Janis Menage

Matrix Metalloproteinases And Aggrecanase : Their Role in Disorders of the Human Intervertebral Disc

Study Design. A comprehensive immunohistochemical study of matrix metalloproteinase activity in discs from patients with different disc diseases.— Objectives. To identify individual matrix metalloproteinase enzymes that could contribute to the degeneration of the matrix of the intervertebral disc, to identify the cells that produce matrix metalloproteinases (for example, the endogenous disc cells or invading cells associated with vascularisation), and to determine if “aggrecanase” contributes to degradation of proteoglycans in disc disorders. Summary of Background Data. Matrix disorganization and loss of substance are the most common findings in degenerate discs, and proteinase enzyme activity is one means of causing these changes. Methods. Forty-nine discs from 46 patients with degenerative disc disease, posterior anular tears, spondylolisthesis, or disc herniation were studied immunohistochemically to determine the presence of matrix metalloproteinases 1, 2, 3, 7, 8, 9 and 13, tissue metalloproteinases 1 and 2, and proteoglycan degradation products generated by either matrix metalloproteinases or aggrecanase activity. In addition, in situ zymography was used to confirm matrix metalloproteinase activity. Results. The most extensive staining was seen for matrix metalloproteinases 1, 2, 3, and 9, with 91%, 71%, 65%, and 72% of samples having some immunopositivity for the respective antibodies. In contrast, staining for matrix metalloproteinases 7 and 8 was much less (38% for both). Tissue inhibitor of metalloproteinases 1 and 2 were expressed in 34% and 79% of specimens, respectively. Matrix metalloproteinases were found particularly in cell clusters and blood vessels of degenerate discs, with staining correlating positively with macroscopic degenerative grade. For all of the enzymes, there was most staining in the herniation specimens and least in the autopsy samples. The opposite was true of staining for the matrix metalloproteinases inhibitor, tissue inhibitor of metalloproteinases 2, with most found in the autopsy specimens. Enzyme activity was confirmed by in situ zymography and staining for matrix metalloproteinase degradation products of proteoglycans. In addition, there was staining with antibodies demonstrating aggrecanase degradation products. Conclusions. Matrix metalloproteinase activity is more prevalent in herniated discs than in other disc disorders studied, although matrix metalloproteinases may have been more common earlier in the disease progression. Matrix metalloproteinases can be produced by invading blood vessels and associated cells, as well as by indigenous disc cells. Aggrecanase activity, although present in some samples, was not as obvious as that of matrix metalloproteinases. In addition to altered matrix metalloproteinase production, there appears to be a change in the balance between enzymes and endogenous inhibitors, tissue inhibitors of metalloproteinases. This study highlights specific matrix metalloproteinases that might be most efficient to target in developing therapeutics for minimizing degradation of the extracellular matrix of the disc.

Histology and pathology of the human intervertebral disc.

The intervertebral disc is a highly organized matrix laid down by relatively few cells in a specific manner. The central gelatinous nucleus pulposus is contained within the more collagenous anulus fibrosus laterally and the cartilage end plates inferiorly and superiorly. The anulus consists of concentric rings or lamellae, with fibers in the outer lamellae continuing into the longitudinal ligaments and vertebral bodies. This arrangement allows the discs to facilitate movement and flexibility within what would be an otherwise rigid spine. At birth, the human disc has some vascular supply within both the cartilage end plates and the anulus fibrosus, but these vessels soon recede, leaving the disc with little direct blood supply in the healthy adult. With increasing age, water is lost from the matrix, and the proteoglycan content also changes and diminishes. The disc-particularly the nucleus-becomes less gelatinous and more fibrous, and cracks and fissures eventually form. More blood vessels begin to grow into the disc from the outer areas of the anulus. There is an increase in cell proliferation and formation of cell clusters as well as an increase in cell death. The cartilage end plate undergoes thinning, altered cell density, formation of fissures, and sclerosis of the subchondral bone. These changes are similar to those seen in degenerative disc disease, causing discussion as to whether aging and degeneration are separate processes or the same process occurring over a different timescale. Additional disorders involving the intervertebral disc can demonstrate other changes in morphology. Discs from patients with spinal deformities such as scoliosis have ectopic calcification in the cartilage end plate and sometimes in the disc itself. Cells in these discs and cells from patients with spondylolisthesis have been found to have very long cell processes. Cells in herniated discs appear to have a higher degree of cellular senescence than cells in nonherniated discs and produce a greater abundance of matrix metalloproteinases. The role that abnormalities play in the etiopathogenesis of different disorders is not always clear. Disorders may be caused by a genetic predisposition or a tissue response to an insult or altered mechanical environment. Whatever the initial cause, a change in the morphology of the tissue is likely to alter the physiologic and mechanical functioning of the tissue.

