STUDY DESIGN: In vitro and in vivo studies to determine the anabolic effects of intervertebral disc (IVD) to adenovirus-mediated therapeutic gene transfer. OBJECTIVES: To quantify the anabolic effect of human IVD cells in vitro and rabbit IVD in vivo to therapeutic gene transfer. SUMMARY OF LITERATURE REVIEW: An alternative possibility to delivery of growth factors, in continuous manner, is the genetic modification of disc cells through gene transfer. Contemplating to extend this approach to treatment of disc degeneration, it is necessary to demonstrate anabolic effect of human IVD cells and rabbit disc to therapeutic gene transfer. MATERIALS AND METHODS: In vitro: IVD tissue was obtained from twelve patients. IVD cells were then isolated, cultured, and transduced with Ad/TGF-beta1. Genetically modified disc cells were incorporated into alginate beads and cultured. In vivo: Fifteen skeletally mature New Zealand white rabbit were used. 15ul of saline containing Ad/TGF-beta1 were injected into the nucleus pulposus of the disc in six rabbits. All rabbits were sacrificed 6 weeks after surgery. Nucleus pulposus tissues were harvested, weighted, and cultured. Conditioned medium of alginate bead and rabbit disc tissue cultures were subjected to ELISA to detect TGF-beta1 production. Newly synthesized proteoglycan were analyzed using chromatography on Sephadex G-25 in PD-10 columns after S35-sulfate incorporation. RESULTS: Concentration of TGF-beta1 increased over time in alginate beads cultures transduced with Ad/TGF- beta1. At 6 weeks nucleus pulposus tissue from the disc injected with Ad/TGF-beta1 exhibited 200% (p<0.05) increase in TGF- beta1 production. There was statistically significant 290% increase in newly synthesized proteoglycan in alginate cultures transduced with Ad/TGF- beta1 (p<0.05) compared to control. At 6 weeks nucleus pulposus tissue from the disc injected with Ad/TGF- beta1 exhibited 85% increase in proteoglycan synthesis (p<0.05) over that of intact control. CONCLUSION: In this study, we observed the robust upregulation of proteoglycan synthesis in gene transferred disc cells in vitro and in vivo - indicating good prospects for biologic effects of therapeutic gene therapy in the disc using adenovirus-mediated approach.
Anabolic Agents ; Chromatography ; Culture Media, Conditioned ; Enzyme-Linked Immunosorbent Assay ; Genetic Therapy* ; Humans ; Intercellular Signaling Peptides and Proteins ; Intervertebral Disc ; Intervertebral Disc Degeneration ; New Zealand ; Proteoglycans ; Rabbits ; Transforming Growth Factor beta1 ; Up-Regulation

PURPOSE: To elucidate host immune responses to intradiscal gene transfer. MATERIALS AND METHODS: Twenty rabbits were utilized. Ad/luciferase (adenovirus construct) were injected into nucleus pulposus of lumbar vertebrae. Group 1 received intradiscal injection of Ad/luciferase only, Group 2 received subcutaneous and intradiscal injection simultaneously, Group 3 received subcutaneous injection then intradiscal injection with 2 weeks interval. Blood samples were obtained serially after injection. Animals were sacrificed at 7 weeks. Antibody to adenovirus in peripheral blood was measured with ELISA and transgene expression was measured with standard luciferase kits. RESULTS: All rabbits in the Group 2 and 3 exhibited increased production of neutralizing antibody. There were clearly two subgroups in Group 1, three rabbits exhibited production of antibody but remaining three rabbits showed little or no production of antibody. All rabbits showed robust increase in transgene expression regardless of titer of neutralizing antibody. CONCLUSION: The intervertebral disc is favorable site for adenovirus-mediated gene transfer escaping from systemic immunity.
Adenoviridae ; Animals ; Antibodies, Neutralizing ; Enzyme-Linked Immunosorbent Assay ; Injections, Subcutaneous ; Intervertebral Disc ; Luciferases ; Lumbar Vertebrae ; Rabbits ; Transgenes ; United Nations

STUDY DESIGN: In vitro experiment to determine the matrix synthesis of intervertebral disc (IVD) cell to various biologic interventions and conditions. OBJECTIVES: To elucidate biologic responses in terms of matrix synthesis of human IVD cells in vitro to various factors i.e. concentration of adenoviral vector and exogenous growth factor, duration of incubation, and type of culture methods. SUMMARY OF LITERATURE REVIEW: Sophisticated method to delivery of growth factors, in continuous manner, is the genetic modification of disc cells through gene transfer. Direct comparison of gene transfer and exogenous growth factor on matrix synthesis has not been reported. MATERIALS AND METHODS: IVD tissue was obtained from twenty three patients. Isolation and preparation of disc cells in monolayer (D) and alginate beads (3D) culture were performed. Disc cells in 2D and 3D were treated with either Ad/TGF-beta1 or exogenous TGF-beta1. Control cultures were treated with either saline or Ad/luciferase. Matrix synthesis (newly synthesized proteoglycan) was measured in various conditions (concentration of adenoviral vector and exogenous growth factor, duration of incubation, and type of culture methods). Newly synthesized proteoglycan were analyzed using chromatography on Sephadex G-25 in PD-10 columns after S35-sulfate incorporation. RESULTS: Ad/TGF-beta1 showed increase in proteoglycan synthesis (plateau at 75MOI) in 3D culture, (plateau at 25MOI) in 2D culture. In 3D culture, Ad/TGF-beta1 showed significant increase in proteoglycan synthesis on day 1, 2, and 3 of incubation. In 2D culture, Ad/TGF-beta1 showed significant increase in proteoglycan synthesis on day 2 of incubation with significant loss of anabolic effect on day 3. In 3D culture, exogenous TGF-beta1 showed increase in proteoglycan synthesis (plateau at 2ng/ml) while in 2D culture, there is no synthetic response to exogenous TGF-beta1 CONCLUSION: Therapeutic gene transfer provided sustained and increased anabolic responses than exogenous growth factor.
Anabolic Agents ; Chromatography ; Genetic Therapy ; Humans* ; Intercellular Signaling Peptides and Proteins ; Intervertebral Disc* ; Proteoglycans ; Transforming Growth Factor beta1