Gabriella Cs-Szabo

Suppression of Osteoblast Function by Titanium Particles

In order to understand the effect of particulate debris on osteoblast function, we studied the effect of different particles, including titanium and polystyrene, on bone collagen mRNA (messenger RNA) with the use of Northern blot hybridization analysis, and we studied the effect of the particles on the biosynthesis of bone collagen with analysis of 3H-proline incorporation and with the Western blot technique. The steady-state levels of mRNA for procollagens &agr;1(I) and &agr;1(III) were markedly suppressed in human MG-63 osteoblast-like cells exposed to phagocytosable titanium particles that were smaller than three micrometers. Both titanium and polystyrene particles smaller than three micrometers suppressed the expression of the gene that codes for collagen, and the suppression of the expression of the gene was related to the size but not to the composition of the particles. The biosynthesis of both type-I and type-III collagen also was decreased in cells that had been treated with titanium particles. Neither the viability nor the proliferation of cells was affected by particulate debris. These data indicate that phagocytosable titanium particles can significantly suppress the expression of the gene that codes for collagen in osteoblast-like cells (p < 0.05).

Human Articular Chondrocytes Express Osteogenic Protein-1

This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms.

AGE-RELATED CHANGES IN THE EXTRACELLULAR MATRIX OF NUCLEUS PULPOSUS AND ANULUS FIBROSUS OF HUMAN INTERVERTEBRAL DISC

Study Design. 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. Objective. 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). Summary of Background Data. 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. Methods. 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. Results. 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. Conclusion. 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.

Prostaglandin E2 and its cognate EP receptors control human adult articular cartilage homeostasis and are linked to the pathophysiology of osteoarthritis.

OBJECTIVE To elucidate the pathophysiologic links between prostaglandin E(2) (PGE(2)) and osteoarthritis (OA) by characterizing the catabolic effects of PGE(2) and its unique receptors in human adult articular chondrocytes. METHODS Human adult articular chondrocytes were cultured in monolayer or alginate beads with and without PGE(2) and/or agonists of EP receptors, antagonists of EP receptors, and cytokines. Cell survival, proliferation, and total proteoglycan synthesis and accumulation were measured in alginate beads. Chondrocyte-related gene expression and phosphatidylinositol 3-kinase/Akt signaling were assessed by real-time reverse transcription-polymerase chain reaction and Western blotting, respectively, using a monolayer cell culture model. RESULTS Stimulation of human articular chondrocytes with PGE(2) through the EP2 receptor suppressed proteoglycan accumulation and synthesis, suppressed aggrecan gene expression, did not appreciably affect expression of matrix-degrading enzymes, and decreased the type II collagen:type I collagen ratio. EP2 and EP4 receptors were expressed at higher levels in knee cartilage than in ankle cartilage and in a grade-dependent manner. PGE(2) titration combined with interleukin-1 (IL-1) synergistically accelerated expression of pain-associated molecules such as inducible nitric oxide synthase and IL-6. Finally, stimulation with exogenous PGE(2) or an EP2 receptor-specific agonist inhibited activation of Akt that was induced by insulin-like growth factor 1. CONCLUSION PGE(2) exerts an antianabolic effect on human adult articular cartilage in vitro, and EP2 and EP4 receptor antagonists may represent effective therapeutic agents for the treatment of OA.

Identification of multiple loci linked to inflammation and autoantibody production by a genome scan of a murine model of rheumatoid arthritis.

