The influence of nutrient deprivation on nucleus pulposus cells in vivo and in vitro

Intervertebral disc degeneration is one of the causes of low back pain with a multifactorial etiology of which lack of nutrition for the nucleus pulposus cells has been proposed as one of the causes. During ageing, the cartilaginous endplates of the disc calcify and become less permeable to nutrients. Since the disc is a large avascular structure the cells inside the disc depend on diffusion for solute transport. For the nucleus pulposus the most important route is the route through the endplate. In this research project we want to see if partial blockage of this endplate route (mimicking endplate calcification in humans) results in nutrient deprivation, and if this nutrient deprivation could cause disc degeneration. This study has an in vivo and an in vitro part. In the in vivo part we test the endplate perfusion inhibition, and how the cells respond in a short term to the nutrient deprivation caused by this perfusion inhibition. In the in vitro part we test the sensitivity of isolated nucleus pulposus cells to low glucose conditions with or without serum and with or without cyclic hydrostatic loading. Also we test the capability of bone marrow derived stromal cells (BMSCs) to survive in hydrogels which is no problem to nucleus pulposus cells and articular chondrocytes, this with respect to their potential for disc and cartilage repair for future development of biological treatments. In vivo: Under general anesthesia cuts were made in sheep vertebrae, close and parallel to the endplate to disrupt the blood supply to the disc. Immediately or 4 weeks later, diffusion of N2O into the disc was measured amperometrically. Post-mortem a fluorescent dye (procion red) was infused into the lumbar vasculature to visualize the vascular buds in histological section. In the 4 week study gene expression, DNA quantification and cell viability assessment were added as outcome measures. The results of the acute study confirmed that disrupting the blood supply decreased the perfusion of the endplate and inhibited diffusion of N2O into the disc. There was a significant correlation between the amount of perfused vascular buds and the amount of diffused N2O inside the disc. In the 4 week study there were problems with the diffusion measurements, but the cell viability was lower and Collagen I and MMP13 gene expression were significantly up-regulated in the experimental discs compared to the control discs. In vitro : Isolated nucleus pulposus cells were cultured in 3D cell pellets under high (4.5 g/l, only serum group) low (1.0 g/l) and ultra low (0.1 g/l) glucose conditions with and without cyclic hydrostatic load (sine wave, 0.5 - 1.5 MPa, 1Hz, 4 hours per day) and with or without serum. At days 0, 7 and 14 pellets were harvested for DNA, GAG, gene expression analysis and histology (only d14). In all serum groups DNA content per pellet increased over the 14 day culture period. In the serum groups, GAG increased over time in the ultra low glucose group. In the low and high glucose groups GAG content increased till d7, but had dropped again at d14. A similar trend was found in aggrecan and collagen II gene expression. In the serum free groups GAG content was low at d7, but had increased at d14. Histology also showed more GAG in the ultra low glucose groups and the serum free groups than in the low and high glucose groups when pellets were cultured with serum. In none of the groups an effect of load on any of the outcome measures was found. BMSCs, articular chondrocytes and nucleus pulposus cells cast in 1.2% alginate or 2% agarose were cultured for 21 days in serum containing media or (only BMSCs) in medium with 1% ITS+ and 10ng/ml TGF??1. By day 21, nucleus pulposus cells and articular chondrocytes proliferated, maintained up-regulation of aggrecan and collagen type II, produced GAGs and stained positively for collagen type II in both scaffolds. In contrast, number of living BMSCs and DNA content of their constructs decreased in both scaffolds. Addition of TGF??1 resulted in cell survival and behavior more similar (gene expression, GAG production and collagen type II synthesis) to articular chondrocytes and nucleus pulposus cells. In conclusion: Nucleus pulposus cells are very tough cells that survived very low glucose conditions. But when perfusion has been inhibited in vivo, they have problems surviving and maintaining their gene expression. When considering BMSCs as a strategy for disc (or cartilage) repair, chondrogenic differentiation is advised in order to maintain their viability. This research shows that glucose deprivation alone or combined with load does not result in degenerative changes in nucleus pulposus cells and that in order to study disc de- and regeneration in vitro oxygen concentration and pH should also be included.