors were isolated by enzymatic digestion, then expanded in monolayer culture. At confluency, a portion of those cells were seeded at high density onto a semipermeable membrane and cultured for 14, 21, or 28 days (monolayer group). The remaining cells were suspended in alginate and cultured until a cell-associated matrix was observed (10-17 days). Cells and their associated matrix were released from alginate (ARC group), seeded onto a semipermeable membrane, and cultured as already described. DNA (Hoechst 33258 Assay), glycosaminoglycan (GAG; dimethylmethylene blue assay), and collagen (hydroxyproline assay) were analyzed biochemically. Immunohistochemistry was performed to assess expression of collagens type I and type II. Histochemistry was performed to localize cells accumulating sulfated GAG (Alcian blue stain). Results: The ARC constructs, in contrast to the monolayer constructs, had substantial structural stability and the histologic and gross appearance of cartilaginous tissue. ARC constructs demonstrated significantly greater GAG and collagen accumulation than monolayer constructs (P

OBJECTIVE: To evaluate the effect of calcitonin (CT) on the histology and biochemistry of articular cartilage from unstable operated and nonoperated knee in a canine model of experimental osteoarthritis (OA). METHODS: Eighteen dogs underwent anterior cruciate ligament transection (ACLT) of the right knee and were randomly distributed into three groups of six dogs each. From day-1 after surgery until sacrifice 84 days post-ACLT, each dog received a daily nasal spray that delivered the placebo, 100 units of CT or 400 units of CT. Histologic lesions were scored. Hyaluronan (HA) and antigenic keratan sulfate (AgKS) were quantified by enzyme-linked immunosorbent assays (ELISAs), whereas aggrecan molecules extracted under nondissociative conditions were characterized by velocity gradient centrifugation. RESULTS: All canine cruciate-deficient knees developed OA. At a daily dose of 400 units, CT had no effect on the size of osteophytes but significantly reduced the severity of cartilage histologic lesions in unstable knees. CT also enhanced the HA content as well as the size distribution and relative abundance of fast-sedimenting aggrecan aggregates in cartilage from both operated and nonoperated knees. On the other hand, in the CT-treated group, the cartilage content of AgKS increased in operated joints, but not in nonoperated joints. CONCLUSIONS: Because CT delivered as a nasal spray markedly reduced the severity of most OA changes, both at the histological and biochemical level, this form of therapy may have benefits for humans who have recently experienced a traumatic knee injury, and as well as for dogs who spontaneously rupture their ACL.

Objective: This study examined if viscosupplementation from intra-articular administration of a commercially available form of hyaluronan (HA) could promote the restoration of proteoglycan (PG) depletion induced by chymopapain and then if the repair could be maintained once HA treatment was discontinued. Methods: Animals received cartilage injury with intra-articular chymopapain (2.0 mg) followed by weekly treatment with intra-articular HA. HA treated animals were compared to injured animals with no treatment, contralateral untreated joints and joints from normal controls. The effect of intra-articular HA alone on articular cartilage was also examined. Results: Serum keratan sulfate levels confirmed degradation of the cartilage PGs in the chymopapain-injected knees. Intra-articular chymopapain resulted in marked loss of PGs. There were no significant differences among the control groups (untreated control, HA/800 treatment only). HA treatment did not affect the loss of PGs caused by chymopapain after 42 days. However, in animals receiving chymopapain injury followed by weekly HA treatment for 42 days and then 42 days of free cage activity without HA, cartilage PG contents were significantly increased. Intra-articular HA alone had no effect on the articular cartilage. Conclusion: The results in the present study suggest a potential protective effect of HA on chymopapain-induced acute articular cartilage injury in rabbits that, in time, permits damaged cartilage to resynthesize matrix PGs after the HA treatment is discontinued.

