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The increasing incidence of disc degeneration and its correlation with lower back pain is an alarming trend in modern society. The research of intervertebral disc degeneration and low back pain would greatly benefit from additional methods to study its etiology and possible treatment methods. A large animal model that maintains the biological and mechanical environment that is most similar to the human lumbar spine could provide substantial improvements in understanding and resolving the problem of intervertebral disc related low back pain.This dissertation presents my doctoral work of investigating the potential for the camelid cervical spine to serve as a suitable animal model for advancing biomechanical research of low back pain and intervertebral disc degeneration in the human lumbar spine. Specifically, this work identifies the cellular, morphological and biomechanical characteristics of the camelid cervical spine and intervertebral disc as compared to the human lumbar spine. My results demonstrate that there are remarkable similarities in all aspects. Many of the similarities with respect to the cellular environment of the intervertebral disc are a consequence of the camelid status as a large mammal. Additional testing of the cellular makeup of the camelid intervertebral disc cells revealed that many human qRT-PCR primers associated with disc degeneration are suitable for use in alpacas without modification. From a biomechanics standpoint, the camelid cervical spine also has a vertically oriented spinal posture and is unsupported near the end in an open kinetic chain, providing a mechanical parallel with the human lumbar spine. The camelid cervical intervertebral disc size is closer to the human lumbar intervertebral disc than all other currently used animal models available for comparison in the literature. Average flexibility (range of motion) of a camelid spinal motion segment showed similarities in all modes of loading. Based on magnetic resonance imaging and radiologic grading of the intervertebral disc, almost 90% of elderly camelids exhibited advanced degeneration (Pfirrmann grade 3 or higher) in their cervical spine, and about half of aged camelids have developed severe degeneration (Pfirrmann grade 4 or higher) in at least one or more of their cervical segments, most commonly within the two lowest cervical segments (e.g. c6c7 and/or c7t1). Thus, while there remain differences, the remarkable similarities between the camelid and human spine strengthen the case for using camelids as a model for human disc degeneration, normal and pathological biomechanics and fluid transport, and potentially as a pre-clinical model for investigating the efficacy of novel spinal devices.

Objective - To investigate the effects of injected protease solution on the mechanical advancement of disc degeneration, and to establish test protocol for future pre-clinical validation of spinal arthroplasty devices. The hypothesis that injection of a protease into a cadaveric lumbar disc will mimic advanced degeneration mechanics was the subject of this study. Summary of Background Information - Spinal disc degeneration is a universal condition that progresses in adults due to aging, disease, or injury. Stages of disc degeneration have been categorized in cadaver specimens, with each degeneration level exhibiting characteristic changes in flexibility parameters. Spinal disc tissue can be compromised through introduction of proteolytic enzymes into the collagenous fibers of the annulus fibrosus. Methods - 18 motion segments from 8 human lumbar spines were subjected to flexibility testing. Each specimen was either injected with 0.600 mL of trypsin solution in the annulus fibrosus, 0.600 mL of phosphate-buffed saline, or a fluid-less needle-stick. Motion testing followed with rotations applied in all three major spinal motions. Test sections were transected mid-disc after testing to characterize initial degeneration severity, and acquired motion data was analyzed to show flexibility traits over time. Results - Trypsin, saline, and control injections all caused changes in motion from pre-injection baselines. Saline injections were slightly more effective at mimicking the mechanics of higher grades of degeneration with more fidelity than trypsin injections. All motion parameters were altered by the study treatments, with hysteresis and neutral zone parameters experiencing changes similar to that seen in natural degeneration with greater fidelity. Lateral Bending motion showed the greatest magnitude response to injections, with Flexion-Extension tests showing the smallest change. Discussion - Unexpectedly, fluid-less control injections caused changes to hysteresis and neutral zone parameters, suggesting an alteration to viscoelastic properties due to simple needle puncture. Fluid injections (Trypsin and Saline) caused an immediate transient post-injection change to biomechanics that dissipated over time, except in Axial Rotation. Saline injections provided the highest fidelity in mimicking the motion of more advanced stages of degeneration.

Currently, forensic scientists are only able to determine time since death (or postmortem interval) up to the first 60 hours. This is based largely on insect activity. Herein, it is proposed to use the degradation of the intervertebral disc (IVD) after death to determine a relationship between the mechanical properties of cadaveric tissue and time since death in order to extend the 60-hour window. To that end, 1 fresh human spine and 6 pig spines were each separated into sections (6 human and 48 pig), with each section having one intact disc. The sections were buried, unearthed, and cleaned, leaving only the disc and bone. To determine the mechanical properties, each disc underwent three different tests: cyclic conditioning, compression, and stress relaxation testing. The Schapery collocation method was used to create a theoretical curve from the data for the experimental curve. Observations were made involving the corresponding k values of the curve. Although there are trends in the data for k values that approximate the experimental stress relaxation curve, a correlation could not be determined.