OBJECTIVE Proteoglycan-induced arthritis (PGIA) is a murine model of rheumatoid arthritis (RA), both in terms of its pathology and its genetics. PGIA can only be induced in susceptible murine strains and their F2 progeny. As with RA, the genetics are complex, containing both major histocompatibility complex (MHC)-related and non-MHC-related components. Our goal was to identify the underlying non-MHC-related loci that confer PGIA susceptibility. METHODS We used 106 polymorphic markers to perform simple sequence-length polymorphism analysis on F2 hybrids of susceptible (BALB/c) and nonsusceptible (DBA/2) strains of mice. Because both strains of mice share the H2d haplotype, this cross permits identification and analysis of non-MHC-related genes. RESULTS We identified a total of 12 separate quantitative trait loci (QTL) associated with PGIA, which we have named Pgia1 through Pgia12. QTLs associated with the inflammatory symptoms of PGIA were linked to chromosomes 7, 9, 15 (2 separate loci), 16, and 19. QTLs associated with autoantibody production were identified on chromosomes 1, 2, 7, 8, 10, 11, 16, and 18. QTLs on chromosomes 7 and 16 showed linkage to both inflammation and autoantibody production, suggesting a shared regulatory component in arthritis induction. The first inflammation QTL on chromosome 15 and the autoantibody QTL on chromosome 7 originate from the DBA/2 background, which indicates that as in RA, susceptibility genes can originate from heterogeneous backgrounds. CONCLUSION These data demonstrate the complexity of PGIA, where QTLs may be involved in multiple traits or even originate from a genetic background previously determined to be resistant.

The Effects of Collagen Fragments on the Extracellular Matrix Metabolism of Bovine and Human Chondrocytes

Cartilage matrix degradation generates collagen type II fragments. The objective of this study is to explore the possibility that these collagen fragments may be part of an endogenous metabolic feedback. Initially, collagen fragments were extracted from normal or osteoarthritic cartilage, as part of a matrix fragment preparation. Later, collagen fragments were generated by digestion of bovine collagen type II with bacterial collagenase (col2f). These fragments were added to cultures of isolated chondrocytes (bovine and human) and cartilage explants (human). In a dose-dependent manner, col2f caused inhibition of cell attachment to collagen, inhibition of collagen synthesis, and induction of matrix degradation. In addition, when col2f were added to human cartilage explants, an induction of gelatinase activity was detected in the media. These data sets present first evidence that degradation products of collagen may be directly involved in the regulation of cartilage homeostasis.

The characterization of versican and its message in human articular cartilage and intervertebral disc

Splicing variation of the versican message and size heterogeneity of the versican core protein were analyzed in human articular cartilage and intervertebral disc. Splicing variation of the message was studied by PCR analysis to detect the presence or absence of exons 7 and 8, which encode large chondroitin sulfate attachment regions. At all ages in normal cartilage from the third trimester fetus to the mature adult, the presence of the versican isoform possessing exon 8 but not exon 7 (V1) could be readily detected. The message isoforms possessing neither exon 7 nor 8 (V3) or both exons 7 and 8 (V0) were only detectable in the fetus, and the isoform possessing only exon 7 (V2) was never detected. In osteoarthritic cartilage and in adult intervertebral disc the versican message pattern was the same as that observed in the normal adult with only the isoform possessing exon 8 being detected. Core protein heterogeneity was studied by immunoblotting following enzymic removal of the glycosaminoglycan chains from the proteoglycan, using an antibody recognizing the globular G1 region of versican. All articular cartilage extracts from the fetus to the mature adult contained multiple core protein sizes of greater than 200 kDa. The adult cartilage extracts tended to have an increased proportion of the smaller sized core proteins and osteoarthritic cartilage possessed similar core protein sizes to the normal adult. In contrast, intervertebral disc at all post‐natal ages showed a greater range of size heterogeneity with a prominent component of about 50 kDa. The abundance of this component increased if the samples were treated with keratanase prior to analysis, suggesting that the G1 region of versican in disc can be substituted with keratan sulfate. The increased presence of versican in the disc relative to articular cartilage may suggest a more pronounced functional role for this proteoglycan, particularly in the nucleus pulposus.

The effects of age, sex, ethnicity, and spinal level on the rate of intervertebral disc degeneration: A review of 1712 intervertebral discs

