(F-205) Effects of intervertebral disc-like pressure and pH on Human Adipose Derived Mesenchymal Stem Cells

Introduction:: Low back pain (LBP) affects approximately 12% of adults in the US, and of these cases, between 26 and 42% are caused by intervertebral disc degeneration (IVDD)¹. Current treatment approaches for LBP and IVDD mainly focus on reliving pain and do not address the degeneration¹. For this reason, the use of tissue engineered IVD combining scaffolds and stem cells to regenerate the damaged structures has been proposed. There are several types of stem cells that have shown potential for regenerative treatments, primarily adipose and amnion derived mesenchymal stem cells². However, IVD cells, specifically those in the nucleus pulposus, experience elevated pressures caused by supporting the mechanical loads applied to the spine and lower pH due to the avascular nature of the IVD. These conditions could affect the viability of any stem cells that are placed in the IVD as part of a treatment³. The aim of the current study is to determine the response of human adipose derived mesenchymal stem cells (hADMSCs) to simulated IVD-like conditions in vitro.



Materials and Methods:: hADMSCs were cultured in DMEM supplemented with 10% FBS and 1% pen-strep. After they reached 80% confluency, cells were seeded onto 15mm cell culture treated coverslips at 50,000 cells/coverslip. Cells were then exposed to one of six conditions: control pH (7.4), low pH control (6.5), normal pH low pressure (0.03MPa), low pH low pressure, normal pH high pressure (0.275MPa), or low pH high pressure for six days. Cells were kept in the same pH media for the duration and exposed to pressure conditions via a custom bioreactor for 1hr/day. Additional experiments were done to determine the effect of gradual pH and pressure changes. Over the six day experimental period, the control group was kept at pH7.4, one was kept at pH6.5, and the pH for another group was decreased by 0.3 every day from 7.4 to 6.5, then kept at 6.5 for the next three days. For the gradual pressure experiment, the control group was maintained at atmospheric pressure, one group was exposed to 0.275MPa for one hour/day, and the third group was exposed to 0.05MPa on the first day, which was increased by 0.05MPa every day through day five, then 0.275MPa on day six. At the end of the prescribed time periods, the cells were fixed and stained with either Coomassie Blue or DAPI to analyze cell morphology or the number of cells, respectively. Microscopy images (10 per group) were analyzed using the analyze particles feature of NIH Image-J software.

Results, Conclusions, and Discussions:: Results: The results of the present study provided evidence that hADMSC proliferation decreased when they were exposed to either low pH or high pressure from the combined pressure and pH experiments, although none of the differences are statistically significant. In contrast, when these cells were exposed to gradually changing media pH the number of cells was similar to that of the control group and significantly greater compared to those exposed to a sudden change in pH. When the cells were exposed to gradual increases in pressure over time, the number of cells was similar to the group that was exposed to high pressure for all 6 days.

Discussion and Conclusions: Based on the results of the present studies, both the low pH and higher pressure conditions that exist in IVDs could cause a decrease in hADMSC proliferation. The results also indicate that gradually introducing hADMSCs to the more stressful environment may lessen the negative effects. Future studies are needed to test hADMSCs seeded on a decellularized bovine NP scaffold under long-term pressure cycling in order to determine if they are a viable option for a regenerative treatment for IVDD.




Acknowledgements (Optional): : Funding: NIH GM103444

References (Optional): : 1. Mohd Isa IL et al. Int J Mol Sci. 2023 2. Topoluk N et al. J Tissue Eng Regen Med. 2018 3. Raj PP. Pain Practice. 2008