Abstract
Introduction
Nutritional supply of cells within the avascular intervertebral disk occurs mainly by diffusion from the blood vessels ending at the endplate of the vertebral body. With proceeding disk degeneration and calcification of the endplate the main route of transport of nutrients and metabolites is impaired. The consequential reduced nutrient supply is supposed to be a key contributor to disk degeneration. In short-term approaches using undifferentiated nucleus pulposus (NP) cells, gene expression of matrix proteins was decreased after glucose reduction but mechanosensitivity was not altered. Aim of this study was to investigate effects of long-term differentiation of NP cells regarding their responsivity to glucose reduction and mechanical stimulation.
Materials and Methods
NP cells isolated from bovine caudal discs (n = 7) were cultured in alginate beads for 3 weeks for differentiation in high glucose (25 mM) chondrogenic medium supplemented with 10 ng/mL TGF-β1. After differentiation, part of the samples was exposed to reduced glucose concentration (5 mM) for 3 days. Afterwards glucose concentration was further reduced to either 0 mM, 0.5 mM, or 5 mM for 24 hours. Subsequently, half of all samples were mechanically loaded by hydrostatic pressure for 30 minutes at 0.1 Hz and 2.5 MPa or maintained at unloaded conditions. Gene expression of aggrecan, collagen type II and type I, MMP2, MMP3, MMP13, and aggrecanase was analyzed by real-time RT-PCR. Statistical significance was tested by Wilcoxon test (p < 0.05).
Results
Total 3 weeks of differentiation in high-glucose (25 mM) chondrogenic medium caused a slight upregulation of aggrecan (2.7×), collagen type II (1.9×), and collagen type I (2.6×) expression in NP cells compared to day 0. While MMP2 (3.2×) expression increased significantly, a significant decrease of MMP3 (0.06×), MMP13 (0.006×), and aggrecanase (0.5×) expression was determined. Subsequent reduction of glucose concentration from high-glucose medium to medium containing 5 mM glucose decreased significantly expression of collagen type II (0.01×) and collagen type I (0.003×) to gene expression levels below that of day 0. Further reduction of glucose concentration revealed minor influences on gene expression.
Effects of mechanical stimulation depended strongly on the medium in which NP cells were cultured. In NP cells, which were maintained in high-glucose chondrogenic medium without glucose reduction, mechanically induced effects were strongest. Within this group, mechanical loading increased significantly expression of c-FOS (3.3×) and collagen type I (4.6×), whereas expression of collagen type II (1.4×) increased only moderately. Furthermore, a decrease of gene expression of MMP2 (0.6×), MMP3 (0.02×), MMP13 (0.0005×) and aggrecanase (0.003×) after mechanical stimulation was observed. In cells exposed to glucose reduction, the mechanically induced changes in gene expression were minimal and no distinct correlation with glucose concentration could be pointed out.
Conclusion
After successful in vitro differentiation of NP cells, subsequent glucose deprivation revealed a strong downregulating impact on gene expression of matrix molecules and increased expression of matrix-degrading enzymes. Application of mechanical stimulation could not change these effects of glucose reduction. However, in high glucose chondrogenic medium supplemented with TGF-β1, hydrostatic pressure increased expression of matrix molecules and decreased expression of matrix-degrading enzymes. These findings indicate that mechanical sensitivity of NP cells depends on environmental conditions, as NP cells were less mechanosensitive under glucose deprivation. These interactions of glucose supply, presence of TGF-β1, and mechanical loading in the regulation of disk matrix turnover are investigated in further studies.
Yes
None declared