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Study Design In vivo randomized controlled study in the goat intervertebral disc (IVD) injury model.

To confirm that primary intervertebral disc cells cultured in monolayer transduced with adenovirus maintained their phenotype, hence is an appropriate system to test gene therapy agents.Adult bovine nucleus pulposus and anulus fibrosus cells cultured in monolayer were transduced with adenoviruses expressing human bone morphogenetic proteins (AdBMPs) or Sox9 (AdSox9), or green fluorescence protein (AdGFP, as control). Chondrocyte phenotypic markers (e.g., type II collagen and aggrecan) and the chondrocyte hypertrophy marker (type X collagen) were measured 6 days after viral transduction by reverse-transcription polymerase chain reaction.Primary nucleus pulposus and anulus fibrosus cells transduced with AdBMPs, AdSox9, or adenovirus-expressing green fluorescence protein only (AdGFP, as control) continue to express healthy chondrocyte phenotypic markers and showed no evidence of the expression of the chondrocyte hypertrophy marker (type X collagen gene). Thus, we have shown that bovine nucleus pulposus and anulus fibrosus cells transduced with adenovirus overexpressing 12 different bone morphogenetic proteins or Sox9 maintain their phenotype in short-term culture.In this study, primary bovine intervertebral disc cells transduced with adenovirus overexpressing 12 bone morphogenetic proteins or Sox9 preserved their phenotype in short-term culture. These cells did not express the type X collagen gene, an undesirable chondrocyte hypertrophic gene that could lead to ossification. Therefore, low-passage intervertebral disc cells cultured in monolayer is an appropriate culture system to test therapeutic genes. We further suggest that these cells may also be appropriate for engineering tissues or for cell therapy for degenerative disc diseases.

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.

One of the advantages of chemonucleolysis for the treatment of a herniated intervertebral disc is the potential for the disc to self-repair. It has been suggested that the enzymes used for chemonucleolysis differentially affect the potential of the disc cells to promote repair.To test the ability of nucleus pulposus and anulus fibrosus cells to repair the extracellular matrix degraded in vitro by either chondroitinase ABC or chymopapain.An alginate cell culture system was used to monitor the progress of matrix repair after chemonucleolysis in vitro.Rabbit nucleus pulposus or anulus fibrosus cells precultured for 10 days in alginate gel were briefly exposed to low concentrations of chondroitinase ABC or chymopapain and then returned to normal culture conditions for up to 4 weeks. At each time point, the contents of DNA and matrix macromolecules and proteoglycan synthesis were measured.The DNA content of enzyme-treated alginate beads during the following 4 weeks of culture was higher in the chondroitinase ABC group than in the chymopapain group (NP, p.01, and AF, p.05). The content of proteoglycan in beads containing nucleus pulposus and anulus fibrosus cells in the chondroitinase ABC group was higher than that in the chymopapain group (NP and AF, p.001). The rate of proteoglycan synthesis and the content of collagen did not, however, differ between those two groups.Intervertebral disc cells exposed to chondroitinase ABC reestablish a matrix richer in proteoglycan than cells exposed to chymopapain. This may be because of differences in the substrate spectrum of each enzyme. Although these results cannot be translated directly to the in vivo situation, they suggest the possibility that cells in discs subjected to chondroitinase ABC-induced chemonucleolysis retain a greater ability to replenish their extracellular matrix with proteoglycans than cells in discs exposed to chymopapain.