The alginate bead culture system has unique properties that make it possible to study the accumulation and turnover of macromolecules in two distinct matrix compartments of the cartilage matrix: the cell-associated matrix (CM) and the further removed matrix (FRM). Taking advantage of this culture system, the purpose of this study was to examine age-related changes in the metabolism of hyaluronan (HA) in these two compartments. Bovine chondrocytes, isolated from fetal, young adult, and old adult articular cartilage, were cultured in alginate beads. On Days 7 and 14 of culture, the alginate gel was solubilized, the CM and FRM were separated and macromolecules in both compartments were analyzed. When compared to the cells from fetal and old adult animals, the young adult cells proliferated at the fastest rate. Fetal cells produced a more abundant CM that was richer in proteoglycans (PGs) than the CM of young or old adult cells. With increasing age, there was an increased tendency for PG, collagen, and HA to escape incorporation into the CM and to become immobilized in the FRM. Very striking changes also were observed in the ratio of HA to PG, which increased markedly with age, and in the size of the HA molecules, which decreased markedly with age. The results suggest that the metabolism of HA in cartilage undergoes pronounced age-related changes, some of which are retained during culture in alginate gel. The findings also suggest that the previously documented age-related decrease in the size of HA in native bovine cartilage reflects, at least in part, a biochemical process occurring at the time or at least soon after the glycosaminoglycan chain is synthesized. It does not appear to simply be the result of age-related changes occurring slowly with time after synthesis, as was previously suggested to be the case for human articular cartilage.

Concentrations of the bone resorption markers pyridinoline and deoxypyridinoline and the bone formation marker osteocalcin were measured in 24-h urine collections from 30 subjects who underwent unilateral total hip replacements for monoarticular symptomatic osteoarthrosis and 10 controls. The patient groups were divided based on the femoral implant type (cemented cobalt alloy stem, cementless porous coated cobalt alloy stem, and cementless porous coated titanium alloy stem). Urine collections were performed before surgery and then at 3, 6, 12, 24, and 36 months. There were significant changes over time in the three patient groups for pyridinoline, deoxypyridinoline, and the ratio of osteocalcin to deoxypyridinoline (p < or = 0.01), but the control group values did not change over time. The resorption markers tended to peak at 3 months and the osteocalcin to deoxypyridinoline ratio was more variable, having depressed values in the cementless cobalt alloy group and elevated values in the other two groups compared with baseline. The cementless cobalt alloy group had higher resorption marker levels than the cemented cobalt alloy group at 6, 12, 24, and 36 months and higher levels than the cementless titanium alloy group at all postoperative times (p < 0.05). The osteocalcin to deoxypyridinoline ratio was lower in the cementless cobalt alloy group than in the cemented cobalt alloy group at 3, 6, and 24 months and the cementless titanium alloy group at 6, 12, and 24 months (p < 0.05). For the cemented cobalt chrome group, the baseline-normalized resorption marker values at 3 months and 6 months were correlated with the severity of radiographically assessed bone loss at 36 months (0.749 < r < 0.840; p < 0.05). For the cementless titanium alloy group, baseline-normalized osteocalcin/ deoxypyridinoline ratios at 3 months and 6 months were related inversely to radiographic bone loss at 36 months (0.687 < r < 0.749; p < 0.05). Thus, body fluid markers of bone metabolism change after total hip replacement. In addition, the changes in the marker concentrations were sensitive to implant design and were correlated with subsequent stress-shielding-induced bone loss.

The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis. Normal tissues (n = 11) contained 15 microg of keratan sulfate and 8 microg of chondroitin/dermatan sulfate per mg dry weight. Keratan sulfates consisted of approximately 4% unsulfated, 42% monosulfated, and 54% disulfated disaccharides with number of average chain lengths of approximately 14 disaccharides. Chondroitin/dermatan sulfates were significantly longer, approximately 40 disaccharides per chain, and consisted of approximately 64% unsulfated, 28% 4-sulfated, and 8% 6-sulfated disaccharides. The fine structural parameters were altered in all diseased tissues. Keratan sulfate chain size was reduced to 3-4 disaccharides; chain sulfation was absent in MCD type I corneas and cartilages, and sulfation of both GlcNAc and Gal was significantly reduced in MCD type II. Chondroitin/dermatan sulfate chain sizes were also decreased in all diseased corneas to approximately 15 disaccharides, and the contents of 4- and 6-sulfated disaccharides were proportionally increased. Tissue concentrations (nanomole of chains per mg dry weight) of all glycosaminoglycan types were affected in the disease types. Keratan sulfate chain concentrations were reduced by approximately 24 and approximately 75% in type I corneas and cartilages, respectively, and by approximately 50% in type II corneas. Conversely, chondroitin/dermatan sulfate chain concentrations were increased by 60-70% in types I and II corneas. Such changes imply a modified tissue content of individual proteoglycans and/or an altered efficiency of chain substitution on the core proteins. Together with the finding that hyaluronan, not normally present in healthy adult corneas, was also detected in both disease subtypes, the data support the conclusion that a wide range of keratocyte-specific proteoglycan and glycosaminoglycan remodeling processes are activated during degeneration of the stromal matrix in the macular corneal dystrophies.

