In vitro spine testing using a robot-based testing system: comparison of displacement control and "hybrid control".

The two leading control algorithms for in-vitro spine biomechanical testing-"load control" and "displacement control"-are limited in their lack of adaptation to changes in the load-displacement response of a spine specimen-pointing to the need for sufficiently sophisticated control algorithms that are able to govern the application of loads/motions to a spine specimen in a more realistic, adaptive manner. A robotics-based spine testing system was programmed with a novel hybrid control algorithm combining "load control" and "displacement control" into a single, robust algorithm. Prior to in-vitro cadaveric testing, preliminary testing of the new algorithm was performed using a rigid-body-spring model with known structural properties. The present study also offers a direct comparison between "hybrid control" and "displacement control". The hybrid control algorithm enabled the robotics-based spine testing system to apply pure moments to an FSU (in flexion/extension, lateral bending, or axial rotation) in an unconstrained manner through active control of secondary translational/rotational degrees-of-freedom-successfully minimizing coupled forces/moments. The characteristic nonlinear S-shaped curves of the primary moment-rotation responses were consistent with previous reports of the FSU having a region of low stiffness (neutral zone) bounded by regions of increasing stiffness (elastic zone). Direct comparison of "displacement control" and "hybrid control" showed that hybrid control was able to actively minimize off-axis forces and resulted in larger neutral zone and range of motion.

Mesenchymal stem cells injection in degenerated intervertebral disc: cell leakage may induce osteophyte formation.

Recent studies have shown that mesenchymal stem cell (MSC)-based therapy might be an effective approach for the treatment of intervertebral disc degeneration (IDD). However, many unanswered questions remain before clinical translation, such as the most effective stem cell type, a reliable transplantation method, including the carrier choice, and the fate of stem cells after misdirected delivery, among others. The objective of the study was to evaluate the fate and effect of allogenic bone marrow MSCs after transplantation into an IDD model. The L2-3, L3-4 and L4-5 intervertebral discs (IVDs) of four rabbits were stabbed to create IDD. Rabbit MSCs were expanded in vitro and in part transduced with retrovirus/eGFP. After 3 weeks, 1 × 10(5) MSCs were injected into the IVDs. The rabbits were followed by X-ray and MRI 3 and 9 weeks after injection. Then the animals were sacrificed and the spines analysed histologically. MRI showed no signs of regeneration. X-ray and gross anatomy inspection demonstrated large anterolateral osteophytes. Histological analysis showed that the osteophytes were composed of mineralized tissue surrounded by chondrocytes, with the labelled MSCs among the osteophyte-forming cells. The labelled MSCs were not found in the nucleus. Inflammatory cells were not observed in any injected IVDs. These results raise concern that MSCs can migrate out of the nucleus and undesirable bone formation may occur. While cause cannot be inferred from this study, the presence of MSCs in the osteophytes suggests a potential side-effect with this approach. IVD regeneration strategies need to focus on cell carrier systems and annulus-sealing technologies to avoid pitfalls.

Bone densitometry within titanium lumbar interbody fusion cages: a computed tomography feasibility study.

STUDY DESIGN: The use of lumbar fusion cages to augment interbody arthrodesis has become popular. Previous studies have shown that titanium cages may obscure information within and around the cage, confounding assessments of fusion. Densitometry within cages, if possible, could be an important outcome variable for comparing different cage augmented fusion treatments. OBJECTIVE: The purpose of this feasibility study was to (1) validate CT-based (computer tomography-based) density evaluations within titanium cages in a phantom model, and (2) demonstrate that intrametal cage lucencies can be produced by CT reconstruction artifacts and do not necessarily represent bone fragmentation or fibrous and cartilaginous tissue. METHODS: An anthropomorphic lumbar spine phantom was used. Dual titanium cages were inserted into 6 anterior-middle column replicas, and the cages were filled with 10, 100, or 400 mg/cc hydoxyapatiite equivalent material. The column replicas were composed of either fibrous/cartilaginous tissue equivalent material or trabecular bone equivalent material. Column replicas were placed within the phantom and imaged with CT. Direct axial and reformatted sagittal and coronal images were evaluated for artifact. Region of interest analysis was performed for intracage density. RESULTS: Intracage CT values calculated from axial, sagittal, and coronal images were directly related to the true CT values of the materials (r = 0.99, P < 0.0001). CT was used to distinguish between fibrous tissue and bone within cages. CT values within cages overestimated the intracage materials' true values (paired t test, P < 0.001). Intracage CT values were correctable and could be converted to bone mineral content. Intracage CT values were not affected by adjacent anterior-middle column material differences (fibrous/cartilaginous tissue vs. trabecular bone) (paired t test, P > 0.05). Lucent streaking within and adjacent to cages was similar to clinical images. CONCLUSION: In this phantom-based study, densitometry was performed within titanium cages, even in the presence of CT artifact. These results support further pursuit of this potential assessment tool.