Autologous chondrocyte implantation for cartilage repair: monitoring its success by magnetic resonance imaging and histology

Autologous chondrocyte implantation is being used increasingly for the treatment of cartilage defects. In spite of this, there has been a paucity of objective, standardised assessment of the outcome and quality of repair tissue formed. We have investigated patients treated with autologous chondrocyte implantation (ACI), some in conjunction with mosaicplasty, and developed objective, semiquantitative scoring schemes to monitor the repair tissue using MRI and histology. Results indicate repair tissue to be on average 2.5 mm thick. It was of varying morphology ranging from predominantly hyaline in 22% of biopsy specimens, mixed in 48%, through to predominantly fibrocartilage, in 30%, apparently improving with increasing time postgraft. Repair tissue was well integrated with the host tissue in all aspects viewed. MRI scans provide a useful assessment of properties of the whole graft area and adjacent tissue and is a noninvasive technique for long-term follow-up. It correlated with histology (P = 0.02) in patients treated with ACI alone.

Biochemical and structural properties of the cartilage end-plate and its relation to the intervertebral disc.

The cartilaginous end-plate is thought to play an important role in the nutrition of the intervertebral disc, and hence may be of significance in the etiology of back pain. The present study describes the biochemical and histologic properties of the end-plate and adjacent tissues in the young human adult lumbar spine. Thus, a datum is established in which to demonstrate variation with location and relationships between properties of different tissues. Results of the chemical analyses show a change in composition through the end-plate with that at the outer annulus and nearer the bone, having higher collagen but lower proteoglycan and water contents, than the end-plate nearest the disc at the nucleus. Histology demonstrated numerous disruptions along the end-plate, with Schmorls nodes being present in several specimens. Where these occurred, the disc, and in some cases the end-plate, showed loss of proteoglycan compared with the surrounding tissue.

1991 Volvo Award in Basic Sciences: Collagen Types Around the Cells of the Intervertebral Disc and Cartilage End Plate: An Immunolocalization Study

Several types of collagen are known to exist in the intervertebral disc in addition to the fibrillar collagens, Types I and II. Although they constitute only a small percentage of the total collagen content, these minor collagens may have important functions. This study was designed to investigate the presence of Types I, II, III, IV, VI, and IX collagens in the intervertebral disc and cartilage end plate by immunohistochemistry, thereby establishing their location within the tissues. Types III and VI collagen have a pericellular distribution in animal and human tissue. No staining for Type IX collagen was present in normal human disc, but in rat and bovine intervertebral disc, it was also located pericellularly. These results show that cells of the intervertebral disc and cartilage end plate sit in fibrous capsules, forming chondrons similar to those described in articular cartilage. In pathologic tissue the amount and distribution of the collagen types, and the organization of the pericellular capsule, differ from that seen in control material.

Mechanoreceptors in intervertebral discs. Morphology, distribution, and neuropeptides.

Study Design The present study investigated the occurrence and morphology of mechanoreceptors in human and bovine intervertebral discs and longitudinal ligaments. Objective To determine the type and frequency of mechanoreceptors present in intervertebral discs and anterior longitudinal ligaments in two patient groups, those with low back pain and those with scoliosis. Bovine coccygeal discs were examined. Summary of Background Data Nerves have been described in intervertebral tissues, but there is little information on the endings of these nerves and their receptors, stimulation of which can cause a nerve impulse. Methods The presence of mechanoreceptors were investigated by immunolocalization of nerves and neuropeptides.By examining sequential sections, the frequency of receptors was assessed. Results Immunoreactivity to neural antigens showed mechanoreceptors in the anulus fibrosus and longitudinal ligaments of bovine and human specimens. Their morphology resembled Pacinian corpuscles, Ruffni endings, and, most frequently, Golgi tendon organs. They were found in 50% of discs investigated from patients with low back pain and in 15% of those with scollosis. Conclusions Mechanoreceptors were found in the outer 2–3 lamellae of the human intervertebral disc and anterior longitudinal ligament. Physiologic studies in other tissues indicate that these provide the individual with sensation of posture and movement, and in the case of Golgi tendon organs, of nociception. In addition to providing proprioception, mechanoreceptors are thought to have roles in maintaining muscle tone and reflexes. Their presence in the intervertebral disc and longitudinal ligament can have physiologic and clinical implications.

