Browsing by Subject "Intervertebral disc"

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.

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.

Lumbar intervertebral discs (IVD) play a critical role in facilitating the mobility and load-bearing functionality of the spine. Consequently, degeneration of the IVDs has been linked to the development of low back pain (LBP), a leading cause of disability in the world. While the pathomechanisms leading to the development of IVD degeneration and LBP are heterogeneous and often difficult to discern, it is believed that the changes in IVD function (i.e., mechanics, composition, tissue structure) may be closely related to the development of discogenic LBP. Specifically, because the IVD has a limited capacity to repair itself, disruptions to IVD tissue structures and biochemical composition may enable nervous tissue innervation into the IVD, potentiating the development of discogenic LBP. However, because our ability to study these changes in vivo remains limited, it remains unknown whether or not we can leverage the study of IVD function to identify risk factors associated with the development of LBP prior to their transition to a painful state. Accordingly, the overarching goal of this work is to develop non-invasive imaging techniques which may be used to perform spatiotemporal analyses of IVD kinematics and composition in vivo. Building upon prior work in our lab, Specific Aim 1 of this proposed research first seeks to develop a controlled methods to investigate the links between IVD function, composition and LBP by examining the in vivo response of IVDs to controlled dynamic loading in asymptomatic individuals. Using data generated in the prior aim, Specific Aim 2 then seeks to first develop and validate an image-segmentation method which enables precise kinematic analysis of the IVD to be carried out in an automated fashion, in vivo. Subsequently, Specific Aim 2 then seeks expand our current ability to characterize IVD function in response to dynamic activity by developing and validating a novel methodology for evaluating three-dimensional (3D) internal spatiotemporal changes in IVD kinematics using a novel deep-learning-based deformable image registration network. This dissertation is organized as a collection of original research articles which were conducted during my time as a PhD student in the Musculoskeletal Bioengineering Laboratory. The first of these (Chapter 3 - Increasing BMI Increases Lumbar Intervertebral Disc Deformation Following A Treadmill Walking Stress Test) was published in the Journal of Biomechanics (Coppock et al., 2021) in May 2021. The second of these (Chapter 4 - In vivo Intervertebral Disc Mechanical Deformation Following a Treadmill Walking “Stress Test” is Inversely Related to T1rho Relaxation Time) was published in the Osteoarthritis and Cartilage (Coppock et. al, 2022). The third, and fourth manuscripts are currently under review (Chapter 5 - Automated Segmentation and Prediction of Intervertebral Disc Morphology and Uniaxial Deformations from MRI; Chapter 6 - In Vivo Analysis of Intervertebral Disc Mechanics Using a Diffeomorphic Deep-Learning Approach. Chapter 7 - The Effects of a 6-month Weight Loss Intervention on Physical Function and Serum Biomarkers in Older Adults with and without Osteoarthritis - is published in Osteoarthritis and Cartilage, Open.

The intervertebral discs (IVD) contribute to structural stability of the spinal column, attenuate the impact of compressive loads, and enable a wide spectrum of motions. As a consequence of aging, the majority of the adult population experiences painful symptoms associated with IVD degeneration - a condition characterized by diminished integrity of tissue components. Current treatment options unfortunately cannot restore IVD structure and function. At the present, an avenue of great interest involves autologous or allogeneic cell delivery to the degenerated IVD. Induced pluripotent stem cells (iPSCs) have demonstrated their capacity to differentiate into various cell types. A posited strategy for regenerative medicine applications entails deriving iPSCs from a patient's own somatic cells and directing them toward a specific lineage.

The overall objective of this study is to assess the potential of mouse iPSCs to regenerate nucleus pulposus (NP) cells of the IVD. Previous work identified CD24 as an NP marker, while recent data from our lab noted its expression in mouse iPSCs. The first portion of this thesis employed magnetic activated cell sorting (MACS) to isolate a CD24high iPSC population. Notochordal gene expression was analyzed in this undifferentiated cell fraction via real time RT-PCR. Mouse iPSCs were then cultured in a laminin-rich, 3D culture system for up to 28 days, and NP phenotype was assessed by immunostaining.

The latter half of this work focused on producing a more conducive environment for NP differentiation of mouse iPSCs. This involved the addition of low oxygen tension and notochordal conditioned medium (NCCM) to the culture platform. Mouse iPSCs were evaluated for ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Furthermore, they were compared to NIH 3T3 mouse embryonic fibroblasts cultured under the same conditions.

