This item is under embargo and not available online per the author's request. For access information, please visit http://libanswers.wustl.edu/faq/5640.
Date of Award
Doctor of Philosophy (PhD)
Low back pain and degenerative conditions of the intervertebral disc (IVD) represent major global socioeconomic and medical burdens. The structures that comprise the IVD including the anulus fibrosis and the nucleus pulposus (NP) work together to stabilize the axial skeleton and distribute mechanical forces. However, the degenerative cascade, which is thought to begin with changes to the NP, results in alterations to the disc that can be seen across length scales including elongated cell shapes, tissue dehydration, and loss of disc height. Patients who present clinically with these changes may also experience altered biomechanics, pain upon motion, impairments to their physical function, or associated disabilities. The NP has a limited intrinsic ability for repair, and therefore, biomaterial strategies are of interest as disease modifying, therapeutic options for repairing the tissue and delivering cell sources to defect sites. Cells, including NP cells, are able to sense and respond to cues from both native and engineered microenvironments. As a function of IVD degeneration and with the stiffening of the surrounding extracellular matrix, NP cells shift from an anabolic to a catabolic state and concomitantly become more elongated, lose their characteristic vacuoles, and demonstrate altered gene expression profiles. Previous studies have demonstrated an ability for laminin-presenting hydrogels to promote NP cells to assume behaviors characteristic of the juvenile phenotype including robust clustering of rounded cells, biosynthesis of sulfated glycosaminoglycans amongst other extracellular matrix proteins, and expression of NP cell phenotypic markers. While these effects have previously been observed, the mechanisms underlying the NP cell-laminin interactions have remained unclear. Understanding the interactions between NP cells and laminins provides insights into the mechanobiology of degenerative NP cells and can be used to inform the design of biomaterials for intervertebral disc regeneration. The studies presented in this dissertation aimed to elucidate the integrin subunits which facilitate the interactions between laminin-presenting biomaterials and degenerative human NP cells and the role of integrins in modulating NP cell behaviors. The results demonstrate that integrin α3 is a primary mediator of NP cell interactions to full-length laminin and that blocking this integrin subunit reduces intracellular signaling, biosynthesis, expression of NP cell markers, and promotes altered cytoskeletal organization. Integrin subunits were also shown to mediate cell attachment to a library of hydrogels formulated by conjugating laminin-mimetic peptides to a poly(ethylene) glycol (PEG) backbone. Data presented herein also demonstrate that stiff (10.5 kPa) peptide-functionalized hydrogels were able to promote similar cell behaviors as soft (0.3 kPa) PEG gels presenting full-length laminin. This finding indicates that biomaterials can be formulated that leverage the advantages of both short peptides (controlled sites of cellular interaction and economic value) and stiff in situ forming hydrogels (improved injection and mechanical properties, no UV crosslinking) while eliciting similar effects as soft gels made with the full-length protein. Furthermore, culture on a PEG-peptide gel induced global transcriptomic changes in degenerative NP cells including an upregulation of genes associated with notochordal morphogenesis and a concomitant downregulation of genes related to cell cycle progression and cellular contractility. Overall, this dissertation serves to identify the integrin subunits by which NP cells interact with laminin-presenting substrates and the signaling pathways that transduce cues from the extracellular microenvironment to regulate cell behaviors and drive the juvenile NP cell phenotype. Additionally, the results of both mechanistic and exploratory studies characterize clinically relevant biomaterials which could be utilized to deliver cells to the degenerative disc space and to promote tissue regeneration and reversal of pathology.
Lori A. Setton
Nathaniel Huebsch, Gretchen A. Meyer, Amit Pathak, Simon Y. Tang,
Available for download on Sunday, April 16, 2023