| dc.description.abstract |
<p>Juvenile nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large,
vacuolated cells that form cell clusters with numerous cell-cell interactions. With
maturation and aging, NP cells lose their ability to form these cell clusters, with
associated changes in NP cell phenotype, morphology and proteoglycan synthesis that
may contribute to IVD degeneration. Studies demonstrate healthy, juvenile NP cells
exhibit potential for preservation of multi-cell clusters and NP cell phenotype when
cultured upon soft, laminin-containing substrates; however, the mechanisms that regulate
metabolism and phenotype of these NP cells are not understood. N-cadherin is a cell
adhesion molecule that is present in juvenile NP cells, but disappears with age. The
goal of this dissertation was to reveal the role of N-cadherin for NP cells in multi-cell
clusters that contribute to the maintenance of the juvenile NP cell morphology and
phenotype in vitro, and to evaluate the potential for laminin- functionalized poly(ethylene
glycol) (PEG-LM) hydrogels to promote human NP cells towards a juvenile NP cell phenotype.
</p><p>In this dissertation, juvenile porcine IVD cells were promoted to form cell
clusters in vitro, and analyzed for preservation of the juvenile NP phenotype on soft,
laminin-rich hydrogels. In the first part of this dissertation, preservation of the
porcine juvenile NP cell phenotype and presence of N-cadherin was analyzed by culturing
porcine NP cells on soft, laminin-rich or PEG-LM hydrogels. Secondly, cadherin-blocking
experiments were performed to prevent cluster formation in order to study the importance
of cluster formation in NP cell signaling. Finally, human IVD cells were cultured
on PEG-LM hydrogels to investigate the potential to revert degenerate, human NP cells
toward a juvenile NP cell phenotype and morphology. </p><p>Findings reveal soft (<500
Pa), laminin-rich substrates promote NP cell clustering, a key feature of the juvenile
NP cell that is associated with N-cadherin positive expression. Additionally, N-cadherin-mediated
cell-clustering regulates NP cell matrix production and gene expression of NP-specific
and NP-matrix related markers. Inhibition of N-cadherin-mediated contacts resulted
in decreased expression of juvenile NP cell features. Finally, juvenile human NP cells
are also able to form N-cadherin positive cell clusters on soft, PEG-LM hydrogels
with higher expression of juvenile NP cell features compared to culturing on stiff
PEG-LM hydrogels. Some degenerate, human NP cells are also able to form N-cadherin
positive cell clusters with some features of the juvenile NP cell. </p><p>The studies
presented in this dissertation support the proposed hypothesis and establish the importance
of soft, laminin-rich substrates in promoting NP cell clustering behaviors with associated
features of a juvenile cell phenotype and morphology. Additionally, these studies
establish a regulatory role for N-cadherin in juvenile NP cells and suggest that preservation
of N-cadherin-mediated cell-cell contacts is important for preserving the juvenile
NP cell phenotype and morphology. Furthermore, findings from this dissertation reveal
the ability to promote degenerate, mature human NP cells towards a juvenile NP cell
phenotype, demonstrating the potential to use PEG-LM hydrogels as a means for autologous
cell delivery for the restoration of healthy IVD.</p>
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