Regulation of Human Nucleus Pulposus Cell Phenotype and Behavior by Laminin-Mimetic Peptide Substrates

Abstract

<p>Intervertebral disc (IVD) disorders can cause pain and disability for affected individuals. A subset of IVD disorders are thought to originate in the nucleus pulposus (NP) region of the IVD, due to alterations in tissue structure and composition with age and injury. Cells of the NP undergo a phenotypic and behavioral shift with age, changes that are thought to disrupt tissue homeostasis and lead to disc degeneration. There is significant interest in developing biomaterials and strategies to revert adult degenerate NP cells to healthy, young NP cell phenotypes with increased biosynthesis and cell clustering. Studies demonstrate that healthy porcine NP cells interact with laminin proteins through specific integrin subunits on soft substrates in a process that regulates cell morphology and biosynthesis. The central hypothesis of this dissertation is that the engagement of cell-surface adhesion receptors, using laminin-mimetic peptides on a controlled stiffness material, can revert adult degenerate NP cellular phenotype and behaviors to their healthy, biosynthetically active form.</p><p>In the first part of this dissertation, integrin subunits used by adult degenerate human NP cells to attach to laminin were revealed using flow cytometric analyses, function blocking antibodies, and immunohistochemistry. Secondly, NP cell recognition peptides were identified by screening laminin-mimetic peptides for cell attachment. Finally, human NP cells were cultured on peptide functionalized polyacrylamide gels to examine the effect of ligand and substrate stiffness in regulating adult human NP cell phenotype and biosynthesis. </p><p>Findings reveal that adult human NP cells express and utilize integrin subunits α3, α5, and β1 to attach to laminins, in contrast to integrin α6β1 found previously for healthy porcine NP cells. This difference between current and previous findings likely arises from aging-associated difference in NP cells between human and porcine tissues. Select laminin-mimetic peptides, chosen from the literature and informed by NP cell integrin expression, were found to promote significant NP cell attachment in a concentration dependent manner. Finally, a subset of laminin mimetic peptides were found to promote a young NP cell phenotype by increasing cell clustering on soft (0.3 kPa) and stiff (14 kPa) substrates as well as increasing proteoglycan synthesis on soft substrates. </p><p>The studies presented in this dissertation demonstrate that adult degenerate human NP cells attach to laminin proteins in an integrin dependent manner. Furthermore, laminin-mimetic peptides are able to mediate human NP cell attachment at levels comparable to full-length laminin, increase cell clustering when presented on soft and stiff substrates, and can increase NP cell biosynthesis when presented on soft substrates. Utilizing laminin-mimetic peptides may allow for the design of biomaterials that promote a healthy young NP phenotype for a variety of disc therapies.</p>