Three-dimensional analysis of cervical spine motion: reliability of a computer assisted magnetic tracking device compared to inclinometer.

We aimed to investigate the reliability and reproducibility of a magnetic tracking technique for the assessment of overall cervical spine motion (principal and coupled movements). Ten asymptomatic male volunteers with a mean age of 29.3 years (range 20-37 years) were included in the study. Flexion, extension, left and right lateral bending and left and right axial rotation were measured using a magnetic tracking device (MTD) mounted onto a custom head-piece. For rotational movements in the frontal and sagittal planes the results were compared with the measurements of two standard inclinometers. Intra-observer, inter-observer and intra-instrument reliability was assessed with the intraclass correlation coefficient method. There were no significant differences for all motion measurements between the MTD and the inclinometer. High inter-observer reliability was found in flexion, extension, axial rotation and lateral bending indicating that the testing routine is applicable for different examiners. The intra-observer variability was high in flexion and extension, whereas in lateral bending the reliability coefficients were lower and displayed a fair to good reliability for most of the measurements with the MTD. The results of the MTD were found to be highly comparable with the inclinometer results with an inter-instrument correlation coefficient ranging from 0.88 to 0.99. The MTD is a reliable, reproducible method for three-dimensional motion analysis of the cervical spine and therefore a valuable method both for the clinical assessment of various degenerative and traumatic disorders and as a supplement of different therapeutic procedures and rehabilitation.

Characterization of the performance of a custom program for image processing of pressure sensitive film.

A custom program for the processing of pressure sensitive (Fuji) film data is presented and validated in this paper. Some of the shortcomings of previous descriptions of similar programs in literature are addressed. These shortcomings include incomplete descriptions of scan resolution, processing technique, and accuracy of results. Of these, the accuracy of results is the most important and is addressed in this study by using Fuji film calibration data. In Fuji film calibration, known loads are applied to forms with known area. The accuracy of this program and that of the two commercially available image processing programs were determined. The results of the custom program are found to be within 10% of the results from the commercial programs and from experimental data. This level of accuracy is the same reported level of accuracy of Fuji film, verifying the custom program for use in Fuji film contact pressure and area measurements.

Minor crashes and 'whiplash' in the United States.

In the United States there is currently a paucity of available real world minor rear crash data with struck vehicle delta-V, or speed change, less than or equal to 15 kilometers per hour. These data are essential as researchers attempt to define 'whiplash' injury risk potential in these minor crashes. This study analyzed a new set of 105 U.S. minor rear aligned crashes between passenger vehicles. Mean struck vehicle delta-V and acceleration were 6.3 km/h (s.d. = 2.1 km/h) and 1.4 g (s.d. = 0.5 g), respectively. A total of 113 struck vehicle occupants were diagnosed within five weeks post-crash with 761 ICD-9-CM complaints and 427 AIS injuries (99.5% AIS1) attributed to the crashes. No striking vehicle occupants reported complaints. The main ICD-9-CM diagnoses were 40.6% cervical, 22.5% lumbar/sacral and 10.2% thoracic and the main AIS1 diagnoses were 29.7% cervical, 23.2% lumbar/sacral and 14.3% thoracic. The diagnosis disparity was mainly due to coding for pre-existing degenerative diagnosis in ICD-9-CM. Degenerative spine conditions were not significant for increased AIS1 injury risk. Surprisingly, many non-'whiplash' diagnoses were found. The AIS injury diagnosis distribution and frequency in these minor delta-V crashes did not correspond with previous minor rear crash studies. A prospectively collected and unbiased minor rear crash databank in the model of CIREN or NASS is highly desirable to verify or refute these results for the U.S. population since the current study cohort may have been influenced by litigation.

