Date of Graduation

2017

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Ming Pei

Committee Co-Chair

Stephen E Alway

Committee Member

Christopher F Cuff

Committee Member

Ivan Martinez

Committee Member

Emidio E Pistilli

Abstract

Low back pain is one of the most common reasons for doctor visits each year in the United States and consistently represents one of the leading areas of US health care spending in recent decades. Located at the inner space of the intervertebral discs (IVDs) of the spinal column, a soft, shocking absorbing tissue known as nucleus pulposus (NP) is a frequently implicated target of patient symptoms and comorbidities related to back pain. IVDs are quite susceptible to both acute injury, such as disc herniation, and chronic age-related degeneration known as intervertebral disc degeneration (IVDD). These conditions ultimately lead to degeneration and deterioration of the IVD tissue and resident NP cells (NPCs) at the biochemical and cellular level. In recent years, orthopaedic therapies have aspired to utilize cell-based therapies in combination with surgical intervention strategies for the replenishment of NP tissue, however, this approach presents several challenges. Studies which seek to use autologous NPCs (i.e. the patients' own cells) must consider the potential implications and poor efficacy associated with harvesting NPCs from a damaged, typically inflamed environment. Furthermore, initial yield from such procedures is typically limited to the herniated portion of the NP tissue. This avoids compromising the remaining healthy tissue in the disc space. Ultimately, this increases the demand for preconditioning strategies which can effectively: (1) increase cell proliferation and yield, (2) rejuvenate harvested NPCs to a healthy NP phenotype to increase cell redifferentiation/regenerative potential for therapeutic utilization, and (3) accomplishes these goals without causing the preconditioned cells to have any adverse immune responses. In our laboratory, we have previously demonstrated that some of these goals can be accomplished by expanding harvested NPCs on decellularized extracellular matrix (dECM) substrates from various sources, however, several key factors remained unexplored. The signaling roles of Wnt (Wingless-Type integration) proteins of the Wnt-signaling pathways in NP redifferentiation (Wnt3A, Wnt5A, and Wnt11) have been previously associated with dECM, however, they have not been fully elucidated or compared; the modulation of these signals could provide more effective and exploitable means for NPC preconditioning. Additionally, it is unknown whether the use of dECM from allogeneic and xenogeneic sources have immunological consequences in the expansion of human NPCs. To demonstrate these concepts, we evaluated several preconditioning strategies for proliferative and redifferentiation effects in multiple cell lines of NPCs and SDSCs in our studies. We also transduced NPCs with lentiviral vectors to overexpress or knockout key WNT genes and evaluated them for redifferentiation gene and protein expression. For dECM studies, we expanded NPCs on allogeneic and xenogeneic dECMs, evaluated NPC redifferentiation, as well as their immunophenotypes by flow cytometry, and assayed T-cell proliferation in a mixed lymphocyte reaction with expanded cells. In these studies, we have shown that it is possible to increase redifferentiation potential of human NPCs and synovial-derived stem cells (SDSCs), a chondrocytic stem cell population, by preconditioning methods including FGF-2 supplementation, dECM expansion, and hypoxic preconditioning. We have demonstrated key roles for Wnt signals in the redifferentiation of NPCs and its relationship with cell preconditioning. Furthermore, we have provided new data and insights surrounding the immunological implications of NPC expansion on allogeneic and xenogeneic-derived dECM substrates. Overall, these findings will allow researchers and clinicians to effectively target Wnt signals, increase redifferentiation capacity, and to understand potential underlying implications of dECM preconditioning for future cell-based orthopaedic therapies.

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