Corey J. Wallach

A slowly progressive and reproducible animal model of intervertebral disc degeneration characterized by MRI, X-ray, and histology

Study Design. The progression of intervertebral disc degeneration following anterolateral “stab” of adult rabbit lumbar discs by 16-gauge hypodermic needle to a limited (5-mm) depth was studied for up to 24 weeks using magnetic resonance imaging, radiograph, and histologic outcome measures. Objectives. To develop a slowly progressive, reproducible rabbit model of intervertebral disc degeneration suitable for studying pathogenesis and pathophysiology of intervertebral disc degeneration and testing safety and efficacy of novel approaches to the treatment of intervertebral disc degeneration (e.g., growth factors, gene therapy, cell therapy, and tissue engineering). Summary of Background Data. Numerous animal models of intervertebral disc degeneration have been proposed in the literature, each with attendant advantages and disadvantages. The classic “stab model,” involving full-thickness stab of anterior anulus fibrosus of adult rabbit lumbar discs by a number 11 scalpel blade, appears to produce changes in certain biochemical and histologic outcome measures that are similar to changes seen in human intervertebral disc degeneration. However, the immediate herniation of nucleus pulposus on full-thickness stab renders this model less suitable for 1) studying effects of less precipitous changes in nucleus pulposus and anulus fibrosus that may be important in the onset and progression of intervertebral disc degeneration and 2) testing novel therapeutic approaches that target the processes of early intervertebral disc degeneration. Methods. The L2–L3, L3–L4, and L4–L5 lumbar intervertebral discs of 18 skeletally mature female New Zealand White rabbits were stabbed by 16-gauge hypodermic needle to a depth of 5 mm in the left anterolateral anulus fibrosus. Serial magnetic resonance imaging scans of the stabbed discs and intact L1–L2 and L5–L6 control discs were performed at 3, 6, 12, and 24 weeks post surgery and compared with preoperative magnetic resonance images. Supplemental radiograph and histologic analyses were performed. Results. The stabbed discs exhibited a progressive decrease in “magnetic resonance imaging index” (the product of nucleus pulposus area and signal intensity from T2-weighted midsagittal plane images) starting at 3 weeks post stab and continuing through 24 weeks, with no evidence of spontaneous recovery or reversal of magnetic resonance imaging changes. Radiograph findings included early osteophyte formation by 6 weeks post stab and extensive, bridging osteophytes by 24 weeks. Histologic analysis revealed progressive loss of notochordal cells from the nucleus pulposus, filling of the nucleus pulposus space with fibrocartilage, and derangement of anulus fibrosus. Conclusions. Stabbing the anterolateral anulus fibrosus of adult rabbit lumbar discs with a 16-gauge hypodermic needle to a limited (5-mm) depth results in a number of slowly progressive and reproducible magnetic resonance imaging, radiograph, and histologic changes over 24 weeks that show a similarity to changes seen in human intervertebral disc degeneration. This model would appear suitable for studying pathogenesis and pathophysiology of intervertebral disc degeneration and testing safety and efficacy of novel treatments 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.

Safety Assessment of Intradiscal Gene Therapy II: Effect of Dosing and Vector Choice

Study Design. Clinical, biochemical, and histologic analysis was performed after in vivo delivery of cDNA encoding various anabolic cytokines and marker genes to the lumbar epidural space of New Zealand white rabbits, using both adenoviral and adeno-associated viral vectors. Objective. To mimic errant or misplaced doses of gene therapy to better ascertain the potential risks associated with alternative vectors and transgene products with regard to their application to problems of the intervertebral disc. Summary of Background Data. Work done with several anabolic cytokines including bone morphogenic proteins and transforming growth factors, has demonstrated the potential of gene therapy. Recently, data has been published demonstrating that improperly dosed or delivered adenoviral-mediated gene therapy within the subarachnoid space can result in significant morbidity in rabbits. There are currently no studies examining the effect of these errors within the epidural space or using an adeno-associated viral (AAV) vector. Methods. Using either adenoviral or AAV vectors, complementary DNA (cDNA) encoding anabolic cytokines bone morphogenic protein-2 (BMP-2) and transforming growth factor-beta 1 and marker proteins LacZ and green fluorescent protein were injected into the epidural space of 37 New Zealand white rabbits at the L5/6 level. Rabbits were then observed clinically for up to 6 weeks, after which the rabbits were sacrificed in order to perform a comprehensive biochemical and histologic analysis. Results. Following adenoviral-mediated delivery of anabolic cytokine cDNA, up to eighty percent of rabbits demonstrated significant clinical, biochemical, and histologic morbidity. Conversely, AAV-mediated delivery of any cDNA and adenoviral-mediated delivery of marker protein cDNA resulted in no clinical, histologic, or biochemical morbidity. Conclusion. Properly dosed and directed gene therapy seems to be both safe and potentially efficacious. This study suggests that side effects of gene therapy may be due to a combination of dosing, transgene product, and vector choice, and that newer AAV vectors may reduce these side-effects and decrease the risk of this technology.