Biologic response of human intervertebral disc cells to gene therapy cocktail.

STUDY DESIGN: In vitro experiment using human intervertebral disc (IVD) cells and adenovirus-therapeutic gene constructs. OBJECTIVE: To examine the biologic effect of "cocktail" therapeutic gene transfer to human IVD cells in three-dimensional cultures. SUMMARY OF BACKGROUND DATA: Gene therapy is regarded as a potential option for the treatment of degenerative disc disease. Although various anabolic genes have previously been introduced for this purpose, cocktail gene transfer of anabolic genes to IVD cells has never been attempted. METHODS: Human IVDs were harvested during surgical disc procedures and cultured. We prepared recombinant adenovirus constructs bearing the TGF-beta1 gene (Ad/TGF-beta1), the IGF-1 gene (Ad/IGF-1), and the BMP-2 gene (Ad/BMP-2). Transgene expression was detected by luciferase assays, enzyme linked immunosorbent assays, and Western blot analysis. Newly synthesized proteoglycan was measured by S-sulfate incorporation on Sephadex G-25 M in PD 10 columns. Human IVD cells were transduced by single, double, and triple combination of Ad/TGF-beta1, Ad/IGF-1, Ad/BMP-2 with an MOI of 75, then cultured three-dimensionally in alginate beads. RESULTS: Transgene expression was detected at 18 hours after viral transduction. IVD cultures with Ad/TGF-beta1, Ad/IGF-1, Ad/BMP-2 (MOI of 75) showed 2.9, 1.8, and 1.9 fold increases, respectively, in proteoglycan synthesis compared to control. Human IVD cultures with double gene combination (MOI of 75) showed 3.2 to 3.9 fold increases of proteoglycan synthesis. Lastly, Human IVD cultures with triple gene combination (TGF-beta1+IGF-1+BMP-2 genes with an MOI of 75) transfer demonstrated 4.7 fold increase in proteoglycan synthesis compared control. CONCLUSION: Combination or "cocktail" gene therapy offers a promising mechanism for maximizing matrixsynthesis with low dose of adenoviral mixtures, circumventing systemic, local toxic effect, and immune response.

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.

Coculture of bone marrow mesenchymal stem cells and nucleus pulposus cells modulate gene expression profile without cell fusion.

STUDY DESIGN: Changes in gene expression profile and cell fusion of mesenchymal stem cells (MSC) and nucleus pulposus cells (NPC) after coculture were analyzed. OBJECTIVE: To investigate the mechanisms of the interaction between NPC and MSC such us differentiation, stimulatory effect, and cell fusion. SUMMARY OF BACKGROUND DATA: Introduction of exogenous cells to supplement and replenish intervertebral disc cell population offers a potential approach to treat intervertebral disc degeneration (IDD). Recent evidences showed that intradiscal injection of MSC effectively alter the course of IDD in vivo, and the regenerative potential may result from up-regulated extracellular matrix protein synthesis mediated by MSC and NPC interaction. METHODS: Using a double labeling cell system and flow activated cell sorting, we quantitatively analyzed changes in the gene expression profile of human male MSC and female NPC after coculture in a 3-dimensional system that allows short distance paracrine interactions typical of the nucleus pulposus. Furthermore, we analyzed for cell fusion in the cell interaction by fluorescence in situ hybridization (FISH) for X and Y chromosomes, using a 3-dimensional culture system to allow cell-to-cell interactions conducive to cell fusion. RESULTS: Two weeks of coculture cell interaction in a 3-dimensional environment induces a change in MSCs towards a more chondrogenic gene expression profile indicating MSC differentiation, and NPC gene expression changes in matrix and chondrogenic genes demonstrating only a modest trophic effect of MSC on NPC. Moreover, FISH analysis demonstrated that cell fusion is not responsible for MSC plasticity in the interaction with NPCs. CONCLUSION: This study clarifies the mechanism of MSCs and NPCs interaction in a 3-dimensional environment, excluding cell fusion. These data support the use of undifferentiated MSC for stem cell therapy for IDD treatment.

