Helga I. Georgescu

Herniated cervical intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2

Study Design Herniated cervical disc specimens were obtained from patients undergoing surgical discectomy for persistent radiculopathy and cultured in vitro to determine whether various biochemical agents were being produced. Objectives Our hypothesis is that biochemical mediators of inflammation and tissue degradation play a role in cervical intervertebral disc degeneration and in the pathophysiology of cervical radiculopathy. Summary of Background Data Neck pain with or without radiculopathy is a common clinical problem, but the etiology of neck pain and the exact pathophysiology of rediculopathy remain uncertain, We have previously reported the production of various biochemical agents by herniated lumbar disc specimens in vitro. Because of a lack of such studies in the literature with respect to the cervical spine, the purpose of this study was to determine whether similar biochemical agents of inflammation and tissue degradation were being produced by herniated cervical disc specimens. Methods Eighteen herniated cervical dises were obtained from 15 patients undergoing anterior disc surgery. The specimens were cultured and incubated for 72 hours, and the media were subsequently collected for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, prostaglandin E2, and a variety of cytokines were performed. As a control group, six cervical discs specimens were obtained from three patients undergoing anterior surgery for traumatic burst fractures, and similar biochemical analyses were performed. Results The culture media from the herniated cervical disc specimens showed increased levels of matrix metalloproteinase activity compared with the control discs. Similarly, the levels of nitric oxide, prostaglandin E2, and interleukin-6 were significantly higher in the hernlated disc specimens compared with the control discs, Interleukin-1α, interleukin-1β, tumor necrosis factor-α, interleukin-1 receptor antagonist protein, and substance P were not detected in the culture media of the herniated or control discs. Conclusions Herniated cervical disc specimens were making spontaneously increased amounts of matrix metalloproteinases, nitric oxide, prostaglandin E2, and interleukin-6. These results were similar to those obtained in herniated lumbar disc specimens that we have previously reported. These products may be intimately involved in the biochemistry of disc degeneration and the pathophysiology of radiculopathy.

Toward a biochemical understanding of human intervertebral disc degeneration and herniation: Contributions of nitric oxide, interleukins, prostaglandin E2, and matrix metalloproteinases

Study Design. Normal and herniated human intervertebral disc specimens were cultured to study the effects of interleukin‐1β on the production of nitric oxide, interleukin‐6, prostaglandin E2, and matrix metalloproteinases. The effects of endogenously produced nitric oxide on the synthesis of other mediators also were studied. Objectives. To test the hypothesis that the cells of the intervertebral disc are metabolically active and are capable of responding to biochemical stimuli such as interleukin‐1β in a manner that could engender degenerative changes. As part of this study, the authors also investigated some of the possible autocrine regulatory mechanisms that may operate during the biochemical responses of disc cells. Summary of Background Data. The authors previously showed, for the first time, that herniated cervical and lumbar disc specimens spontaneously produce increased amounts of nitric oxide, interleukin‐6, prostaglandin E2, and certain matrix metalloproteinases. These results suggest that these biochemical agents are in some manner involved with degenerative processes in the intervertebral disc. This novel hypothesis merits further evaluation; the current communication reports the results of experiments designed to do so. Methods. Fourteen normal, nondegenerated discs (control group) were obtained from seven patients undergoing anterior spinal surgery for trauma or lumbar scoliosis. Thirty‐six herniated discs (18 lumbar and 18 cervical) were obtained from 30 patients undergoing surgery for persistent radiculopathy. The specimens were placed into tissue culture and incubated for 72 hours in the presence or absence of interleukin‐1β and NG‐monomethyl‐L‐arginine, an inhibitor of nitric oxide synthases, and the media were subsequently collected for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, interleukin‐6, and prostaglandin E2 were performed. Results. Normal, control disc specimens significantly increased their production of matrix metalloproteinases, nitric oxide, interleukin‐6, and prostaglandin E2 in response to interleukin‐1β. Herniated lumbar and cervical discs, which were spontaneously releasing increased levels of these biochemical agents, further increased their production of nitric oxide, interleukin‐6, and prostaglandin E2 in response to interleukin‐1β. Blocking the biosynthesis of nitric oxide in interleukin‐1β‐stimulated disc cells provoked a large increase in the production of interleukin‐6. Conclusions. Cells of the intervertebral discs are biologically responsive and increase their production of matrix metalloproteinases, nitric oxide, interleukin‐6, and prostaglandin E2 when stimulated by interleukin‐1β. The effect is more dramatic in normal, nondegenerated discs where spontaneous synthesis of these mediators is low. Nevertheless, cells of the herniated degenerated discs where spontaneous production was high were still capable of further increasing their synthesis of several of these biochemical agents in response to interleukin‐1β. Endogenously produced nitric oxide appears to have a strong inhibitory effect on the production of interleukin‐6, which suggests that autocrine mechanisms play an important role in the regulation of disc cell metabolism.