Study Design. A gross anatomic and magnetic resonance imaging study of intervertebral disc (IVD) degeneration in fresh cadaveric lumbar spines. Objective. The purpose of this study was to find the rate of IVD degeneration. Summary of Background Data. Age, sex, race, and lumbar level are among some of the factors that play a role in IVD degeneration. The rate at which IVDs degenerate is unknown. Methods. Complete lumbar spine segments (T11/T12 to S1) were received within 24 hours of death. The nucleus pulposus, anulus fibrosus, cartilaginous and bony endplate, and the peripheral vertebral body were assessed with magnetic resonance imaging and IVD degeneration was graded by two observers from grade 1 (nondegenerated) to grade 5 (severely degenerated) on the basis of a scale developed by Tanaka et al. The specimens were then sectioned and gross anatomic evaluation was performed according to Thompson et al. Results. A total of 433 donors and 1712 IVDs were analyzed. There were 366 whites, 47 Africans, 16 Hispanics, 4 Asian. There were 306 male and 127 female donors. The age range was 14 to 81 years, (average: 60.5 ± 11.3). For donors greater than age 40, the L5/S1 IVD degenerated at a significantly faster rate of 0.043 per year compared to 0.031, 0.034, 0.033, 0.027 for L1/L2, L2/L3, L3/L4, L4/L5, respectively. For donors younger than 40, L5/S1 IVD degenerated at a significantly faster rate of 0.141/y compared to 0.033, 0.021, 0.031, 0.050 for L1/L2, L2/L3, L3/L4, L4/L5, respectively. Multiple regression analysis revealed that sex had no significant effect on IVD degeneration whereas African ethnicity was associated with lower Thompson score at L1/L2, L2/L3, L3/L4, L4/L5 when compared with whites. Conclusion. The relatively early degeneration at L5–S1 in all races and lower Thompson grade in donors of African ethnicity needs further investigation. Factors such as sagittal alignment, facet joint arthritis, and genetics potentially play a role in IVD degeneration.

Changes with age in the structure of fibromodulin in human articular cartilage

An anti-peptide antibody was raised in a rabbit against the carboxy terminal region of the human fibromodulin core protein. The antibody was purified from other components of the resulting antiserum by affinity chromatography using the immobilized peptide, and was used to study the structural heterogeneity of fibromodulin extracted from human articular cartilage of different ages by the use of immunoblotting following sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the extracted macromolecules. In the fetus and neonate, fibromodulin was visualized as a diffuse component with a relative molecular weight of 70-110 kDa, whereas in the mature adult a more discrete component of smaller size was apparent with a relative molecular weight of 67 kDa. The size of the fibromodulin from mature adult cartilage could not be altered by pretreatment of the samples with keratanase II or endo-beta-galactosidase before analysis. In contrast, the size of the fibromodulin from younger cartilage could be decreased with both glycosidases, with the endo-beta-galactosidase yielding a smaller product than the keratanase. The size of the product resulting from endo-beta-galactosidase treatment of the fibromodulin from young cartilage was the same as that of the intact fibromodulin from mature adult cartilage. Thus, fibromodulin is present in human articular cartilage at all ages, but the extracted molecules only appear to exist in a proteoglycan form possessing keratan sulfate chains in the juvenile and young adult, and the size of these chains decreases with age. In the mature adult the fibromodulin does not possess either keratan sulfate or non-sulfated polylactosamine chains, though it appears to possess the same number of N-linked oligosaccharides as its counterparts from the younger tissue, but they are not modified further. The majority of the fibromodulin extracted from arthritic cartilage is of the same size as that found in the normal mature adult, although there is evidence for proteolytic processing. The degree of such processing is greater for the fibromodulin obtained from the cartilage of rheumatoid arthritic joints than osteoarthritic joints.

Spontaneous and experimental osteoarthritis in dog: similarities and differences in proteoglycan levels.

The unilateral canine model is the most commonly used model of experimental osteoarthritis (OA). In this model, the anterior cruciate ligament (ACL) of one knee is transected and the contralateral joint is usually used as a control. However, dogs, similar to humans, can develop OA spontaneously with old age. Additionally, certain breeds of dogs are genetically predisposed to OA and can develop symptoms at a young age. The goal of this study was to compare the pathological changes of proteoglycans in OA cartilage from dogs that developed OA spontaneously to those that underwent ACL transection. For this reason, biglycan, decorin and fibromodulin levels and degradation patterns were compared by Western blot hybridization, and aggrecan contents were quantified by dimethylmethylene blue assay. The changes in proteoglycan levels in the cartilage of dogs with spontaneous OA, regardless of their age, were very similar to those published for human OA cartilage. However, when OA developed as a result of ACL‐surgery, the changes in proteoglycans were different from those of slowly developing spontaneous OA. Therefore, these differences should be taken into consideration when the ACL‐transection model is used.