Results demonstrated that a CD24high fraction of mouse iPSCs could be successfully retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of hypoxia and NCCM generated similar phenotypic results. 3T3 fibroblasts unexpectedly exhibited transdifferentiation potential as well. Altogether, these studies conclude that mouse iPSCs do have potential to differentiate into NP-like cells and may be applied to future cell-based therapies for restoration of the degenerated IVD.

Intervertebral 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.

The defining characteristic of the subphylum Vertebrata is the vertebral column, which is comprised of alternating vertebral bodies and intervertebral discs. In spite of being a highly conserved structure, the morphogenetic events that culminate in building the vertebral column remain poorly understood. In particular, patterning mechanisms underlying how segmentation of the spine is precisely established have not been examined at post-embryonic stages. For several years, vertebral column patterning was thought to hinge upon proper segmentation of the embryo, while the notochord served as a transient scaffold for the vertebral bodies and intervertebral discs. Using genetic, live-imaging, and quantitative approaches, this work illustrates that the notochord sheath in zebrafish, provides a template for osteoblast recruitment and vertebral bone formation in the developing spine. Furthermore, we show that notochord segmentation is influenced by the adjacent muscle segments and connective tissue, which may provide mechanical patterning cues. Insights from this work will better inform how adolescent idiopathic scoliosis and congenital scoliosis arise.

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.

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.

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.

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.

Introduction: 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.

Intervertebral 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.

The notochord is a defining feature of our phylum Chordata and has critical roles in human development that are highly conserved in vertebrates. The notochord functions as a hydrostatic scaffold to provide structural rigidity needed for anterior-posterior axis elongation and later for proper spine development. The notochord’s mechanical properties depend on its unique structure. In zebrafish, the notochord consists of a core of giant vacuolated cells surrounded by an epithelial -like sheath. Previous research from our lab has shown that during early development, the notochord vacuole rapidly accumulates fluid and expands within the inelastic notochord sheath. In this work we first investigated the molecular processes by which large vacuolated cells of the notochord maintain integrity while being subjected to a significant amount of stress. We determined that caveolae play a mechanoprotective role in the zebrafish notochord and are crucial in preserving notochord integrity. Upon loss of caveolae, the vacuolated cell collapses at discrete positions under the mechanical strain of locomotion then sheath cells invade the inner notochord and differentiate into vacuolated cells thereby restoring notochord function and allowing normal spine development. Findings from our caveolae work next allowed us to investigate the arrangement of vacuolated cells within the zebrafish notochord. During notochord morphogenesis, the vacuolated cells in wild-type zebrafish arrange themselves in a staircase pattern. However, in both caveolae and vacuole mutants, this pattern is disrupted. We investigated the basis of this pattern and found that it can be described by simple physical principles. We modeled the arrangement of vacuolated cells using a system composed of silicone tubing and sodium polyacrylate jelly beads demonstrating that what we observe in vivo can be described by the theory developed for the packing of spheres in cylinders. We determined that the organization of vacuolated cells within the zebrafish notochord is controlled by the density of fluid filled vacuoles and the diameter of the notochord tube. Lastly, based on our finding that sheath cells of the notochord can form de novo vacuoles, we wanted to identify key factors contributing to notochord vacuole biogenesis and integrity. We used a two-pronged transcriptomics and proteomics approach to identify proteins involved in de novo vacuole formation. We find that loss of a protein previously linked to lysosome related organelle function, Lyst, leads to fragmentation of notochord vacuoles and impaired axis elongation. Interestingly, upon injury of the notochord, sheath cells fail to form a fully inflated vacuole and continue to grow outside of notochord boundaries, forming a tumor-like mass. The tumor-like mass appears very similar to a rare tumor type called chordoma, which is characterized by overgrowth of intervertebral disc tissue. This work suggests that Lyst is important for notochord vacuole biogenesis in zebrafish and may play an important role in chordoma formation. Our work has elucidated novel mechanisms of cell surface integrity and has shown how proper vacuolated cell inflation leads to a structurally intact notochord. Additionally, we have demonstrated the remarkable regenerative capacity of the zebrafish notochord and identified potential regulators of both vacuole biogenesis and chordoma formation.