The biochemical and histological effects of artificial ligament wear particles: In vitro and in vivo studies:

Growing evidence suggests that biochemical mecha nisms play a role in the pathogenesis of arthritis. Car tilaginous wear particles have been shown to induce destructive enzymes and cytokines. To assess the biocompatibility of artificial ACL replacements, the ef fects of wear particles from the following ligaments were analyzed biochemically and histologically: GORE- TEX, Stryker Dacron Ligament Prosthesis, Versigraft carbon, Kennedy LAD, Xenograft, Leeds-Keio, and hu man patellar tendon allograft. Ligaments were frozen and ground to produce wear particles similar to those seen clinically and were added to lapine synovial cell cultures. The resulting conditioned medium was ana lyzed for collagenase, gelatinase, and chondrocyte ac tivating factor (CAF) production. All of the ligaments induced significantly elevated enzyme and CAF production by the synoviocytes, with Xenograft and carbon inducing significantly higher en zyme levels than those of the other five ligaments. Five milligrams of wear particles were injected into the knees of 4 kg to 5 kg rabbits that were analyzed histologically after 14 weeks. Wear particles accumu lated in the periarticular synovial folds and induced modest to severe macrophage infiltration in the syno vium. A hypothetical model explaining the role of artifi cial ligament wear particles in the pathogenesis of arthritis is presented.

Gene transfer of the catabolic inhibitor TIMP-1 increases measured proteoglycans in cells from degenerated human intervertebral discs.

Study Design. Cells from degenerated intervertebral discs were transduced with an adenoviral vector delivering cDNA of the catabolic inhibitor, TIMP-1, and alterations in the measured proteoglycan were assessed. Objectives. To assess the potential of TIMP-1 to favorably modify the proteoglycan content of degenerated intervertebral disc cells. Summary of Background Data. Gene therapy with anabolic factors has resulted in increased proteoglycan synthesis in intervertebral disc cells. Biochemical analysis of degenerated discs has revealed elevated levels of the catabolic enzymes, matrix metalloproteinase, suggesting an intimate role of these factors in the degenerative process. The use of TIMP-1, an endogenous inhibitor of matrix metalloproteinase, via gene therapy may provide an additional method to alter the degenerative processes occurring in the intervertebral disc. Materials and Methods. Degenerated intervertebral disc were isolated from eight patients undergoing elective surgical procedures. Cells were cultured in monolayer and transduced with different concentrations of either an adenoviral-tissue inhibitor of metalloproteinase-1 (Ad-TIMP-1) or adenoviral-bone morphogenic protein-2 (Ad-BMP-2) construct. Cells were cultured in a three-dimensional pellet and proteoglycan synthesis was assessed via 35S-sulfur incorporation. Results. Gene delivery of TIMP-1 and BMP-2 increased measured proteoglycan synthesis at each concentration assessed. IVD cells treated with Ad-TIMP-1 demonstrated an optimal response at a multiplicity of infection (MOI) of 100. Cells treated with Ad-BMP-2 demonstrated a progressive increase in proteoglycan synthesis with increasing viral concentrations. Conclusions. Successful delivery of the anticatabolic gene, TIMP-1, results in increased measured proteoglycan in cultured degenerated disc cells. This finding supports catabolic inhibition as a promising avenue of research for the treatment of degenerative disc disease via gene therapy.

Genetic enhancement of matrix synthesis by articular chondrocytes: Comparison of different growth factor genes in the presence and absence of interleukin‐1

OBJECTIVE To determine whether articular chondrocytes express growth factor genes delivered by adenoviral vectors and whether expression of these genes influences matrix synthesis in the presence and absence of interleukin-1 (IL-1). METHODS Monolayer cultures of rabbit articular chondrocytes were infected with recombinant adenovirus carrying genes encoding the following growth factors: insulin-like growth factor 1 (IGF-1), transforming growth factor beta1 (TGFbeta1), and bone morphogenetic protein 2 (BMP-2). As a control, cells were transduced with the lac Z gene. Cultures were also treated with each growth factor supplied as a protein. Levels of gene expression were noted, and the synthesis of proteoglycan, collagen, and noncollagenous proteins was measured by radiolabeling. Collagen was typed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The effects of growth factor gene transfer on proteoglycan synthesis in the presence of IL-1 were also measured. RESULTS The expression of all transgenes was high following adenoviral transduction. Proteoglycan synthesis was stimulated approximately 8-fold by the BMP-2 gene and 2-3-fold by the IGF-1 gene. The effects of BMP-2 and IGF-1 genes were additive upon cotransduction. Synthesis of collagen and noncollagenous proteins, in contrast, was most strongly stimulated by the IGF-1 gene. In each case, collagen typing confirmed the synthesis of type II collagen. IL-1 suppressed proteoglycan synthesis by 50-60%. IGF-1 and TGFbeta genes restored proteoglycan synthesis to control levels in the presence of IL-1. The BMP-2 gene, in contrast, elevated proteoglycan synthesis beyond control levels in the presence of IL-1. CONCLUSION Transfer of growth factor genes to articular chondrocytes can greatly increase matrix synthesis in vitro, even in the presence of the inflammatory cytokine IL-1. This result encourages the further development of gene therapy for the repair of damaged cartilage.