Study Design. In vitro assessment of the effects of recombinant human osteogenic protein-1 (rhOP-1) on the proteoglycan metabolism of human intervertebral disc cells. Objectives. To determine whether rhOP-1 is effective in stimulating the cell proliferation and proteoglycan metabolism of human intervertebral disc cells cultured in alginate beads. Summary of the Background Data. OP-1 has been shown to stimulate the proteoglycan and collagen synthesis of rabbit intervertebral disc cells in vitro. In vivo, a single injection of rhOP-1 restored the disc height of a degenerated disc in the rabbit anular-puncture model. The effect of rhOP-1 on human intervertebral disc cells remains unknown. Methods. Human nucleus pulposus and anulus fibrosus cells were isolated from the discs of 4 cadaveric spines and one surgical specimen. After preculture for 7 days, alginate beads containing nucleus pulposus and anulus fibrosus cells were cultured for 21 days in media containing 10% fetal bovine serum with 0, 100, or 200 ng/mL rhOP-1 and supplements. The synthesis and accumulation of proteoglycans and the DNA content were biochemically assessed. Results. The addition of rhOP-1 to the media resulted in the prevention of a decreased cell number during culture. Treatment with rhOP-1, compared with the control condition (10% fetal bovine serum), significantly upregulated proteoglycan synthesis and accumulation in alginate beads in all cases tested. A longer exposure over 14 days to rhOP-1 resulted in a pronounced response. The retention of newly-synthesized proteoglycan was higher in the rhOP-1-treated cells than in the control. Conclusions. rhOP-1 was effective in stimulating the cell proliferation and proteoglycan metabolism of human intervertebral disc cells in vitro. The results supported the hypothesis that an in vivo injection of rhOP-1 may increase the metabolic activity of disc cells or prevent apoptosis of disc cells in a degenerated disc. However, the requirement for a long exposure to rhOP-1 for human cells may suggest the need for a prolonged supply of rhOP-1 by a drug delivery system or by repeated injections.

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.

Study design An in vitro biologic study of the effects of adenovirus expressing bone morphogenetic proteins (BMPs) and adenovirus expressing Sox9 on extracellular matrix metabolism by bovine nucleus pulposus cells. Objective To compare the effects of recombinant adenoviral vectors expressing various BMPs (2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15) and Sox9 on extracellular matrix accumulation by bovine nucleus pulposus cells. Summary of background data Nucleus pulposus matrix production may be promoted by transducing the cells with genes that permit the sustained expression of growth factors. The choice of the particular factors or BMPs to be studied for these applications has been largely based on the commercial availability of such products. To our knowledge, this study is the first effort to evaluate systematically the relative effectiveness of the various members of the BMP family in promoting intervertebral disc matrix repair. Methods Adult bovine nucleus pulposus cells cultured in monolayer were transduced with adenoviruses expressing human BMP-2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15, and adenovirus expressing Sox9. Proteoglycan and collagen accumulation, and cell proliferation were measured 6 days after viral transduction. As a positive control, cells were cultured without any exogenous gene in the presence of recombinant human (rh)BMP-7. Results Nucleus pulposus cells transduced with adenoviruses expressing BMP-2, 3, 4, 5, 7, 8, 10, 13, 15, and Sox9 accumulated more proteoglycans than nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein (control). It is noteworthy that nucleus pulposus cells transduced with adenoviruses expressing BMP-2 and 7 resulted in essentially as great a stimulation of proteoglycan accumulation as nucleus pulposus cells maintained in the presence of rhBMP-7 (adenoviruses expressing BMP-2: 104% increase; adenoviruses expressing BMP-7: 162% increase; and rhBMP-7: 120% increase). Nucleus pulposus cells transduced with BMP-2, 4, 5, 7, 8, 10, 14, 15, and Sox9 accumulated significantly more collagen compared to nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein; adenoviruses expressing BMP-4 and 14 were the most effective (552% and 661% increase, respectively). Nucleus pulposus cells also proliferated, as measured by deoxyribonucleic acid content, when transduced with adenoviruses expressing BMP-2 and 8. Conclusions To our knowledge, for the first time, we have shown the relative effectiveness of 12 different BMPs and Sox9 in stimulating proteoglycan and collagen production by nucleus pulposus cells. Adenoviruses expressing BMP-2 and 7 were the most effective in stimulating proteoglycan accumulation, while adenoviruses expressing BMP-4 and 14 were the most effective in stimulating collagen accumulation. To our knowledge, this study is the first to compare the relative effectiveness of various BMPs and Sox9 on extracellular matrix accumulation by nucleus pulposus cells, and could help to develop more efficacious approaches to the treatment of degenerating intervertebral discs.

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.

