Browsing by Author "Hwang, Priscilla Y"
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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 Changes in midbrain pain receptor expression, gait and behavioral sensitivity in a rat model of radiculopathy.(Open Orthop J, 2012) Hwang, Priscilla Y; Allen, Kyle D; Shamji, Mohammed F; Jing, Liufang; Mata, Brian A; Gabr, Mostafa A; Huebner, Janet L; Kraus, Virginia B; Richardson, William J; Setton, Lori AIntervertebral disc herniation may contribute to inflammatory processes that associate with radicular pain and motor deficits. Molecular changes at the affected dorsal root ganglion (DRG), spinal cord, and even midbrain, have been documented in rat models of radiculopathy or nerve injury. The objective of this study was to evaluate gait and the expression of key pain receptors in the midbrain in a rodent model of radiculopathy. Radiculopathy was induced by harvesting tail nucleus pulposus (NP) and placing upon the right L5 DRG in rats (NP-treated, n=12). Tail NP was discarded in sham-operated animals (n=12). Mechanical allodynia, weight-bearing, and gait were evaluated in all animals over time. At 1 and 4 weeks after surgery, astrocyte and microglial activation was tested in DRG sections. Midbrain sections were similarly evaluated for immunoreactivity to serotonin (5HT(2B)), mu-opioid (µ-OR), and metabotropic glutamate (mGluR4 and 5) receptor antibodies. NP-treated animals placed less weight on the affected limb 1 week after surgery and experienced mechanical hypersensitivity over the duration of the study. Astroctye activation was observed at DRGs only at 4 weeks after surgery. Findings for pain receptors in the midbrain of NP-treated rats included an increased expression of 5HT(2B) at 1, but not 4 weeks; increased expression of µ-OR and mGluR5 at 1 and 4 weeks (periaqueductal gray region only); and no changes in expression of mGluR4 at any point in this study. These observations provide support for the hypothesis that the midbrain responds to DRG injury with a transient change in receptors regulating pain responses.Item Open Access Photocrosslinkable laminin-functionalized polyethylene glycol hydrogel for intervertebral disc regeneration(Acta Biomaterialia, 2014-03-01) Francisco, Aubrey T; Hwang, Priscilla Y; Jeong, Claire G; Jing, Liufang; Chen, Jun; Setton, Lori AIntervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to lower 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. Previous studies have shown that NP cell-laminin interactions promote cell adhesion and biosynthesis, which suggests a laminin-functionalized biomaterial may be useful for promoting or maintaining the NP cell phenotype. Here, a photocrosslinkable poly(ethylene glycol)-laminin 111 (PEG-LM111) hydrogel was developed. The mechanical properties of PEG-LM111 hydrogel could be tuned within the range of dynamic shear moduli values previously reported for human NP. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, 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 3-D 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. Overall, these findings suggest that soft, LM111-functionalized hydrogels may promote or maintain the expression of specific markers characteristic of an immature NP cell phenotype. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.Item Open Access Response of Midbrain Pain Receptors in a Rodent Model of Radiculopathy(2012) Hwang, Priscilla YIntroduction: Intervertebral disc herniation may contribute to nerve root compression or inflammatory processes that are associated with radicular pain and motor deficits. Molecular changes at the affected dorsal root ganglion (DRG), spinal cord, and even midbrain, have been documented in rat models of radiculopathy or nerve injury. The objective of this study was to evaluate gait mechanics and the expression of key pain receptors in the midbrain of rats after induced radiculopathy in order to test the hypothesis that DRG injury can promote molecular changes in the midbrain. Materials and Methods: Radiculopathy was induced by harvesting tail nucleus pulposus (NP) and placing upon the right L5 DRG in Sprague-Dawley rats. Tail nucleus pulposus (NP) was harvested and discarded in sham-operated rats. At 1 and 4 weeks after surgery, DRGs were sectioned and tested for immunoreactivity to astrocytes and microglial. Also at 1 and 4 weeks after surgery, midbrains were sectioned and tested for immunoreactivity to serotonin (5HT2B), mu-opioid (μ-OR), and metabotropic glutamate (mGluR4 and 5) receptor antibodies. Quantitative analysis was performed on all midbrain immunostained images and compared to naïve controls. Cerebral spinal fluid was also extracted at 1 and 4 weeks after surgery for monocyte-chemoattractant protein (MCP-1) assessment. Results: NP-treated animals placed less weight on the affected limb 1 week after surgery and experienced mechanical hypersensitivity over the entire time of the study. Astroctye activation was observed at the DRG 4 weeks after surgery. An increased expression of 5HT2B was observed in NP-treated rats at 1, but not at 4 weeks. Increased expression of μ-OR and mGluR5 was observed in the periaqueductal gray (PAG) region of NP-treated rat midbrains at 1 and 4 weeks post-surgery. By contrast, increased expression levels of mGluR5 in the PAG region of sham animals reverted to naïve levels by 4 weeks after surgery. No changes were observed in expression levels of mGluR4 in either sham or NP-treated animals at any point in this study. MCP-1 levels were higher in NP-treated animals at 4 weeks compared to sham animals. Conclusion: These observations support the hypothesis that the midbrain responds to injury at the DRG with a transient and adaptive change in receptors regulating pain mechanisms.
Item Open Access The role of extracellular matrix elasticity and composition in regulating the nucleus pulposus cell phenotype in the intervertebral disc: a narrative review.(J Biomech Eng, 2014-02) Hwang, Priscilla Y; Chen, Jun; Jing, Liufang; Hoffman, Brenton D; Setton, Lori AIntervertebral disc (IVD) disorders are a major contributor to disability and societal health care costs. Nucleus pulposus (NP) cells of the IVD exhibit changes in both phenotype and morphology with aging-related IVD degeneration that may impact the onset and progression of IVD pathology. Studies have demonstrated that immature NP cell interactions with their extracellular matrix (ECM) may be key regulators of cellular phenotype, metabolism and morphology. The objective of this article is to review our recent experience with studies of NP cell-ECM interactions that reveal how ECM cues can be manipulated to promote an immature NP cell phenotype and morphology. Findings demonstrate the importance of a soft (<700 Pa), laminin-containing ECM in regulating healthy, immature NP cells. Knowledge of NP cell-ECM interactions can be used for development of tissue engineering or cell delivery strategies to treat IVD-related disorders.