Effect of growth factors on the proliferation of ligament fibroblasts from skeletally mature rabbits

Growth factors have been shown to stimulate fibroblast proliferation during wound and ligament healing. In this study, we analyzed individual effects of eight growth factors in vitro on the proliferation of fibroblasts from the medial collateral (MCL) and anterior cruciate (ACL) ligaments of skeletally mature rabbits. We compared the proliferative response of growth factor-treated and nontreated fibroblasts of both ligaments. The growth-factor treated fibroblasts of the MCL and ACL were also compared. We found that the fibroblasts exposed to epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-BB proliferated significantly more than untreated fibroblasts. Acidic fibroblast growth factor at a dose of 1.0 ng/ml caused significant increases in fibroblast proliferation only in the MCL. Transforming growth factor-beta 1, insulin-like growth factor-1, platelet-derived growth factor-AA, and interleukin-1 alpha did not significantly stimulate fibroblast proliferation. MCL fibroblasts generally did not proliferate significantly more than ACL fibroblasts with the exception of MCL fibroblasts exposed to the highest doses of basic fibroblast growth factor, acidic fibroblast growth factor and platelet-derived growth factor-BB. The data were also compared with those obtained earlier using fibroblasts from skeletally immature rabbits (Schmidt et al., JOR 1995). The proliferative response of both the MCL and the ACL fibroblasts was found to decrease with skeletal maturation. Thus, our findings suggest that animal age and fibroblast origin are important factors in determining the proliferative response to growth factors.

HIG-82: an established cell line from rabbit periarticular soft tissue, which retains the "activatable" phenotype.

SummaryWe have isolated a continuous cell line from soft tissue lining the knee joints of rabbits. Designated HIG-82, this line was produced by spontaneous establishment of an aging, late-passage culture of primary cells. Like unpassaged, primary cells, HIG-82 cells can be activated by a number of stimuli, including phorbol myristate acetate (PMA), interleukin-1 (IL-1), and the endocytosis of latex beads. Activated cells secrete collagenase, gelatinase, caseinase (stromelysin), and prostaglandin E2 (PGE2) into their culture medium. Pseudodiploid, HIG-82 cells combine a high plating efficiency with a doubling time of approximately 24 h. As primary tissue of this origin is difficult to obtain in large quantities and shows cellular heterogeneity, the HIG-82 cell line should facilitate research into the biology and biochemistry of the fibroblastic cells that line the diarthrodial joints of mammals. Such cells are likely to be important in the pathophysiology of various arthritides.

p38 MAPK Inhibition Modulates Rabbit Nucleus Pulposus Cell Response to IL-1

Analysis of disc gene expression implicated IL‐1 in the development of intervertebral disc degeneration (IDD) in a rabbit stab model. The purpose of these studies is to determine the role of p38 Mitogen Activated Protein Kinase (p38 MAPK) signaling in nucleus pulposus cell response to IL‐1, and to compare rabbit nucleus pulposus (rNP) cell responses to IL‐1 activation with those in a stab model of disc degeneration. NP cells maintained in alginate bead culture were exposed to IL‐1, with or without p38 MAPK inhibition. RNA was isolated for reverse transcription polymerase chain reaction (RT‐PCR) analysis of gene expression, conditioned media analyzed for accumulation of nitric oxide (NO) and prostaglandin E‐2 (PGE‐2), and proteoglycan synthesis measured after 10 days. IL‐1 upregulation of mRNA for cycloxygenase‐2 (COX‐2), matrix metalloproteinase‐3 (MMP‐3), IL‐1, and IL‐6, was blunted by p38 inhibition while downregulation of matrix proteins (collagen I, collagen II, aggrecan) and insulin‐like‐growth‐factor I (IFG‐1) was also reversed. mRNA for tissue inhibitor of matrixmetalloproteinase‐1 (TIMP‐1) was modestly increased by IL‐1, while those for Transforming Growth Factor‐β (TGF‐β) SOX‐9, and versican remained unchanged. Blocking p38 MAPK reduced IL‐1 induced NO and PGE‐2 accumulation and partially restored proteoglycan synthesis. p38 MAPK inhibition in control cells increased mRNA for matrix proteins (aggrecan, collagen II, versican, collagen I) and anabolic factors (IGF‐1, TGF, and SOX‐9) from 50% to 120%, decreased basal PGE‐2 accumulation, but had no effect on message for TIMP‐1, MMP‐3, or COX‐2. Inhibition of p38 MAPK in cytokine‐activated disc cells blunts gene expression and production of factors associated with inflammation, pain, and disc matrix catabolism while reversing IL‐1 downregulation of matrix protein gene expression and proteoglycan synthesis. The results support the hypothesis that IL‐1 could be responsible for many of the mRNA changes seen in rabbit NP in the stab model of disc degeneration, and uphold the concept that development of molecular techniques to block p38 MAPK could provide a therapeutic approach to slow the course of intervertebral disc degeneration.