Study design In vitro assessment of the effects of platelet-rich plasma on the extracellular matrix metabolism of porcine intervertebral disc cells. Objectives To determine whether platelet-rich plasma is effective in stimulating cell proliferation and extracellular matrix metabolism by porcine disc cells cultured in alginate beads. Summary of background data Platelet-rich plasma is used to accelerate wound healing and tissue regeneration. Activated platelets release multiple growth factors that regulate cell proliferation, differentiation, and morphogenesis. Individual growth factors present in platelet-rich plasma have been demonstrated to affect the metabolism of intervertebral disc cells. Methods Platelet-poor and platelet-rich plasma was isolated from fresh porcine blood using a commercially available platelet concentration system. After preculture for 7 days and serum starvation for 24 hours, the beads containing nucleus pulposus and anulus fibrosus cells were then cultured for another 72 hours in serum-free medium, 10% fetal bovine serum, 10% platelet-poor plasma, or 10% platelet-rich plasma. The synthesis of proteoglycans and collagen, the accumulation of proteoglycans, and the DNA content were biochemically assessed. Results Platelet-rich plasma had a mild stimulatory effect on cell proliferation of intervertebral disc cells. Platelet-rich plasma treatment significantly upregulated proteoglycan and collagen synthesis and proteoglycan accumulation when compared with platelet-poor plasma. Conclusions Platelet-rich plasma was effective in stimulating cell proliferation and extracellular matrix metabolism. The response to platelet-rich plasma was greater in the case of anulus fibrosus cells than of nucleuspulposus cells. The local administration of platelet-rich plasma might stimulate intervertebral disc repair. In addition, given the risks of using animal serum for tissue engineering, autologous blood may gain favor as a source of growth factors and serum supplements needed for stimulating cells to engineer intervertebral disc tissues.

STUDY DESIGN In vitro study on the effects of pulsed low intensity ultrasound on the cellular metabolism of bovine intervertebral disc cells. OBJECTIVE To determine whether pulsed low intensity ultrasound has effects on cell proliferation and extracellular matrix metabolism by bovine intervertebral disc cells. SUMMARY OF BACKGROUND DATA The application of pulsed low intensity ultrasound is known to be effective in stimulating fracture and cartilage repair. However, the effects of pulsed low intensity ultrasound on intervertebral disc cells are not known. METHODS Cells of the nucleus pulposus and inner and outer anulus fibrosus were enzymatically isolated from bovine coccygeal tissue and precultured in alginate beads for 14 days. In the ultrasound group, pulsed low intensity ultrasound was administered to the culture for 20 minutes daily for an additional 20 days. The control group was cultured in the same way but without administration of ultrasound. Cell viability, DNA content, proteoglycan and collagen synthesis, and proteoglycan content at days 10 and 20 after the initiation of treatment were evaluated. Characterization of newly synthesized collagen and proteoglycan was performed. RESULTS No significant differences in cell viability and DNA content were observed between the two groups. On day 20, proteoglycan synthesis was increased by the application of pulsed low intensity ultrasound in nucleus pulposus and inner and outer anulus fibrosus cells (24%-26% increase, P < 0.001). The application of pulsed low intensity ultrasound increased proteoglycan content in alginate beads containing inner and outer anulus fibrosus cells (P < 0.05). Collagen synthesis by cells isolated from all three zones of the intervertebral disc was increased by the application of pulsed low intensity ultrasound (16%-19% increase, P < 0.05-0.0001). CONCLUSIONS The application of pulsed low intensity ultrasound stimulated extracellular matrix metabolism in intervertebral disc cells. Pulsed low intensity ultrasound may prove useful for the physical stimulation of cell metabolism for tissue engineering of intervertebral disc tissue.

The purpose of this study was to determine if acetabular articular cartilage damage occurs in the presence or absence of changes in subchondral plate thickness or porosity and trabecular bone architecture after hip hemiarthroplasty. Eight canines were sacrificed 6 months after receiving unilateral hemiarthroplasties in which a cobalt chrome alloy femoral head was used. The acetabular cartilage, subchondral plate, and trabecular bone were quantitatively evaluated. Although the articular cartilage in the treated hip showed gross and histologic degenerative changes, there were no differences in the treated and contralateral hips in any of the trabecular bone parameters or subchondral plate thickness. However, the subchondral plate porosity was increased 2.6-fold in the treated hip. Therefore, degradation of cartilage can occur in the absence of thickening of the subchondral plate or alterations in the supporting trabecular bone architecture. These observations provide a better understanding of the role that periarticular bone has in the degenerative process after hemiarthoplasty.