Cells from different regions of the intervertebral disc: effect of culture system on matrix expression and cell phenotype.

Study Design. This study examined how the culture system and region of cellular origin affect disc cell morphology and extracellular matrix production. Objective. To determine the role of the cell populations in the different regions of the adult intervertebral disc in maintaining gradients in composition across the disc. Summary of Background Data. It is not known whether the steep profiles in composition across the intervertebral disc are maintained by distinct cell populations or whether differences in cell metabolism are determined by changes in the physical environment across the disc. Very little information exists on the matrix produced by cells from the mature, non-notochordal nucleus pulposus. Methods. Cells were extracted from articular cartilage, nucleus pulposus, and the inner and outer anulus fibrosus of caudal discs from 18- to 24-month-old steers cultured in alginate or collagen gels or in monolayer. The effect of culture system and cell origin on cell morphology and matrix synthesis was measured using 35S-sulphate labeling and indirect immunolocalization. Results. Distinct morphologic differences between cells from different regions cultured in monolayer were retained through two passages. The rate of sulfate incorporation varied with cell type. Immediately after isolation, it was two- to threefold greater for nucleus cells than for cells from the disc inner anulus or articular cartilage. The rate was lowest for outer anulus cells. It also varied with culture system. For all cell types, the incorporation rate was highest in alginate and lowest in monolayer. Immunolocalization showed that nucleus cells stained strongly for all proteoglycan epitopes, whereas outer anulus cells stained least and in monolayer produced little proteoglycan. Conclusions. The disc has at least three distinct cell populations, which differ in morphology and in amount and type of matrix they produce. Cells from mature nucleus pulposus produced sulfated glycosaminoglycans at a high rate in contrast to reported results for notochordal nucleus cells. Alginate, although an appropriate culture system for inner anulus and nucleus cells, may not be a suitable medium for outer anulus cells.

Increased Nerve and Blood Vessel Ingrowth Associated With Proteoglycan Depletion in an Ovine Anular Lesion Model of Experimental Disc Degeneration

Study Design. Nerves and blood vessel distribution in discs were localized immunohistochemically and correlated with the proteoglycan contents of normal and degenerate disc tissues. Objective. The aim of the present study was to systematically evaluate whether nerve and blood vessel ingrowth was associated with depletion of disc proteoglycans and degenerative changes in an established experimental model of disc degeneration. Summary of Background Data. Animal models of disc degeneration, allowing longitudinal study of pathogenic mechanisms, are limited. The ovine model enables systematic monitoring of blood vessel and nerve ingrowth during the development of disc degeneration after injury to the anulus fibrosus. Methods. Merino sheep received a controlled left anterolateral surgical defect in the outer anulus fibrosus of the L1–L2 and L3–L4 discs (lesion group); sham-operated controls received the retroperitoneal anterolateral approach only. Animals were killed 3, 6, 12, and 26 months postoperation, and the discs were collected for histology and compositional and morphologic analyses. Sagittal tissue sections were stained with toluidine blue and hematoxylin and eosin; Type IV collagen immunolocalization visualized blood vessel ingrowth, and nerves were immunolocalized using monoclonal antibodies to growth-associated protein (GAP-43), protein gene product 9.5, and glial fibrillary acidic protein. Results. Compositional and histologic results demonstrated early focal depletion 3–12 months postoperation of glycosaminoglycan associated with lesion development, increased blood vessel and nerve ingrowth, and infiltration of cells from the outer anulus fibrosus along the plane of the original defect. Blood vessel numbers in the outer to mid third of the anulus fibrosus were elevated in the lesion discs 3–6 months postoperation reaching a maximum at 12 months postoperation; nerves immunoreactive with protein gene product 9.5 (also maximal at 12 months postoperation) were often found associated (but not exclusively) with blood vessels, and some nerves were also reactive with GAP-43 and glial fibrillary acidic protein, but only at 12 months postoperation. Conclusions. Nerve and blood vessel ingrowth into the anulus fibrosis were strongly associated with proteoglycan depletion. The ovine anular lesion model of disc degeneration is a useful experimental model for the systematic evaluation of nerve and blood vessel development after anular injury.