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.

Feasibility of a stem cell therapy for intervertebral disc degeneration.

BACKGROUND CONTEXT: Different strategies to supplement/replenish the disc cell population have been proposed. Recently, adult stem cells have shown promise as a cell source for a variety of tissue engineering and cell therapy applications. A stem cell can renew itself through cell division and can be induced to develop into many different specialized cell types. Moreover, stem cells have shown ability to migrate and engraft within various tissues, as well as to exert stimulatory effects on other cell types through various mechanisms (eg, paracrine effects, cell-cell interactions). These characteristics make stem cells worthy of investigation as a source of cells for intervertebral disc (IVD) tissue engineering and cell therapy. PURPOSE: To determine feasibility of a stem cell therapy of IVD degeneration. STUDY DESIGN: In vitro studies of adult human cells to examine interactions between nucleus pulposus cells (NPCs) and mesenchymal stem cells (MSCs) at different ratios in 3-D pellet culture. In vivo studies of healthy adult rabbit discs injected with allogenic adult rabbit MSCs to examine stem cell survival and engraftment in living disc tissue. METHODS: In vitro study: Human NPCs were cocultured with human MSCs in different ratios (75:25, 50:50, 25:75) for 2 weeks in pellet culture, for comparison with pure NPC (100:0) and pure MSC (0:100) pellet cultures. Proteoglycan synthesis rate and glycosaminoglycan (GAG) content were measured by radioactive sulfate incorporation and dimethylmethylene blue assay, respectively. In vivo study: MSCs were isolated from the bone marrow of a New Zealand White (NZW) rabbit, retrovirally transduced with the lacZ marker gene, and injected into the nucleus pulposi of the L2-3, L3-4, and L4-5 lumbar discs of 12 other NZW rabbits. Three rabbits each were sacrificed at 3, 6, 12, or 24 weeks after cell implantation, and X-Gal staining was done to assess survival and localization of MSCs in the disc tissues. RESULTS: In vitro study: the 75:25 and 50:50 NPC:MSC cocultures yielded the greatest increases in extracellular matrix (ECM) production. In vivo study: MSCs were detected in histological sections of rabbit discs up to 24 weeks after allogenic stem cell implantation, without evidence of systemic illness in the recipient rabbits. The 24-week results in particular suggested the possibility of stem cell migration and engraftment into the inner annulus fibrosus. CONCLUSIONS: These encouraging results support feasibility of a stem cell therapy approach toward supplementation/replenishment of IVD cells and synthesis/maintenance of a more functional ECM in a degenerated disc. Moreover, the in vivo results demonstrate that transplanted MSCs survive and successfully engraft into the IVD tissue, and are effective vehicles for exogenous gene delivery to the IVD--thus there appear to be multiple mechanisms whereby stem cells might able to confer therapeutic effects in a stem cell therapy of IVD degeneration.

In vitro interaction between muscle-derived stem cells and nucleus pulposus cells.