Background After serial passages in monolayer, chondrocytes dedifferentiate into a fibroblast-like phenotype. Our objective was to determine if culture in alginate affects the phenotype of dedifferentiated human nasal septal chondrocytes. Study design Human nasal septal chondrocytes were seeded at low density and passaged in monolayer culture. At passages (P) 1, 2, and 3 a portion of cells were cultured in alginate. Collagen, glycosaminoglycan (GAG), and DNA production were assessed. Results Chondrocytes in alginate proliferated less yet produced higher levels of GAG and collagen than those in monolayer culture. Alginate encapsulated P1 chondrocytes stained strongly for GAG and collagen type II, and minimally for collagen type I. Monolayer cells at P0 and P1 stained positively for collagen type II. All monolayer passages stained positive for collagen type I with minimal GAG staining. Conclusions Compared with monolayer culture, alginate stimulates deposition of GAG and collagen type II, and supports the chondrocyte phenotype through P1, but does not promote redifferentiation.

Objective: Low back pain associated with degenerative disk disease is a common clinical problem that has enormous socioeconomic impact in today's aging population. As an alternative to the surgical removal of the diseasedintervertebral disk, the direct application of a purified growth factor into the intervertebral disk may provide physiatrists a valuable tool to halt or slow down disk degeneration. Our goal here is to determine if low levels of interleukin-1 (IL-1), a proinflammatory cytokine present in the degenerating disk, could interfere with the potentially beneficial effects of growth factors on proteoglycan synthesis. New knowledge gained from this study will prove useful in the development of new treatment modalities that take advantage of the stimulatory effects of growth factors such as osteogenic protein-1 (OP-1). Design: This was an in vitro study of proteoglycan accumulation and synthesis by cells from the nucleus pulposus, inner annulus fibrosus, and outer annulus fibrosus in the bovine intervertebral disk. Results: In cells cultured with serum and no additional exogenous growth factor, treatment with tow-dose IL-1 does not result in a significant decrease in proteoglycan synthesis. However, in the case of cells stimulated with OP-1, treatment with IL-1 resulted in a statistically significant decrease in sulfur-35-proteoglycan synthesis by cells derived from all three zones of the bovine intervertebral disk (nucleus pulposus, 60.3% [P < 0.0001]; inner annulus fibrosus, 18.4% [P = 0.0330]; outer annulus fibrosus, 12.3% [P = 0.0255]). Proteoglycan accumulation over the 12-day culture period also decreased significantly (nucleus pulposus, 26.8% [P < 0.0001]; inner annulus fibrosus, 15.8% [P = 0.0276]; outer annulus fibrosus, 16.8% [P = 0.0102]). It is worth noting that cells cultured in the presence of both OP-1 and IL-1 synthesized proteoglycan at a faster rate than cells cultured in the presence of IL-1 alone (nucleus pulposus, 58.5% [P < 0.0001]; inner annulus fibrosus, 39.7% [P = 0.0055]; outer annulus fibrosus, 45.1% [P = 0.0164]). Likewise, cells treated with OP-1 and IL-1 accumulated more proteoglycan in their newly formed matrix than cells cultured in the presence of IL-1 alone (nucleus pulposus, 65.3% [P < 0.0001]; inner annulus fibrosus, 60.5% [P = 0.0034]; outer annulus fibrosus, 19.4% (P = 0.0840]). Conclusions: Intervertebral disk cells that are stimulated by the growth factor OP-1, to to increase the rate at which they produce a proteoglycan-rich matrix become more susceptible to the inhibitory effects of the proinflammatory cytokine IL-1 on the rate of proteoglycan synthesis and accumulation in the matrix over time. This notwithstanding, IL-1 at the low dose used did not totally obliterate the stimulatory effects of OP-1 on matrix formation. Consequently, this growth factor may remain partially effective in stimulating disk repair in vivo even when proinflammatory cytokines such as IL-1 are present.