Gene therapy applications for intervertebral disc degeneration

Study Design. Review of current approaches to gene therapy as a potential treatment modality for regeneration of the degenerating intervertebral disc. Objectives. Describe recent advances in the field of gene therapy pertaining to treatment of the degenerating intervertebral disc. Summary of Background Data. The potential application of gene therapy to the treatment of degenerative disc disease has been an area of exciting and active research. Several in vitro studies have documented the capacity of gene therapy to favorably modify the biologic functions of the intervertebral disc cells with the delivery of the cDNA for various growth factors, whereas in vivo studies have reinforced this potential in animal models. Currently, investigators are exploring the therapeutic potential of gene delivery of the cDNA for several promising factors. Methods. Literature review. Results/Conclusions. The application of gene therapy to the treatment of the degenerating intervertebral disc holds much clinical promise and continues to expand as an area of active research. The therapeutic potential of multiple agents, as well as possible vectors, are currently being investigated with encouraging results.

4:21 Gene delivery of the catabolic inhibitor tissue inhibitor of metalloproteinase–1 increases proteoglycan synthesis in human intervertebral disc cells

Abstract Purpose of study: Intervertebral disc degeneration is characterized by a progressive loss of proteoglycan content and subsequent disc dehydration. Previous studies demonstrate increased matrix metalloproteinases in degenerated discs compared with controls and propose an intimate role of these catabolic proteins in the degenerative process. Gene therapy has been investigated as a method to induce endogenous synthesis of therapeutic growth factors to stimulate proteoglycan synthesis. Although many anabolic growth factors increase matrix synthesis, the use of catabolic inhibitors has yet to be explored. The objective of this study was to evaluate the effect of adenoviral-mediated delivery of the catabolic inhibitor tissue inhibitor of metalloproteinase–1 (TIMP–1), as well as the anabolic potential of bone morphogenic protein–2 (BMP–2), on proteoglycan synthesis. Methods used: Human nucleus pulposus cells were isolated by means of enzymatic digestion and cultured in monolayer. Patient samples were pooled and experimental groups normalized for cell number before transduction with either Ad-TIMP-1 or Ad-BMP-2 at 50, 75, 100 or 150 multiplicity of infection (MOI). After 48 hours of incubation, cells were incorporated into a three-dimensional “pellet” culture system. After an additional 48 hours of incubation, active proteoglycan synthesis was assessed with 35S radioactive sulfate incorporation using chromatography and scintillation count. of findings: Both Ad-TIMP-1 and Ad-BMP-2 increase proteoglycan synthesis compared with controls (p Relationship between findings and existing knowledge: Although anabolic factors have previously been shown to increase proteoglycan synthesis in cultured disc cells, this is the first report demonstrating a similar upregulating capacity with the use of catabolic inhibitors. Overall significance of findings: This study demonstrates that both catabolic inhibitors (TIMP–1) and anabolic growth factors (BMP-2) are successful at increasing measured proteoglycan synthesis in cultured human intervertebral disc cells. The optimal regulation of matrix synthesis may be achievable with gene therapy using a combination of both anabolic factors and catabolic inhibitors. Further studies are warranted to assess the potential of combination therapy with these growth factors to upregulate proteoglycan synthesis, as well as to determine if such combinations can minimize the total vector load required to elicit a beneficial, and potentially therapeutic, response (Table 1) Table 1 . Stimulatory effect on proteoglycan synthesis 50 MOI 75 MOI 100 MOI 150 MOI TIMP–1 65% 289% 378% 249% BMP–2 190% 212% 310% 466% . Disclosures: No disclosures. Conflict of interest: No conflict.