p38 MAPK inhibition in nucleus pulposus cells: a potential target for treating intervertebral disc degeneration.

Study Design. Human nucleus pulposus cells were cultured in alginate beads and activated with IL-1&bgr; or TNF-&agr;, with and without inhibition of p38 mitogen activated protein kinase (p38 MAPK) activity. Cell production of factors modulating the anabolic/catabolic balance of the disc was determined. Objective. To determine the role of signaling through p38 MAPK in nucleus pulposus cells response to inflammatory cytokines and whether it might be a valid target for the development of molecular therapies for disc degeneration. Summary of Background Data. Multiple factors contribute to intervertebral disc degeneration (IDD), and development of effective therapies depends on understanding the underlying cellular pathophysiology. Interleukin-1&bgr; and tumor necrosis factor-&agr; are implicated in the development of IDD, and p38 MAPK is part of cytokine and mechanical stress signal pathways in other cells. These studies determine whether inhibiting p38 MAPK can decrease factors that negatively affect the metabolic balance and viability of nucleus pulposus cells. Materials and Methods. Degenerated intervertebral disc tissue was obtained from patients undergoing elective surgical procedures. Nucleus pulposus cells in alginate bead culture were exposed to IL-1 or TNF-&agr;, with or without p38 MAPK inhibition, and conditioned media analyzed for accumulation of nitric oxide (NO), prostaglandin E2 (PGE2), IL-6, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) through 10 days. Results. Inhibition of p38 MAPK decreased PGE2 in conditioned medium of control, unstimulated cells while not affecting TIMP-1 accumulation. Blocking cytokine activation of p38 MAPK reduced IL-1 and TNF-&agr; induced PGE2 and IL-6 accumulation. p38 MAPK inhibition increased the ratio of TIMP-1 to MMP-3 in conditioned medium of cells activated by IL-1 or TNF-&agr;. Conclusion. Inhibition of p38 MAPK in cytokine-activated disc cells blunts production of factors associated with inflammation, pain, and disc matrix catabolism. The data support further analysis of these effects on the anabolic/catabolic balance of nucleus pulposus cells and suggest that molecular techniques blocking this signal could provide a therapeutic approach to slow the course of intervertebral disc degeneration.

Characterization of intervertebral disc aging: longitudinal analysis of a rabbit model by magnetic resonance imaging, histology, and gene expression.

Study Design. A cohort of young, healthy New Zealand White rabbits was followed longitudinally with serial magnetic resonance imaging (MRI) analysis and terminal analysis of histologic changes and gene expression. Objective. To examine the changes observed during normal aging in the intervertebral disc. Summary of Background Data. Although there is a correlation between aging and the onset of intervertebral disc degeneration (IDD), evidence suggests that distinct pathways are involved in these processes. Our group has characterized a reproducible rabbit model of IDD by MRI, radiograph, histology, and mRNA expression. However, no similar analysis has been performed longitudinally for intervertebral disc aging to allow comparison of these 2 important processes. Methods. Four skeletally mature female NZW rabbits were housed for 122 weeks, and lumbar spine MRIs were characterized serially. Histologic and quantitative gene expression analysis of the nucleus pulposus of these aging animals was performed, and compared with adult and young rabbits. Results. Mean MRI index decreased by <25% through 120 weeks. The histologic analysis showed changes in cell composition, with abundant notochordal cells in the young, chondrocyte-like cells and notochordal cells in the adult, and clusters of hypertrophic chondrocytes in the aging discs. The PCR analysis of the nucleus pulposus showed that gene expression of collagen decreased, whereas that for proteoglycans increased with aging. BMP-2, TIMP-1, and SOX-9 expression was significantly lower in the young compared with adult discs and TGF-&bgr;1 demonstrated lower gene expression in young and aging animals. Conclusion. Although dramatic cellular changes were observed, age-related MRI changes occurred in this rabbit model of normal aging at a much slower rate than in a previous injury model of degeneration. In addition, the gene expression analysis of the nucleus pulposus demonstrated remarkable differences between aging and injury induced degeneration. These results suggest that aging and injury contribute uniquely to the process of IDD.