The Effect of Extracellular Collagen on Synthesis of Extracellular Matrix in a 3-Dimensional Culture of Intervertebral Disc Cells

Study Design In-vitro experimental study. Objectives To determine the proteoglycan synthesis of the rabbit nucleus pulposus cells in various concentration of extracellular collagen type I and II under the stimulation of TGF-β1. Summary of Literature Review Therapeutic effect of growth factor and gene therapy can be altered by composition of extracellular matrix. However, the effect of extracellular collagen types I and II on synthetic activity of intervertebral disc cells is not thoroughly studied before. Materials and Methods The nucleus pulposus cells were isolated and cultured from 10 skeletally mature rabbits. Cultures were trypsinized and incorporated into alginate beads with different concentration of extracellular collagen type I and II (0.5%, 1.0% and 1.5%). Those cultures with TGF-β1 (10 ng/ml) served stimulated condition of matrix synthesis. Newly synthesized proteoglycans were assessed by 35S-sulfate incorporation using chromatography on Sephadex G-25 in PD-10 columns. Scintillation count was normalized with DNA content by Hoechst dye method. Results In basal condition, difference in proteoglycan synthesis in given concentration of extracellular collagen type I and II were statistically insignificant. In stimulated condition with TGF-β1, difference in proteoglycan synthesis in given concentration of extracellular collagen type I and II was also statistically insignificant. However, cultures in stimulated condition with TGF-β1 showed increased amount of newly synthesized proteoglycans compared to those of basal condition regardless of the concentration of extracellular collagen type I and II (p < 0.05). Conclusion Anabolic response of rabbit nucleus pulposus cells is relatively insensitive to extracellular matrix composition, which facilitates application of gene therapy in various conditions of disc degeneration.ope