A study 1 on the efficacy of intradiscal biologic therapy, where new cells or genes are implanted into the degenerated disc matrix to reduce inflammation and increase matrix cell production, found that degenerated discs may not have the necessary nutrient transport capabilities to ensure proper disc nutrition during this form of therapy. The authors of the study emphasize the importance of research into the determining factors influencing disc cell nutrient transport in informing targeted treatments and strategies to improve disc nutrition in degenerated discs.
What’s at Stake?
Disc degeneration (DD) is a chronic condition that causes spinal pain in aging adults worldwide. The process of DD involves biomechanical modeling of the entire disc matrix and frequently leads to surgical intervention to remove the offending disc and restore functionality to the spine. For many patients, surgical procedures are unsuccessful, however. A noninvasive treatment that has demonstrated recent promise involves regenerating the DD by injecting it with genes, growth factors, small molecules, or implanted cells. These procedures are intended to reduce inflammation and catabolism and assist in the creation of a new disc matrix. But a cell-rich disc requires increased nutrients, and the cartilage endplate (CEP) of the DD may not have the capacity to deliver these nutrients to the matrix. In this study, researchers examined the effects of CEP transport properties in DD on nutrient diffusion and cell function and survival.
In order to isolate the variable of how nutrient supply affects the nucleus pulposus (NP) cell function, the researchers involved in this study mimicked the in vivo, diffusion-poor disc environment by creating diffusion chambers with similar parameters to isolate the NP nutrient supply mechanics. The cells of the NP receive nutrients that are diffused through the CEP matrix. Cells at the center of the lumbar discs can be up to 10mm from a capillary, while other cells can be just beside a CEP.
Researchers provided glucose and oxygen to cultured NP cells within the chambers. These nutrients were delivered through diffusion from human CEP’s from the open sides of the chamber. Metabolites were expelled into the culture medium by CEP diffusion. The functioning and survival of the cells require a balance between CEP transport properties and cell density, allowing for the request and supply of nutrients. The researchers reproduced the disc matrix environment and physiologic transport conditions in their CEP tissue cultures and diffusion chambers to monitor the effects of NP cell viability and gene expression across the different conditions of nutrient transport.
Specifically, intact human CEP’s from human cadaveric lumbar spines were used for the study. Full-thickness samples of the CEP’s and surrounding calcified cartilage were frozen and sectioned. The researchers calculated the diffusivity of each full-thickness CEP sample through fluorescence and photo-bleaching and using the Axelrod method. They measured each CEP’s biochemical composition spatially via imaging. They created special maps of the collagen, aggrecan, and mineral-to-matrix ratio of the CEP samples with the highest and lowest diffusivities. They measured CEP thickness with photomicrographs and then determined the average measurement across the five chambers.
Bovine NP cells were used in the study (similar to human NP cells). Post-incubation cell viability was determined using a cytotoxicity assay involving gel-stains and low-magnification imagery. Each L4-L5 donor CEP was analyzed for cell density and the anabolic and catabolic gene expressions were examined after chamber incubation. A regression model of fluorescence intensity was used to determine the NP cell gene expression and distance from the CEP. Spatial fluctuations of the CEP composition were described based upon regression models.
The diffusive transport of nutrients varied widely between the CEP samples, affecting the function, health, and survival potential of the NP cells. In fact, there was a four-fold variation in small solute diffusivity in our human CEP sample array. Those allowing less diffusive transport reduced the supply of nutrients to the NP and shortened the viable distance within the diffusion chambers up to 51 percent with typical cell density. Those permitting poor diffusion seemed to downregulate anabolic and catabolic NP cell gene expression. This may mean that a reduced number of disc cells are capable of being sustained through low nutrient CEP diffusion, and the cell’s ability to retain its matrix homeostatic condition is hindered.
When we increased cell density, there was a reduction in cell viability caused by the CEP transport properties, though increasing cell density should raise nutritional demands and shorten the viable distance. The CEP’s in our study that exhibited low diffusive transport were unresponsive to doubling the cell density, perhaps because they did not provide enough nutrient diffusion to nurture the cell.
We imaged the CEP’s to identify any differences between those with low or high intradiscal diffusivity. Our data found that those with low-diffusivity (and shortened viable distance) contained more collagen and aggrecan, mineral, and lower cross-link maturity. This could explain the blockage of solute penetration and diffusion. At any rate, there appears to be a strong correlation between NP cell survival or function and the availability and mobility of the nutrient supply in the CEP. Compositional defects with the CEP matrix can inhibit nutrient diffusion and undermine biologic therapies that depend upon an increased supply of nutrients to the cell matrix to succeed.
Our findings suggest that the composition of CEP can contribute to or detract from the function and viability of NP cells. Deficits within the CEP matrix can cause poor nutrient diffusion and block solute passages. This can cause an abundance of collagen and aggrecan, as well as mineral, and lower cross-link maturity. When cell density is increased, CEP’s developed transport deficits, decreasing the cell’s viability. It appears NP function and survival are dependent on the proper CEP composition, as an imbalance in this makeup can reduce the supply of nutrients to the cells, reducing the success rates of biologic therapies.