The goal of this study was to examine the effects of mechanical compression on chondrocyte biosynthesis of extracellular matrix (ECM) components during culture in a new alginate disk culture system. Specifically, we have examined chondrocyte biosynthesis rates, and the structure of aggrecan core protein species present in the cell-associated matrix (CM), in the further removed matrix (FRM) and in the surrounding culture medium. In this alginate disk culture system, chondrocytes can be subjected to mechanical deformations similar to those experienced in vivo. Our results show that over an 8-week culture period, chondrocytes synthesize a functional ECM and can respond to mechanical forces similarly to chondrocytes maintained in native cartilage. In the alginate disk system, static compression was shown to decrease and dynamic compression to increase synthesis of aggrecan of bovine chondrocytes. Western blot analysis of the core proteins of aggrecan molecules identified a number of different species that were present in different relative amounts in the CM, FRM, and medium. Over 21 days of culture, the predominant form of aggrecan found in the ECM was a full-length link-stabilized species. In addition, our data show that the application of 40 h of static compression caused an increase in the proportion of newly synthesized aggrecan molecules released into the medium. However, this was not accompanied by a significant change in the size and composition of aggrecan and aggrecan fragments in the different compartments, suggesting that mechanical compression did not alter the catabolic pathways. Together, these data show that chondrocyte function is maintained in an alginate disk culture system and that this culture system is a useful model to examine chondrocyte ECM assembly and some aspects of catabolism normally found in vivo.

Phenotypically stable young adult bovine articular chondrocytes suspended in beads of alginate gel were first cultured for 5 days, using daily changes of medium containing 10% fetal bovine serum and supplements. The cells in the beads were then maintained in culture for a further 3 days in the presence or absence of interleukin-1alpha at 1 ng/ml in the daily change of medium. The exposure to interleukin-1alpha caused the incorporation of (35)S-sulfate into the predominant cartilage proteoglycan, aggrecan, to decrease by approximately 60%. In addition, proteoglycans that had accumulated into the cell-associated matrix during the first 5 days of culture in the absence of interleukin-1alpha moved into the matrix further removed from the cells and from there into the medium. In contrast, the exposure to interleukin-1alpha was found to markedly promote the rate of synthesis of hyaluronan, especially during the first 24 h. Over the 3 days of culture in the presence of interleukin-1alpha, a large proportion of the newly synthesized hyaluronan molecules, as well as those that had previously become residents of the cell-associated matrix, moved out of this compartment and appeared to become permanent residents of the further removed matrix. These results demonstrate that exposure of young adult articular chondrocytes to interleukin-1alpha has profound effects on the metabolism of hyaluronan, a molecule that plays a critical role in the retention of proteoglycan molecules in the matrix. Importantly, the results suggest that exposure of chondrocytes to interleukin-1 in inflamed joints, such as occurs in rheumatoid arthritis, leads to the rapid loss of coordination of the synthesis of aggrecan and hyaluronan, two of the critical constituents of the proteoglycan aggregate. In addition, we present evidence that these interleukin-1-induced effects differentially alter the metabolism of hyaluronan in the metabolically active cell-associated matrix and the metabolically inactive matrix further removed from the chondrocytes.

Sodium alginate, which gels in the presence of calcium ions, is commonly used for culture of anchorage-independent cells, such as chondrocytes. Normally, the gel appears microscopically homogeneous but, depending on the conditions of gelation, it may contain a varying number of small channels that extend inward from the surface. We have examined the influence of these channels on the morphology of cultured chondrocytes entrapped in alginate beads. Growth-plate or articular chondrocytes cultured in alginate normally proliferate and form rounded cell clusters but, in alginate beads containing numerous channels, many chondrocytes become aligned and form columns similar to those in the growth plate in vivo. As the pattern of cellular growth and morphology in alginate is profoundly influenced by the presence of channels in the gel, further studies were conducted to determine what specific conditions of gelation affect their formation. The channels are especially numerous when both the alginate and the gelling solutions lack sodium ions or other monovalent cations. The channels are cavities in the gel formed by particulate blocking of the rapid diffusion of calcium ions from the gelling solution into the boundary of the calcium alginate solution, and hence they extend inward from cells at the surface of the alginate gel. An understanding of the conditions under which these channels develop makes it possible either to avoid their formation or, alternatively, to enhance the number of channels in order to encourage proliferating cells to grow in radial columns, rather than in a less organized pattern characteristic of most culture systems.