This in vitro study used the alginate bead culture system to probe for differences in the effects of fibronectin fragment on cell proliferation and proteoglycan metabolism by different populations of intervertebral disc cells and articular chondrocytes.To compare the effects of fibronectin fragment on cell proliferation, and proteoglycan synthesis and degradation by cells from the nucleus pulposus, the anulus fibrosus, and articular cartilage.In articular cartilage, the administration of fibronectin fragment stimulates cartilage degeneration. Fibronectin fragment levels were increased in human intervertebral discs with increased disc degeneration. Fibronectin fragment injected into the central region of the rabbit intervertebral disc induced a progressive degeneration of that disc.Bovine tails and metacarpophalangeal joints from 14- to 18-month-old animals were used. Alginate beads containing cells isolated from intervertebral discs and articular cartilage were cultured with (1-100 nmol/L) or without (control) fibronectin fragment in the presence of 10% fetal bovine serum. In these cultures, deoxyribonucleic acid and proteoglycan contents, as well as the rate of proteoglycan synthesis were determined. Proteoglycan degradation was measured in cultures with or without 10 nmol/L fibronectin fragment.In articular chondrocytes, fibronectin fragment strongly suppressed proteoglycan synthesis and stimulated proteoglycan degradation; the total proteoglycan content was diminished in a dose-dependent manner. Compared to articular chondrocytes, nucleus pulposus cells responded to fibronectin fragments in a similar, although less pronounced manner. On the other hand, anulus fibrosus cells treated with fibronectin fragment did not show any significant effects on proteoglycan degradation. A slight but statistically significant up-regulation of proteoglycan synthesis was observed at 10 nmol/L fibronectin fragment in outer anulus fibrosus cells. However, total proteoglycan content was decreased significantly at high concentrations of fibronectin fragment.Fibronectin fragment has different effects on cell proliferation, proteoglycan synthesis, degradation, and accumulation by articular chondrocytes and intervertebral disc cells. The different effects of fibronectin fragment in those different cell types suggest metabolic differences between these cells, and may further suggest differences in pathways of fibronectin fragment signaling as well as a differential need of these cells to be involved in tissue remodeling in which both anabolic and catabolic pathways might be altered.

Objective: The objective of this study was to determine if cells isolated from three distinct zones in the bovine intervertebral disc (IVD) differ in their response to growth factor osteogenic protein-1 (OP-1) because of inherent biological differences. The new knowledge gained will help determine if treatment of degenerative disc disease with purified growth factors is effective and will provide guidance in terms of the injection technique and frequency of treatments required. Design: This was an in vitro study measuring the effects of OP-1 on proteoglycan accumulation and synthesis by cells from the nucleus pulposus, inner-annulus fibrosus (inner-AF), and outer-annulus fibrosus (outer-AF) in the bovine IVD. Results: Growth factor OP-1, at 100 ng/ml, stimulated proteoglycan accumulation and resulted in a statistically significant increase in the proteoglycan content of cells derived from three zones of the bovine IVD: 97% in the nucleus pulposus, 40% in the inner-AF, and 75% in the outer-AF. To elucidate the mechanism of enhanced proteoglycan accumulation in response to OP-1, we studied the rate of proteoglycan synthesis and cell proliferation. OP-1 stimulation resulted in a statistically significant increase in the DNA content in cultures containing cells from all three zones of the IVDs: 79% in the nucleus pulposus, 100% in the inner-AF, and 73% in the outer-AF. After dividing by DNA content, OP-1 resulted in a statistically significant increase in the rate of proteoglycan synthesis in the nucleus pulposus (78%) and outer-AF (17%) cells, but the increase in inner-AF cells (23%) did not achieve statistical significance. Conclusions: OP-1 stimulates proteoglycan accumulation by bovine IVD cells isolated from all three zones of the bovine IVDs. Cells from all three zones proliferated significantly. Individual cells derived from nucleus pulposus and outer-AF, but not those from the inner-AF, synthesized proteoglycans at a significantly faster rate with OP-1 stimulation.

