In vitro and in vivo testing of a novel regulatory system for gene therapy for intervertebral disc degeneration.

STUDY DESIGN: In vitro and in vivo testing of a gene expression control system. OBJECTIVE: The purpose of this study is to establish the ability of controlling gene expression using an adeno-associated viral vector containing a novel control system (AAV-RheoSwitch GFP [Intrexon Corp., Blacksburg, VA]) in intervertebral disc cells for potential use in gene therapy trials. SUMMARY OF BACKGROUND DATA: Gene therapy for disc degeneration shows promise; however, concern remains regarding safety. Careful control of gene expression is needed to facilitate translation into clinical trials. METHODS: Rabbit nucleus pulposus cells were treated in vitro with increasing multiplicities of infection of AAV-RheoSwitch GFP, followed by increasing concentrations of Intrexon's activator ligand, and examined for fluorescence during and after removal of ligand. New Zealand white rabbits were injected with AAV-RheoSwitch GFP and killed either before or after 5 days of daily ligand injection. Tissues were analyzed for the presence of green fluorescent protein (GFP) with fluorescence microscopy and immunohistochemical staining. RESULTS: In vitro, GFP expression was noted to be dose and time dependent, decreased 24 hours after removal of ligand, and was minimally detectable in cells after 48 hours. In vivo, increasing GFP expression was seen in animals treated with viral vector and ligand. No GFP expression was evident in tissues from rabbits that received only virus, or activator ligand alone. In addition, no GFP expression was evident in the adjacent discs, spinal cord, dura, bone, liver, or brain of any animals. CONCLUSION: These data demonstrate that in vitro ligand-induced gene expression can be stimulated and effectively turned off by removal of the ligand. In addition, we demonstrated the in vivo utility of this system through showing up-regulation of GFP without nonspecific gene expression or expression in adjacent tissues. This system, therefore, has the potential to increase the safety of gene therapy in the treatment of intervertebral disc degeneration.

Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro.

STUDY DESIGN: We demonstrated the differentiation of notochordal cells by direct observation using a live automated cell imaging system. We also hypothesized that notochordal cells have characteristics of chondrocyte-like cells. OBJECTIVE: To determine characteristics of notochordal cells by matrix protein expression and their differentiation using a live automated cell imager. SUMMARY OF BACKGROUND DATA: Although notochordal cells are critical to homeostasis of intervertebral disc, their fate has not been extensively studied and there is little evidence of notochordal cells as progenitors. METHODS: Notochordal cells purified from rabbit nucleus pulposus were isolated after serial filtration. Notochordal cells in 3-dimensional culture were compared to chondrocyte-like cells by S sulfate incorporation into proteoglycan and reverse transcription polymerase chain reaction for gene expression(collagen II and aggrecan). Notochordal cells in 2-D culture were used for immunocytochemical staining (collagen II, aggrecan, and SOX9) and time-lapsed cell tracking study. RESULTS: Notochordal cells were capable of proteoglycan production at a rate comparable to chondrocyte-like cells (108% +/- 22.6% to chondrocyte-like cells) and expressed collagen II, aggrecan, and SOX9. In time-lapsed cell tracking analysis, notochordal cells were slower in population doubling time than chondrocyte-like cells and differentiated into 3 morphologically distinct cell types: vacuolated cells (area: 2392 +/- 507.1 microm, velocity: 0.09 +/- 0.01 microm/min); giant cells (area: 12678 +/- 1637.0 microm, velocity: 0.08 +/- 0.01 microm/min) which grew rapidly without cell division; polygonal cells (area: 3053 +/- 751.2 microm, 0.14 +/- 0.01 microm/min) morphologically similar to typical differentiation type of chondrocyte-like cells (area: 2671 +/- 235.6 microm, 0.19 +/- 0.01 microm/min). Rarely, notochordal cells formed clusters analogous to that observed in vivo. CONCLUSION: These studies demonstrate a chondrocyte phenotype of notochordal cells and are the first direct evidence of notochordal cell differentiation, suggesting that they may act as progenitor cells, which has the potential to lead to their use in novel approaches to regeneration of degenerative intervertebral disc.

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-beta1 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.

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-beta (TGF-beta) 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 beta or TNF-alpha, 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 cell's 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 beta and tumor necrosis factor-alpha 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-alpha, 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-alpha 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-alpha. 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.

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.