Browsing by Subject "laminin"
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Item Open Access Cadherin-Mediated Cell-Cell Interactions Regulates Phenotype And Morphology of Nucleus Pulposus Cells Of The Intervertebral Disc(2015) Hwang, Priscilla YJuvenile 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.
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.
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.
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.
Item Open Access Laminin-Functionalized Polyethylene Glycol Hydrogels for Nucleus Pulposus Regeneration(2013) Francisco, Aubrey ThereseIntervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to low back pain. There is significant interest in cell-based strategies for regenerating the nucleus pulposus (NP) region of the disc; however, few scaffolds have been evaluated for their ability to promote or maintain an immature NP cell phenotype. Additionally, while cell delivery to the pathological IVD has significant therapeutic potential for enhancing NP regeneration, the development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell - laminin interactions in the NP region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into biomaterial scaffolds for NP tissue engineering or cell delivery to the disc may be beneficial for promoting NP cell survival and phenotype. In this dissertation, laminin-111 (LM111) functionalized poly(ethylene glycol) (PEG) hydrogels were developed and evaluated as biomaterial scaffolds for cell-based NP regeneration.
Here, PEG-LM111 conjugates with functional acrylate groups for crosslinking were synthesized and characterized to allow for protein coupling to both photocrosslinkable and injectable PEG-based biomaterial scaffolds. PEG-LM111 conjugates synthesized using low ratios of PEG to LM111 were found support NP cell attachment and signaling in a manner similar to unmodified LM111. A single PEG-LM111 conjugate was conjugated to photocrosslinkable PEG-LM111 hydrogels, and studies were performed to evaluate the effects of hydrogel formulation on immature NP cell phenotype in vitro. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, softer LM111 presenting hydrogels were found to promote cell clustering and increased levels of sGAG production as compared to stiffer LM111 presenting and PEG-only gels. When cells were encapsulated in 3D gels, hydrogel formulation was found to influence NP cell metabolism and expression of proposed NP phenotypic markers, with higher expression of N-cadherin and cytokeratin 8 observed for cells cultured in softer (<1 kPa) PEG-LM111 hydrogels.
A novel, injectable PEG-LM111 hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. PEG-LM111 conjugates were crosslinked via a Michael-type addition reaction upon the addition of PEG-octoacrylate and PEG-dithiol. Injectable PEG-LM111 hydrogel gelation time, mechanical properties, and ability to retain delivered cells in the IVD space were evaluated. Gelation occurred in approximately 20 minutes without an initiator, with dynamic shear moduli in the range of 0.9 - 1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 hydrogel carrier, as compared to cells in liquid suspension.
The studies presented in this dissertation demonstrate that soft, LM111 functionalized hydrogels may promote or maintain the expression of specific markers and cell-cell interactions characteristic of an immature NP cell phenotype. Furthermore, these findings suggest that this novel, injectable laminin-functionalized biomaterial may be an easy to use and biocompatible carrier for delivering cells to the IVD.
Item Open Access The Effects of Extracellular Matrix Mechanics and Composition on the Behaviors of Nucleus Pulposus Cells from the Intervertebral Disc(2009) Gilchrist, Christopher LeeIntervertebral disc (IVD) disorders are a major contributor to disability and health costs. Disc disorders and resulting pain may be preceded by changes which first occur in the nucleus pulposus (NP) region of the IVD, where significant alterations in tissue cellularity, composition, and structure begin early in human life and continue with increasing age and degeneration. These changes coincide with the loss of a distinct cell population, notochordally-derived immature NP cells, which may play a key role in the generation and maintenance of this tissue. These cells reside in a gelatinous, highly-hydrated extracellular matrix (ECM) environment and exhibit in situ cell-matrix and cell-cell interactions which are quite distinct from cells in other regions of the disc or in other cartilagenous, including expression of laminin cell-matrix receptors and cell-associated laminin proteins. The ECM environment is known to be a key regulator of cellular behaviors, with ECM ligands and elasticity modulating cell adhesion, organization, differentiation, and phenotype. The primary motivating hypothesis of this thesis is that the unique ECM environment of the NP plays a key role in modulating immature NP cell behaviors, and that laminin ligands, in combination with ECM elasticity, modulate immature NP cell behaviors including adhesion, organization, and phenotype.
To investigate this hypothesis, flow cytometric analyses were performed to examine IVD cell integrin receptor expression over time in culture, including assessment of key laminin-binding integrin subunits. The roles of specific integrin receptors modulating NP cell adhesion to ECM proteins were identified in studies utilizing function-blocking antibodies. NP cell adhesion, spreading, and relative cell adhesion strength was assessed on various ECM constituents, including specific isoforms of laminin. Additionally, studies were performed to examine the roles of ECM ligand and substrate stiffness in modulating NP cellular organization, utilizing polyacrylamide gel substrates with tunable mechanical properties and functionalized with different ECM ligands. Finally, the role of ECM environment was examined on one key measure of NP cell function, proteoglycan production, over time in culture.
NP cells isolated from immature NP tissues were found to express high levels of the laminin-binding integrin subunit alpha 6 ex situ and maintain this expression over time in culture. Function blocking studies revealed this receptor to be a key regulator of NP cell adhesion to laminin, in contrast to cells from the adjacent AF region, which did not express this receptor nor adhere to laminin. Cell adhesion studies demonstrated that NP cells preferentially interact with two laminin isoforms, LM-511 and LM-332, in comparison to other ECM proteins, with enhanced cell attachment, spreading, and adhesion strength on surfaces coated with these ligands. These findings correspond with laminin isoform and receptor expression patterns identified in immature NP tissues. Additionally, NP cell-cell interactions were found to be modulated by both ECM ligand and substrate stiffness, with soft, laminin-functionalized substrates promoting self-assembly of NP cells into cell clusters with morphologies similar to those identified in immature NP tissues. Finally, culture of immature NP cells on soft, laminin-rich substrates was found to promote a key measure of NP cell function, proteoglycan synthesis.
The studies presented here demonstrate that immature NP cells interact uniquely with laminin extracellular matrix proteins, and that laminin ligands and matrix elasticity are two key regulators of NP cell organization and phenotype in the IVD. These findings suggest that alterations in one or both of these factors during IVD aging or degeneration may contribute to the differentiation or loss of this unique cell population. Additionally, these results indicate that soft, laminin-functionalized biomaterials may be appropriate for in vitro culture and expansion of immature NP cells, as well as for use in NP tissue engineering strategies.