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.

STUDY DESIGN A study of the mechanisms involved in matrix repair by intervertebral disc cells cultured in alginate gel was performed. OBJECTIVES To determine the effects of osteogenic protein-1 on the extracellular matrix of intervertebral disc cells previously exposed to interleukin-1, which is an in vitro model for degraded extracellular matrix. SUMMARY OF BACKGROUND DATA Disc degeneration is accompanied by a decrease in the content of negatively charged proteoglycans in the matrix. No previous attempt has been made to repair the degraded matrix of the disc. METHODS Nucleus pulposus and anulus fibrosus cells were isolated from the lumbar discs of New Zealand white rabbits and were separately encapsulated in alginate beads. The alginate beads were cultured with or without osteogenic protein-1 after previous exposure to interleukin-1alpha in the presence of 10% fetal bovine serum. The total contents of proteoglycan, collagen, and DNA in the alginate beads were measured. The rate of proteoglycan synthesis by the encapsulated cells was also determined. RESULTS Treatment with interleukin-1alpha resulted in a significant decrease in proteoglycan and collagen contents in the matrix formed by both the nucleus pulposus and anulus fibrosus. However, subsequent treatment with osteogenic protein-1 led in both cases to rapid recovery of proteoglycans and collagens, whose contents returned to the levels seen in cells not previously exposed to interleukin-1alpha. By the end of the culture period (day 21), those values reached levels higher than those found in beads containing cells never exposed to interleukin-1alpha. Further, the rate of proteoglycan synthesis by both cell types in beads treated with osteogenic protein-1 after previous exposure to interleukin-1alpha was significantly higher than in beads whose cells were not treated with osteogenic protein-1 after previous exposure to interleukin-1alpha. CONCLUSION Disc cells that have been previously exposed to interleukin-1alpha have lost none of their potential to upregulate proteoglycan synthesis in response to stimulation with osteogenic protein-1. On stimulation with osteogenic protein-1, these disc cells not only replenished the matrix with proteoglycans that had been lost during interleukin-1alpha treatment but proceeded to reform a matrix that was richer in these resilient molecules than that formed by disc cells never exposed to interleukin-1alpha.

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.

Objective The adduction moment at the knee during gait is the primary determinant of medial-tolateral load distribution. If the adduction moment contributes to progression of osteoarthritis (OA), then patients with advanced medial tibiofemoral OA should have higher adduction moments. The present study was undertaken to investigate the hypothesis that the adduction moment normalized for weight and height is associated with medial tibiofemoral OA disease severity after controlling for age, sex, and pain level, and to examine the correlation of serum hyaluronan (HA) level with disease severity and with the adduction moment in a subset of patients. Methods Fifty-four patients with medial tibiofemoral OA underwent gait analysis and radiographic evaluation. Disease severity was assessed using the Kellgren-Lawrence (K-L) grade and medial joint space width. In a subset of 23 patients with available sera, HA was quantified by sandwich enzyme-linked immunosorbent assay. Pearson correlations, a random effects model, and multivariate regression models were used. Results The adduction moment correlated with the K-L grade in the left and right knees (r = 0.68 and r = 0.60, respectively), and with joint space width in the left and right knees (r = -0.45 and r = -0.47, respectively). The relationship persisted after controlling for age, sex, and severity of pain. The partial correlation between K-L grade and adduction moment was 0.71 in the left knees and 0.61 in the right knees. For every 1.0-unit increase in adduction moment, there was a 0.63-mm decrease in joint space width. In the subset of patients in whom serum HA levels were measured, HA levels correlated with medial joint space width (r = -0.55), but not with the adduction moment. Conclusion There is a significant relationship between the adduction moment and OA disease severity. Serum HA levels correlate with joint space width but not with the adduction moment. Longitudinal studies will be necessary to determine the contribution of the adduction moment, and its contribution in conjunction with metabolic markers, to progression of medial tibiofemoral OA.

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.