Restoring knee stability through reconstruction, while providing symptomatic relief, has not been shown to decrease the incidence of degenerative changes after rupture of the anterior cruciate ligament. This suggests that posttraumatic osteoarthritis may not be purely bio mechanical in origin, but also biochemical. To test this, we measured the levels of seven cytokine modulators of cartilage metabolism in knee joint synovial fluid after anterior cruciate ligament rupture. We also measured keratan sulfate, a product of articular cartilage catab olism. The sample population consisted of patients with uninjured knee joints ( N = 10), and patients with acute ( N = 60), subacute ( N = 18), and chronic ( N = 8) anterior cruciate ligament-deficient knees. Synovial fluid samples were analyzed by enzyme-linked immu nosorbent assays. Normal synovial fluids contained high levels of the interleukin-1 receptor antagonist but low concentrations of other cytokines. Immediately af ter ligament rupture there were large increases in in terleukins 6 and 8, tumor necrosis factor α, and keratan sulfate. Interleukin-1 levels remained low throughout the course. As the injury became subacute and then chronic, interleukin-6, tumor necrosis factor-α, and keratan sulfate levels fell but remained considerably elevated 3 months after injury. Concentrations of inter leukin-1Ra fell dramatically. Granulocyte-macrophage colony-stimulating factor concentrations were normal acutely and subacutely but by 3 months after injury were elevated 10-fold. Our data reveal a persistent and evolving disturbance in cytokine and keratan sulfate profiles within the anterior cruciate ligament-deficient knee, suggesting an important biochemical dimension to the development of osteoarthritis there.

Cartilage oligomeric matrix protein (COMP) is a high-molecular-weight glycoprotein found at a high concentration in articular cartilage. Recent studies have shown that the joint fluid and serum levels of antigenic COMP, measured by an enzyme-linked immunosorbent assay (ELISA) which uses a polyclonal antiserum raised against bovine COMP, provide important information about metabolic changes occurring in the cartilage matrix in joint disease. In this report, we describe the specificity of three monoclonal antibodies (mAbs) to human COMP and their usefulness in quantifying antigenic COMP fragments in body fluids. Two of the mAbs (16-F12 and 18-G3) recognized both oligomeric and monomeric forms of COMP, but the third (17-C10) reacted positively only with the former. Immunoblots of human COMP, predigested with trypsin for up to 6 h, showed that the three mAbs are directed against different epitopes identified on small tryptic fragments of 30 kDa (16-F12), 25 kDa (17-C10), and 40 kDa as well as 30 kDa (18-G3), respectively. The antibodies also recognized a different pattern of fragments in human pathological synovial fluids. This was particularly striking in the case of the medium size fragments (16-F12: 90 and 110 kDa; 17-C10: 70 and 90 kDa; 18-G3: up to five bands from 70 to 130 kDa). Competitive indirect inhibition ELISAs developed with mAbs 16-F12 and 17-C10 revealed further differences in the specificities of these antibodies. Thus, while mAb 16-F12 can be used only to quantify antigenic COMP in human synovial fluid and serum, mAb 17-C10 is useful in addition when analyzing canine and horse synovial fluid as well as canine serum. The results of analyses of synovial fluid samples from patients with osteoarthritis and rheumatoid arthritis provided preliminary evidence in support of the contention that measurement of the different COMP epitopes recognized by these mAbs in body fluids could prove useful in the clinical assessment of patients with joint disease.

A 56-year-old black woman with full-thickness mosaic pattern central corneal cloudiness, similar in appearance to central cloudy dystrophy and posterior crocodile shagreen, underwent corneal transplantation. Atypical features included decreased vision, photophobia, and epithelial involvement, with occasional foreign body sensation. Numerous 0.5-2.0-micron-diameter lacunae were present in the corneal stroma and Bowman's layer, and a saw-toothed lamellar pattern was often evident in the corneal stroma. Soybean agglutinin (SBA), a lectin that binds N-acetyl-galactosamine residues, bound diffusely to stromal foci exhibiting similar size and distribution to the lacunae observed by electron microscopy. An absence of histochemically detectable lipid associated with these lacunae suggests that SBA reacted with glycoconjugates other than glycolipids. Biochemical analyses revealed similar contents of keratan sulfate, chondroitin/dermatan sulfate, and collagen as in normal controls, suggesting that the SBA binding moieties are associated with a glycoprotein or proteoglycan that is structurally or compositionally different from those found in normal cornea. This patient may represent an extreme variant of Vogt's or Francois central corneal clouding or a previously undescribed corneal dystrophy.