BACKGROUND CONTEXT: Current therapies for intervertebral disc degeneration (IDD) are aimed at treating the clinical symptoms arising from IDD rather than directly treating the underlying problem. Pathophysiology of IDD is characterized by a progressive decrease in proteoglycan content and cell density in the nucleus pulposus (NP). A cell-based therapy is a promising concept that uses various cell types to repopulate the disc in an attempt to slow, stop, or reverse the progressive loss of proteoglycans. Stem cells appear to be an excellent candidate for this purpose, based on their ability to differentiate into various connective tissue lineages. The muscle tissue could serve as a good source of adult stem cells because of its vast abundance through out the human body. PURPOSE: To examine the interaction between the nucleus pulposus cells (NPCs) and the muscle-derived stem cells (MDSCs) in vitro. STUDY DESIGN: NPCs and MDSCs were cocultured and proteoglycan production and cell proliferation were evaluated. METHODS: Various ratios of human NPCs were cocultured for 2 weeks with murine MDSCs (transduced with retro/LacZ) in a monolayer culture. Each well contained an admixture of cells with NPC-to-MDSC ratios of 0:100, 25:75, 50:50, 75:25, 100:0. Glycosaminoglycan (GAG) content (1,9 dimethylmethylene blue [DMMB]), newly synthesized proteoglycan ((35)S incorporation), and DNA content were measured, and cultures were stained with 5-bromo-4-chloro-3-indolyl-beta-D-galactosidase (X-Gal) for cell counting. RESULTS: The NPC-to-MDSC ratio of 75:25 resulted in a significant increase in GAG content compared with NPCs alone. All coculture ratios showed increase in GAG content in comparison with MDSC culture alone. In addition, cocultures showed a significant increase in (35)S incorporation normalized to DNA content in comparison with MDSC alone. CONCLUSIONS: The data from this study shows a synergistic effect between MDSCs and NPCs resulting in an upregulated proteoglycan synthesis and NPCs proliferation in vitro.

Regulation of transgene expression using an inducible system for improved safety of intervertebral disc gene therapy.

STUDY DESIGN: Human nucleus pulposus cells (NPCs) were transduced with an adenoviral vector that expresses Fas Ligand (FasL) and green fluorescent protein (GFP) under the control of a tetracycline-regulated gene expression system to test the transgene control. OBJECTIVES: To describe the application of a Tet-off gene regulation system for intervertebral disc (IVD) gene therapy. SUMMARY OF BACKGROUND DATA: Gene therapy has proven its ability to beneficially modulate the biologic processes of the IVD cells in vitro and in vivo. However, we have observed that expression of transgenic growth factors outside the IVD in the event of a misdirected injection has potentially detrimental consequences (e.g., toxicity). To date, a safety system that allows the control transgene expression has not been produced for intradiscal gene therapy. METHODS: Human NPCs were transduced with Ad/FasL-GFPTET, at 0, 50, 100, and 200 MOI. After 1 day (time 0) cells were cultured in the presence of tetracycline (1, 10, 100 mg/L) for 3 days, and then tetracycline was withdrawn. The transgene expression was evaluated either daily by flow cytometry (from time 0 to day 6) or by imaging the GFP signal (time 0, day 3 and day 9). RESULTS: NPC expression of GFP 1 day after transduction was proportional to the MOI used. GFP expression was decreased after 3 days of tetracycline administration at all concentrations used. The expression of GFP recovered after removal of tetracycline. CONCLUSIONS: The transgene expressed by the transduced NPC was efficiently regulated by inclusion of tetracycline in culture media. The presence of tetracycline turns off the protein expression and the subsequent absence allows it to recover again, demonstrating the ability to control gene expression in NPCs. Therefore, we propose a Tet-off inducible system as an efficient tool for modulating the transgene expression to avoid the toxicity that could result from a missed injection.

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.

Biologic modification of animal models of intervertebral disc degeneration.

Intervertebral disc degeneration is a chronic process that can become manifest in clinical disorders such as idiopathic low back pain, sciatica, disc herniation, spinal stenosis, and myelopathy. The limited available treatment options (including discectomy and spinal fusion) for these and other disabling conditions that arise from intervertebral disc degeneration are highly invasive, achieve limited success, and only address acute symptoms while doing nothing to halt the process of degeneration. Although the precise pathophysiology of intervertebral disc degeneration has yet to be clearly delineated, the progressive decline in aggrecan, the primary proteoglycan of the nucleus pulposus, appears to be a final common pathway. Animal models as well as in vitro studies of the process of disc degeneration have yielded many potentially useful targets for the reversal of disc degeneration. One current research trend is the use of established animal models of disc degeneration to study the role of therapeutic modalities in reversing the process of degeneration, often with use of the delivery of genes or gene products that influence the anabolic and catabolic pathways of the disc. This article reviews the ability of gene-product delivery systems and gene therapy to alter biologic processes in animal models of disc degeneration and examines future trends in this field.

Safety assessment of intradiscal gene transfer: a pilot study.