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

The role of cytokines in the regulation of articular inflammation and cartilage degradation was evaluated in the rabbit model of Haemophilus influenzae type b arthritis. At 6 and 12 h after intraarticular infection, treatment with IB4 monoclonal antibody to the CD18 leukocyte receptor alone or in combination with dexamethasone resulted in significant reduction of synovial fluid (SF) neutrophil concentration. Treatment with dexamethasone alone was associated with lower SF concentrations of interleukin-1 (IL-1), tumor necrosis factor-alpha, and stromelysin than in other groups. At 24 h after infection, increased cartilage degradation was detected in untreated controls and in animals treated with IB4 alone or in combination with dexamethasone compared with those treated with dexamethasone alone. Multiple regression analyses indicated SF concentration of IL-1 and stromelysin as the significant predictors of cartilage degradation. These data suggest that IL-1 mediates cartilage degradation by regulation of metalloproteinases, such as stromelysin, during acute experimental bacterial arthritis.

Various markers of the metabolism of articular cartilage have been identified in synovial fluid, blood, and urine of patients with osteoarthritis (OA). The joint fluid level of a cartilage-derived molecule, or its fragment, can be used as a marker of the synthesis or catabolism of that molecule in the articular surfaces within that joint. In blood and urine, on the other hand, the level of a marker is useful in assessing systemic changes affecting the metabolism of a molecule in all the cartilages in the body. Quantification of specific markers in body fluids already has proved useful in identifying increased catabolic as well as anabolic activities in articular cartilage during preradiologic as well as later stages of OA. The markers also can be sued for monitoring the effect of drugs on cartilage matrix molecules and in differentiating among different subtypes of osteoarthritis. Markers should prove most useful in prospective studies aimed at identifying early changes in cartilage metabolism in humans at high risk for developing OA.

Low back pain carries an enormous socioeconomic burden. Current treatment modalities for symptomatic intervertebral disk (IVD) degeneration have limited and often inconsistent clinical benefits. Novel approaches with the potential to halt or even reverse disk degeneration and restore physiologic disk function, such as biological treatments, are therefore very attractive. The following barriers are impeding the development of successful therapeutic interventions: (1) the biology and pathophysiology of disk degeneration are not well understood, and (2) the precise relationship between IVD degeneration and low back pain remains unclear. This article reviews the structural changes that take place during IVD degeneration and their relationship to diskogenic back pain. It also presents treatment modalities that currently are under laboratory investigation and are being studied in clinical trials. The authors of recent studies have shown that the content of large proteoglycans, such as aggrecan and versican, decreases with aging and IVD degeneration, whereas the content of certain small proteoglycans, such as biglycan, increases. Proinflammatory cytokines such as interleukin-1 and tumor necrosis factor-α also are associated with IVD degeneration and are potential biomarkers of IVD degeneration and repair. Our group of investigators and others have developed in vitro models of IVD cell and explant culture in addition to in vivo animal models to study IVD degeneration and repair. With the use of these models, we have tested candidate therapeutic agents to assess their therapeutic potential for matrix restoration. When a rabbit annular puncture model of IVD degeneration was used, injections of either bone morphogenetic protein-7 (also known as osteogenic protein-1) or bone morphogenetic protein-14 (also known as growth differentiation factor-5) were shown to be effective in restoring IVD structures. On the basis of these data, the Food and Drug Administration has recently allowed the initiation of Investigational New Drug clinical trials on osteogenic protein-1 and growth differentiation factor-5 in the United States. Protein therapies such as other growth factors, inhibitors of degradation enzymes or cytokines, and cell therapies also are being investigated in laboratory settings with the goal of restoring disk function and alleviating back pain symptoms. These therapies may be used by physiatrists with the skills required to administer intradiskal injections and supervise a comprehensive rehabilitation program after the procedures. Ultimately, the clinical use of any biological treatment discussed in this article would require the collective efforts of clinicians and researchers.

