Publication Date

2019-12-12

Availability

Open access

Embargo Period

2019-12-12

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biomedical Engineering (Engineering)

Date of Defense

2019-11-04

First Committee Member

Alicia R. Jackson

Second Committee Member

Chun-Yuh Huang

Third Committee Member

Ramon Montero

Fourth Committee Member

Francesco Travascio

Fifth Committee Member

Weiyong Gu

Abstract

Lower back pain is a major socioeconomic concern in developed nations. Moreover, it is a condition which a majority of people will experience an episode within their lifetime, with a portion becoming chronically afflicted. Degeneration of the intervertebral disc (IVD) is believed to play a critical role in initiating lower back pain. Poor nutrient supplies are implicated as a one of the root causes of this degeneration and much effort has been spent to better elucidate the behavior of IVD cells. The IVD is the largest avascular structure within the body, relying on diffusion to migrate nutrients in and clear wastes out. Due to this limitation, nutrients concentrations are low within the center of the disc and waste products also accumulate in high concentrations. For most cell niches, this is considered a harsh environment, but completely normal for the cells of the IVD. Understanding how these cells behave under these environmental conditions may elucidate what nutritional and environmental factors lead to aberrant IVD cell behavior. This dissertation will explore the relationship between nutrient conditions and the metabolic adaptations of the IVD cells. Additionally, a custom bioreactor was constructed in order to study the complex IVD organ as a whole under varied nutritional conditions. Glucose consumption rate and gene expression of nucleus pulposus (NP) cells was investigated in an agarose gel system over prolonged culture periods with varied oxygen tension and glucose concentration treatments. Glucose consumption rate was found to decrease with increasing oxygen tension and over time but not with changing glucose concentration. Catabolic gene expression increased for all groups over time, with inhibitors of catabolism following suit. Collagen Type I increased in expression with time for the high oxygen tensions while other anabolic genes did not show any consistent trends over the culture period. The optimal reference genes were evaluated and the genes RPL4 and YWHAZ were found to be more stable than the commonly used 18s and GAPDH. Next the glucose consumption rate was modeled using a more sophisticated method, the Michalis-Menten kinetic model, allowing for increased fidelity in computational modeling and optimizing culture conditions. No differences between oxygen tension were found in the GCR. Gene expression was again analyzed but the limited culture time did not allow for differences to be seen between oxygen tensions. A correlational analysis revealed several targets for further genetic studies. A custom-built bioreactor was developed and validated to load whole IVD under simulated physiological loading conditions. This reactor was then used to test whole IVDs under free-swelling and dynamic loading conditions. Tissues were compared for their gross composition as well as gene expression differences. No differences were seen between loaded and free-swelling discs, and both tended toward a catabolic state when examining gene expression. Gross changes in tissue composition were seen briefly in hydroxyproline content before returning to baseline. The contents of this dissertation greatly enhance the knowledge of IVD NP cell metabolism. Given that these cells play a vital role in disc degeneration and will be critical in regenerative strategies to treat said condition, understanding the behavior of these critical cells is of paramount importance

Keywords

intervertebral disc; bioreactor; metabolism; oxygen tension; gene expression

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