BACKGROUND: Several recent in vitro and in vivo studies have reported the beneficial properties of gene delivery of therapeutic factors to the intervertebral disc, as a potential treatment strategy for degenerative disc disease; however, to date, no studies have assessed the safety and toxicity of the practical application of this treatment modality. PURPOSE: To assess the safety of inappropriately dosed or misdirected gene delivery to the spinal column in an in vivo model. STUDY DESIGN: The potential toxicity of gene therapy to the spinal column was assessed in this pilot study by monitoring clinical and histological changes in the spinal cord after intradural injections of an adenoviral vector containing the complementary deoxyribonucleic acid (cDNA) for potentially therapeutic factors in the treatment of degenerative disc disease. METHODS: Fourteen New Zealand White rabbits were divided into experimental groups to receive an intradural injection (<10 microL) of saline alone or saline in combination with recombinant transforming growth factor beta1 (TGF-beta1) or an adenoviral vector containing the cDNA for either TGF-beta1 (at previously established therapeutic or elevated concentrations) or bone morphogenic protein-2 (BMP-2). Animals were monitored clinically and spinal cords were harvested for histological analysis. RESULTS: No neurological deficits developed in any of the animals receiving injections of saline alone or saline in combination with the therapeutic dose of Ad-TGF-beta1, Ad-BMP-2, or with recombinant TGF-beta1. However, animals receiving a higher concentration of Ad-TGF-beta1 developed bilateral lower extremity paralysis with significant histological changes. CONCLUSIONS: Inappropriately dosed or directed gene delivery to the spinal column may result in significant complications. However, with appropriate dosing, a therapeutic window may exist where the potential benefits of gene therapy in the treatment of degenerative disc disease outweigh its risks.

Gene therapy for disc repair.

BACKGROUND CONTEXT: Recent advances in our understanding of the biologic makeup and environment of the intervertebral disc (IVD) have led to increased interest in the development of novel treatments for disc degeneration. Certain genes found to alter rates of matrix synthesis and catabolism within the disc have provided targets for scientists seeking to alter the balance between the two. To this end, gene therapy has emerged as a potential therapeutic option, and recent research efforts have yielded very promising results. PURPOSE: To update and consolidate information regarding the recent advances in gene therapy and its application to IVD degeneration. STUDY DESIGN/SETTING: Review of recent and ongoing research in the field of gene therapy, particularly regarding its application to the treatment of IVD degeneration. METHODS: Literature review. RESULTS: With its unique ability to provide sustained delivery of potentially therapeutic agents, gene therapy has shown much promise with regard to the treatment of IVD degeneration. There have been many exciting developments such as safer and more reliable vector constructs, favorable results using therapeutic transgenes in disc cells both in vitro and in vivo, and improvement in transgene regulation, and investigators continue to explore ways in which gene therapy can become a powerful tool in the future treatment of disc degeneration. CONCLUSIONS: With continued perseverance and dedication, many advances have been made in the application of gene therapy to the IVD, and it continues to demonstrate great potential to become a powerful tool in the future treatment of disc degeneration.

The adeno associated viral vector as a strategy for intradiscal gene transfer in immune competent and pre-exposed rabbits.

STUDY DESIGN: Experimental animal study. OBJECTIVES: This study evaluates the in vitro and in vivo transduction efficacy and transgene expression in immune competent and pre-exposed rabbits. SUMMARY OF BACKGROUND DATA: Degenerative disc disease (DDD) continues to pose a substantial clinical problem. Therapeutic options such as an interbody fusion are highly invasive and result in the loss of the intervertebral disc. In addition, interbody fusion puts the adjacent discs at an even higher risk for disc degeneration. A novel approach to slow DDD is to introduce high levels of growth factors into the degenerating disc by delivering the gene coding for the appropriate growth factor. The most efficient technique to do so to date uses viral vectors. However, viral vectors may be problematic because of their immunogenicity. The adeno-associated virus (AAV) viral vector is known to be less immunogenic than commonly used adenoviral vectors. METHODS: Human nucleus pulposus cells were transduced in vitro. Twenty-four Rabbits were injected with AAV viral vectors carrying different marker genes. Transgene expression and the humoral/cellular immune response to the vector was evaluated. RESULTS: We could show that the AAV viral vector transduces human as well as rabbit nucleus pulposus cells in vitro and in vivo. There is a significant humoral immune response against the AAV vector that decreases transgene expression over 10-fold in preimmunized animals. CONCLUSIONS: AAV is a valuable new vector to achieve transgene expression in the intervertebral disc. In preimmunized animals, its use needs to be further evaluated because of the significant reduction in transgene expression.