The experiments described herein tested the effects of CaCl2 and ZnCl2, added at various concentrations in the culture medium, upon the synthesis of collagen and proteoglycan by adult and fetal (articular, epiphyseal and hypertrophic) bovine chondrocytes maintained in high density multilayer cultures. CaCl2 concentrations below 0.5 mM or the addition of 1-50 microM ZnCl2 to the medium selectively promoted the production of collagen by all four populations of chondrocytes but had no effect on fibroblasts. Further, these changes had no statistically significant effect on the incorporation of 35S-sulfate into macromolecules or on the synthesis of gelatinase A, measured by gelatin zymography. The addition of CaCl2 and ZnCl2 at these concentrations did not result in a change in the relative proportion of non-crosslinked 3H-collagen molecules (synthesized in the presence of beta-aminopropionitrile) partitioning in the cell layer and medium compartments, and did not appreciably alter the pattern of collagens synthesized by any of the cell populations. The hypertrophic cells synthesized high levels of collagen type X in the presence as well as absence of exogenously added cations. However, CaCl2 at 10 mM caused a marked upregulation of collagen type X synthesis by a preparation of chondrocytes derived from the entire growth plate, consistent with the view that calcium at that concentration stimulated the differentiation of some of the cells into hypertrophic chondrocytes.

SummaryThe concentrations of cartilage proteoglycan (aggrecan), stromelysin-1, tissue inhibitor of metalloproteinases-1 (TIMP-1) and procollagen II C-propeptide in knee joint fluid and the levels of aggrecan, hyaluronan and keratan sulfate in serum were measured before and after exercise in 33 healthy athletes. The samples before exercise were obtained after 24 h rest from running or soccer and the samples after exercise were obtained 30–60 min after the exercise. Nine athletes ran on a treadmill for 60 min, 16 ran on road for 80 min and 8 played one soccer game (90 min). A reference group of 28 patients with knee pain but not evidence of joint pathology or injury was used for comparison. In joint fluid no single marker from the degradative processes in cartilage matrix changed significantly with exercise but all showed a rising trend. All markers except stromelysin showed lower concentrations in athletes at rest compared to the reference group. In serum from runners before exercise the concentration of keratan sulfate was significantly higher than in both the soccer and reference groups and further increased after exercise. The increase in markers after exercise may reflect an effect of mechanical loading in combination with a possible high turnover rate of body cartilage matrix in these individuals.

Purpose of study: Chondroitinase-ABC (C-ABC) is commonly used to degrade the chondroitin sulfate and dermatan sulfate chains of proteoglycans. In some countries, C-ABC, injected intradiscally, shows promise as a chemonucleolytic agent. However, the removal of glycosaminoglycans may lead, over the long-term, to significant degradation of the injected intervertebral disc (IVD). Osteogenic protein-1 (OP-1) has been shown to stimulate IVD matrix synthesis in vitro and, thus, may be useful in inhibiting and/or slowing down IVD matrix degradation in vivo. The purpose of this study was to monitor the effect of recombinant human OP-1 (rhOP-1) on the in vivo repair of IVDs co-injected with C-ABC. Methods used: Twenty-four New Zealand White rabbits (3 kg) were divided equally into four groups (control vehicle; C-ABC [10 mU] alone; rhOP-1 [100 μg] alone; or C-ABC + OP-1 [10 mU+100 μg, respectively] co-injected). The assigned agents (10 μl/disc) were injected into three levels of IVDs for each rabbit in each group. Radiographs of the lumbar spine were taken every 2 weeks after in the injections. At 4 or 12 weeks after the injections, three rabbits in each group were euthanized and the IVDs assessed biochemically and histologically. of findings: Significant disc space narrowing was observed 2 weeks after the injection of either C-ABC or C-ABC + OP-1, but not after injection of the control vehicle. In the C-ABC group, this narrowing was sustained for up to 12 weeks (4 weeks, 77 ± 11%; 12 weeks, 75 ± 8%). In the C-ABC + OP-1 group, the disc height index (DHI) had begun to return toward normal after 4 weeks (C-ABC vs. C-ABC + OP-1, p