To characterize age-related changes in the matrix of human intervertebral disc (IVD) specimens, human specimens from the third to the eighth decade of life were collected and analyzed for collagen and proteoglycan (PG) composition.To identify age-related changes in the concentration of matrix macromolecules (collagen and PGs, including the small leucine-rich PGs biglycan, decorin, fibromodulin, and lumican) in human anulus fibrosus (AF) and nucleus pulposus (NP).IVD degeneration is associated with changes in the concentration and fragmentation of matrix molecules. Deciphering age-related matrix alterations may help us to better understand the regulatory mechanisms underlying IVD degeneration.Forty-six whole IVDs were obtained from the thoracolumbar spines (T11-L5) of humans aged between 32 and 80 years. All specimens were classified as Thompson grade 1 or 2 according to MRI criteria. Specimens were separated into (i) outer-and (ii) inner AF, and (iii) NP. DNA, collagen, and PG contents were measured using chemical assays, whereas small nonaggregating PG levels were analyzed by comparative Western blotting.Total PG and collagen contents in both the AF and NP consistently decreased with aging. The concentrations of small nonaggregating PGs varied. In the outer anulus, decorin levels decreased, whereas biglycan and fibromodulin levels increased with age. In the inner anulus and nucleus, biglycan demonstrated a significant increase with aging. These changes differed in most cases from those previously reported for degenerating disc tissues.Collagen and PGs appeared to undergo specific age-related changes in the human IVD. Although the total contents of these 2 families of molecules decreased during aging, individual species of small nonaggregating PGs showed species-specific age-related changes. Interestingly, the level of biglycan rose and remained elevated in all 3 compartments of the disc with aging. The functional significance of these alterations is yet to be determined.

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 structure and content of the extracellular stromal matrix of several oedematous human corneas was investigated using electron microscopy, X-ray diffraction and biochemical techniques. Electron microscopy revealed the presence of wavy lamellae and various sized collagen-free ‘lakes’ within the stroma of the oedematous corneas, with their posterior sections containing by far the largest ‘lakes’. The existence of ‘lakes’ was supported by the equatorial X-ray diffraction evidence. Staining the oedematous corneas with Cuprolinic blue prior to electron microscopical and meridional X-ray diffraction studies demonstrated a loss of stromal proteoglycans normally associated with collagen. Immunochemical evidence demonstrated reduced levels of antigenic keratan sulphate in the oedematous corneas while biochemical techniques revealed constant chondroitin sulphate levels in the same corneas.

Spine care is a fast-growing sector of the outpatient practice for physiatrists. Current nonsurgical treatment modalities and surgical options for severe symptomatic intervertebral disc degeneration have limited and inconsistent clinical results. Thus, the development of novel approaches, such as biological treatments that offer the potential to halt or even reverse disc degeneration and restore physiologic disc function, are very attractive. In this article, we first review the structural changes that occur during intervertebral disc degeneration and their relationship with discogenic back pain. Subsequently, we review the treatment approaches currently under clinical trial and laboratory investigation. Physiatrists specializing in spine care have the skill set required for administering intradiscal injections and supervising a comprehensive rehabilitation program after the procedures. Ultimately, the clinical use of any biological treatment discussed herein would require the collective efforts of physicians (such as physiatrists and surgeons) and researchers (such as chemical and biomedical engineers, biologists, and chemists).

A cartilage growth mixture (CGM) model is proposed to address limitations of a model used in a previous study. New stress constitutive equations for the solid matrix are derived and collagen (COL) remodeling is incorporated into the CGM model by allowing the intrinsic COL material constants to evolve during growth. An analytical validation protocol based on experimental data from a recent in vitro growth study is developed. Available data included measurements of tissue volume, biochemical composition, and tensile modulus for bovine calf articular cartilage (AC) explants harvested at three depths and incubated for 13days in 20% fetal borine serum (FBS) and 20% FBS+β-aminopropionitrile. The proposed CGM model can match tissue biochemical content and volume exactly while predicting theoretical values of tensile moduli that do not significantly differ from experimental values. Also, theoretical values of a scalar COL remodeling factor are positively correlated with COL cross-link content, and mass growth functions are positively correlated with cell density. The results suggest that the CGM model may help us to guide in vitro growth protocols for AC tissue via the a priori prediction of geometric and biomechanical properties.