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.

Quantitative analysis of gene expression in a rabbit model of intervertebral disc degeneration by real-time polymerase chain reaction.

BACKGROUND CONTEXT: Serial analysis of gene expression during the course of intervertebral disc degeneration (IDD) could elucidate valuable insight into pathophysiology and provide a basis for identification of potential targets for the development of novel cellular- and gene-based therapies. However, very few previous studies described the changes in gene expression through the process of IDD using a suitable animal model. PURPOSE: To use a recently developed rabbit annular stab model and the technique of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify the change in expression of key rabbit-specific mRNA sequences encoding for selected extracellular matrix (ECM) products, catabolic, anabolic, and anti-catabolic factors in normal and stabbed discs. STUDY DESIGN: Gene expression analyses were performed to characterize a slowly progressive and reproducible animal model of IDD using real-time RT-PCR. METHODS: Twelve rabbits underwent an annular stab with a 16-gauge needle to the L2-L3, L3-L4, and L4-L5 discs, and three rabbits served as sham controls. Nucleus pulposus tissues were harvested from the stabbed discs at 3, 6, 12 and 24 weeks after confirmation of degenerative changes by magnetic resonance imaging (MRI) scan. Real-time RT-PCR was performed with the use of rabbit-specific primers for 1) extracellular matrix (ECM) component genes: collagen type Ia and IIa, and aggrecan; 2) catabolic genes: matrix metalloprotease-3 (MMP-3), inducible nitric oxide synthase (iNOS), and interleukin-1beta (IL-1beta); 3) anabolic growth genes: bone morphogenic protein-2, and -7 (BMP-2, -7), transforming growth factor-beta1 (TGF-beta1), and insulin-like growth factor-1 (IGF-1); and 4) anti-catabolic gene: tissue inhibitor of metalloprotease-1 (TIMP-1). These data were normalized to mRNA levels of glyceraldehyde phosphate dehydrogenase (GAPDH), a constitutively expressed gene. RESULTS: The MRI images confirmed progressive decline in the nucleus pulposus area of high T2 signal and in the signal intensity of the stabbed discs over the 24-week study period consistent with IDD. The ECM components, aggrecan and collagen type IIa mRNA levels had decreased markedly by week 3 and never recovered, whereas type Ia collagen mRNA gradually increased throughout course of degeneration. BMP-2, BMP-7 and IGF-1 mRNA were relatively decreased from weeks 3 to 6 but then increased at weeks 12 and 24 to end at a level near the preoperative level. The TIMP-1 expression fell dramatically to approximately one tenth of the preoperative level by week 3 and remained low throughout the degenerative process. The remaining results, including those from TGF-beta1 and the catabolic genes (MMP-3, IL-1beta, iNOS) demonstrated a double peak characteristic. The gene expression increased by week 3, decreased to a low level at weeks 6 and 12 and then had a second, late peak at 24 weeks. CONCLUSIONS: The gene expression profiles of ECM components and anabolic, catabolic, and anti-catabolic factors demonstrate many characteristics similar to the findings in human disc degeneration and suggest an inability of the intervertebral disc (IVD) to mount an early anabolic response to injury, thereby offering a possible explanation for the disc's lack of reparative capabilities. Catabolic genes are strongly up-regulated both early and late in degeneration, lending strong support to the hypothesis that an anabolic or catabolic imbalance plays a primary role in IDD. According to the resultant patterns, augmenting early production of BMP-2, BMP-7, IGF-1 or TIMP-1 by gene transfer techniques might possibly alter the progressive course of degeneration as seen in the stab model. The next step will be to transfer these therapeutic genes to regulate the biologic processes and ideally alter the progressive course of disc degeneration.