4:27 Transduction of TGF-B1 inhibits proteoglycan synthesis in intervertebral disc cells in a three-dimensional culture

Abstract

Purpose of study: Intervertebral disc degeneration is characterized by a progressive loss of proteoglycan content and a subsequent decrease in the disc's hydrostatic capacity. Growth factors, such as TGF-B1, administered in recombinant form or synthesized endogenously by means of gene therapy, stimulate proteoglycan synthesis in intervertebral disc cells in monolayer cultures. However, these cultures do not accurately reflect the native environment of intervertebral disc cells, and therefore, conclusions regarding TGF-B1's in vivo potential are difficult to extrapolate. The purpose of this study was to evaluate the effect of adenoviral-mediated delivery of TGF-B1 on proteoglycan synthesis in human intervertebral disc cells cultured in a three-dimensional system.Methods used: Nucleus pulposus cells were isolated from human intervertebral discs by means of enzymatic digestion and cultured in monolayer. Patient samples were pooled and experimental groups normalized for cell number before transduction with Ad-TGF-B1 at either 50, 75 or 100 multiplicity of infection (MOI). After 48 hours of incubation, cells were trypsinized and incorporated into a three-dimensional "pellet" culture system. After an additional 48 hours, proteoglycan synthesis was assessed with 35S radioactive sulfate incorporation using chromatography and scintillation count.of findings: Cultures treated with Ad-TGF-B1 demonstrated a progressive decrease in active proteoglycan synthesis with an increasing MOI. For an MOI of 50, proteoglycan synthesis was 53.4% of control; for an MOI of 75, 24.8% of control and for a MOI of 100, 11.2% of control (p<.05). Additionally, a dose-dependent response was shown to be significant with increasing viral concentrations (p<.001).Relationship between findings and existing knowledge: The inhibition of proteoglycan synthesis by TGF-B1 observed in this study contrasts with the stimulatory effect previously reported in monolayer cultures. The concentrations employed in our work were 80% less than those previously demonstrated to be stimulatory, yet proteoglycan synthesis in the three-dimensional culture was uniformly diminished in a dose-dependent manner (Table 1)Table 1Inhibitory effect of TGF-B1 on proteoglycan synthesis50 MOI75 MOI100 MOITGF-B153.4%24.8%11.2%.Overall significance of findings: Although exogenous administration of the growth factor TGF-B1 has been shown to favorably modify proteoglycan synthesis in human intervertebral disc cells in vitro, its brief half life limits its potential role in the treatment of chronic disorders, such as degenerative disc disease. Experiments with adenoviral-mediated delivery of TGF-B1 in monolayer cultures demonstrate a successful increase in proteoglycan synthesis as well. However, this experimental system does not reflect the in vivo environment, and as a result, conclusions regarding the clinical potential of the gene delivery of TGF-B1 drawn from these experiments may be misleading. As the potential for practical application of gene therapy becomes increasingly feasible, experimental models that accurately simulate the in vivo environment acquire greater significance. The inhibitory response to TGF-B1 seen in our study may result from the increased cell-to-cell interactions within the three-dimensional pellet culture, which in turn may produce higher, and potentially toxic, TGF-B1 concentrations in the microcellular environment. Further experimentation is warranted to clarify whether TGF-B1 possesses therapeutic potential for the future treatment of intervertebral disc disease.Disclosures: No disclosures.Conflict of interest: No conflicts.