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.

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.