Date of Award
8-2025
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Bioengineering
Committee Chair/Advisor
Yongren Wu, PhD
Committee Member
Jeremy Mercuri, PhD
Committee Member
Tong Ye, PhD
Committee Member
Kenneth Tew, PhD, DSc
Abstract
Low back pain consistently ranks among the leading causes of disability worldwide, with degenerative changes in spinal intervertebral discs (IVDs) recognized as a key contributor. Degeneration arises from the disruption of mechanical and biological cues within the disc’s cartilaginous tissue, often exacerbated by aging or injury. In recent decades, cigarette smoking has also been identified as a significant risk factor, as IVD degeneration is more prevalent among individuals with a history of smoking. Smoke inhalation is thought to promote degeneration both directly, by exposing cells to water-soluble toxic chemicals in smoke; and indirectly, through nutrient deficiency resulting from vasoconstriction and carboxyhemoglobin saturation. Both effects may alter disc cellular energy metabolism, which is crucial for cell survival and proliferation, thereby impacting the biosynthesis of IVD extracellular matrix (ECM) components. However, no studies have delineated the direct and indirect effects of smoking through quantitative comparison or examined the regionally heterogeneous impacts on IVD tissue. The objective of this work was to establish an in vitro IVD explant culture model using Sprague Dawley rats to investigate the effects of cigarette smoke extract (CSE) and nutrient deprivation on cellular energy metabolism and ECM biosynthesis in different disc regions. It was hypothesized that both the direct and indirect effects of cigarette smoking impair IVD cellular energy metabolism and ECM biosynthesis, with heterogeneous responses across nucleus pulposus (NP), annulus fibrosus (AF), and cartilage endplate (CEP) regions. To test this hypothesis, two aims were pursued: (1) cellular metabolic activity assessment, using a biochemical analyzer to quantify rates of glucose consumption, lactate production, and their ratio under physiological, CSE-treated, and nutrient-deficient culture conditions in each IVD region; and (2) biosynthetic activity assessment, using metabolic labeling via copper-free click chemistry to measure glycosaminoglycan (GAG) and collagen content in whole-disc samples cultured under the same conditions. In addition, a finite element model incorporating a reaction-diffusion framework was preliminarily developed to investigate the effects of CSE exposure and nutrient deprivation on three-dimensional nutrient gradients and ATP production within the IVD. Cellular metabolic assessment revealed that, in both CSE-treated and nutrient-deprived discs, energy metabolism was primarily driven by glycolysis, indicating a disrupted metabolic profile. Regionally, CEP explants demonstrated greater resistance to metabolic disruption than either NP or AF. Quantitative fluorescence intensity measurements using a plate reader revealed declines in newly synthesized GAG and collagen, which was further supported by fluorescence imaging of the explants. Computational modeling supported these findings by illustrating disruptions in nutrient gradients and ATP production within the disc under both CSE treatment and nutrient deprivation, with the most pronounced reduction observed in the CEP region. Together, these results indicate that both CSE treatment and nutrient deficiency have a cumulative impact on IVD cellular metabolic activity. This study provides new insight into the mechanisms by which cigarette smoking contributes to IVD degeneration and lays groundwork for developing targeted therapies aimed at mitigating its onset and progression.
Recommended Citation
Madden, Avery E., "Effect of Cigarette Smoke Extract and Nutrient Deficiency on Energy Metabolism and Biosynthesis: An in Vitro Intervertebral Disc Explant Culture Study" (2025). All Theses. 4579.
https://open.clemson.edu/all_theses/4579