Changes in collagen cross-linking in degenerative disc disease and scoliosis

Study Design. Biochemical study of the changes in the collagen cross‐link profile of human intervertebral discs collected at surgery from patients with either low back pain associated with disc degeneration or scoliosis. Objective. To determine whether changes occur in the collagen cross‐link profile in the disc of patients with either low back pain associated with disc degeneration or scoliosis, which may well influence matrix integrity. Such changes in the cross‐link profile of a tissue indicates increased matrix turnover and tissue remodeling and may have implications for the progression of these disorders. Summary of Background Data. The diseases of the intervertebral disc, degenerative disc disease and scoliosis, are both characterized by changes in the extracellular matrix components that will affect the mechanical function of the tissue. The stability of the collagenous components and hence the mechanical integrity of connective tissues such as the disc is dependent on the degree and type of cross‐links between the collagen molecules. This article reports results on the distribution of the different cross‐links in the disc and the changes that occur with age, degenerative disc disease, and scoliosis. Methods. Thirty‐three discs were obtained from patients with degenerative disc disease and 29 discs from patients with scoliosis. Samples were acid hydrolyzed and the collagen cross‐links analyzed by either fractionation on an amino acid analyzer configured for cross‐link analysis using ninhydrin postcolumn detection or fractionation by high‐pressure liquid chromatography with fluorescence detection. Results. The reducible cross‐links and the mature cross‐link all increased from the outer anulus fibrosus through into the nucleus pulposus. The highest levels of the mature cross‐link were found in the cartilage endplate. The nonenzymic derived cross‐link, pentosidine, in contrast, showed little difference across the disc, but did show the expected age‐related increase. In degenerative disc disease, no change in the levels of the reducible or mature cross‐links was found, but a decrease was observed in the levels of the age‐related cross‐link pentosidine in the more severe disease samples. In scoliosis, significantly higher levels of the reducible cross‐links were found on the convex than on the concave side of the scoliotic disc. Conclusions. These changes in the cross‐link profile of the intervertebral disc in degenerative disc disease and scoliosis are indicative of increased matrix turnover and tissue remodeling and likely to have implications for the progression of these disorders.

Immunohistochemical study of collagen types I and II and procollagen IIA in human cartilage repair tissue following autologous chondrocyte implantation

This study has assessed the relative proportions of type I and II collagens and IIA procollagen in full depth biopsies of repair tissue in a large sample of patients treated with autologous chondrocyte implantation (ACI). Sixty five full depth biopsies were obtained from knees of 58 patients 8–60 months after treatment by ACI alone (n = 55) or in combination with mosaicplasty (n = 10). In addition articular cartilage was examined from eight individuals (aged 10–50) as controls. Morphology and semi-quantitative immunohistochemistry for collagen types I and II and procollagen IIA in the repair tissue were studied. Repair cartilage thickness was 2.89 ± 1.5 mm and there was good basal integration between the repair cartilage, calcified cartilage and subchondral bone. Sixty five percent of the biopsies were predominantly fibrocartilage (mostly type I collagen and IIA procollagen), 15% were hyaline cartilage (mostly type II collagen), 17% were of mixed morphology and 3% were fibrous tissue (mostly type I collagen). Type II collagen and IIA procollagen were usually found in the lower regions near the bone and most type II collagen was present 30–60 months after treatment. The presence of type IIA procollagen in the repair tissue supports our hypothesis that this is indicative of a developing cartilage, with the ratio of type II collagen:procollagen IIA increasing from < 2% in the first two years post-treatment to 30% three to five years after treatment. This suggests that cartilage repair tissue produced following ACI treatment, is likely to take some years to mature.