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) worldwide which is expected to affect 80% of the world’s population. IVD degeneration (IDD) is a key player in the degenerative cascade associated with LBP. Pro-inflammatory cytokines and mediators, such as nitric oxide, have been shown to be triggers and mediators of IDD. Due to the avascular nature of the adult IVD, the disc is unable to heal or regenerate when damaged. The multi-components of the IVD, namely glycosaminoglycan (GAG)-rich nucleus pulposus (NP), a concentric collagen dense annulus fibrosis (AF), and cartilage endplates (CEPs), further complicate possible regenerative solutions. Cell therapies show promise. This is supported by studies that demonstrate the use of mesenchymal stem cells (MSCs) in animal models showing potential in mitigating inflammatory signaling as well as recovering proteoglycan content. Despite these promising findings, several gaps in knowledge remain. While the biochemical and mechanical properties of an injured disc (via physical or chemical stimulation) have been characterized, the resulting inflammatory signaling cascades remain undefined. A growing body of evidence suggests that TLR4 is involved in the pathogenesis of the IVD. However, it is unknown how TLR4 mediates injury responses of the IVD. Second, it is unknown how mechanical loading of IVDs can influence the transcriptome or secretome of the IVD. The IVD is normally exposed to multimodal loading (e.g., compression, tension, shear, hydrostatic pressure, and osmotic pressure). Both frequency and magnitude regulate whether loading is beneficial or detrimental to disc integrity, which will be explored. Furthermore, the secretome of the IVD, especially during loading, may be essential to creating therapies targeted for regeneration of the IVD. There may be key, distinct paracrine factors that are released in IVD conditioned loading media which can influence the regenerative and anti-inflammatory capabilities of cell-based therapies. To address these gaps, this thesis describes a series of experiments employing novel ex vivo organ culture model to study the response of the IVD to various injury modalities (inflammatory stimulation, puncture injury, compressive loading), and resulting changes in inflammatory, biomechanical, and biochemical responses. Through methods such as RNA sequencing and proteomics, we now have expanded the characterization to beyond candidate genes or proteins, and are more informed on (1) the IVD response to injury, (2) the role of TLR4 signaling in this ex vivo organ culture model, in addition to (3) the downstream effects of loading and how paracrine factors can be used to improve and develop potential cell and molecular therapies. Sex-based differences, in male and female rat caudal IVDs, were also identified and are analyzed in the context of response to injury.

Vertebral fractures affect at least 12-20% of men and women over the age of 50, and the risk of fracture increases exponentially with age. Despite their high prevalence, the failure mechanisms leading to these fractures are not well understood. For example, clinical observations of fractured vertebra often note that one or both vertebral endplates have collapsed, but the precise involvement of the endplates in the initiation and progression of failure has not yet been defined. The mechanisms of failure may also relate to spatial variations in the density and microstructure of the porous trabecular bone within the vertebra as well as to the health of the adjacent intervertebral discs (IVDs) which transfer loads directly to the vertebral endplates. Delineating the contributions of these factors would shed light on the etiology of vertebral fractures and would aid in development of clinically feasible, patient-specific finite element (FE) models of the vertebra. These models are built from a patient's quantitative computed tomography (QCT) scan and have shown tremendous promise for accurate, patient-specific estimates of bone strength and fracture risk. Further validation studies are required to assess the impact of the choices of material properties and boundary conditions, as a prerequisite for broad implementation of these FE models in clinical care. The overall goal of this work was to define the failure processes involved in vertebral fractures and to evaluate the accuracy of patient-specific FE models in simulating these processes. Mechanical testing of human spine segments, in conjunction with micro-computed tomography, enabled the assessment of deformation at the vertebral endplate and deformation throughout the entire bone, as the vertebra was loaded to failure under both axial compression and anterior flexion. These data were compared against predictions of vertebral deformation obtained from QCT-based FE models. The impact of the choice of boundary conditions was specifically examined by comparing the accuracy of the FE predictions between models that simulated applied loads based on measured distributions of pressure within IVDs and models that used highly idealized boundary conditions. The results of these studies demonstrated that sudden and non-recoverable endplate deflection is a defining feature of biomechanical failure of the vertebra, for both compression and flexion loading. The locations of endplate collapse as vertebral failure progressed were associated with the porosity of the endplate and the microstructure of the underlying trabecular bone. FE analyses incorporating the experimentally observed endplate deflections as boundary conditions provided more accurate predictions of displacements throughout the rest of the vertebra when compared to FE models with highly idealized boundary conditions. Under anterior flexion, the use of boundary conditions informed by measurements of IVD pressure mitigated, but did not eliminate, the inaccuracy of the idealized boundary conditions. No further improvement in accuracy was found when using boundary conditions based on pressure measurements corresponding only to IVDs whose level of degeneration matched that observed in the IVDs adjacent to the vertebra being modeled. Overall, the accuracy of the FE predictions of vertebral deformation was only moderate, particularly near the locations of endplate collapse. The outcomes of this work indicate that the vertebral endplate is principally involved in vertebral fractures and that current methods for QCT-based FE models do not adequately capture this failure mechanism. These outcomes provide a biomechanical rationale for clinical diagnoses of vertebral fracture based on endplate collapse. These outcomes also emphasize that future studies of patient-specific FE models should incorporate physiologically relevant loading conditions and also material properties that more accurately represent the vertebral endplate in order to obtain higher fidelity predictions of vertebral failure.

Le rôle de l’inflammation dans la discopathie dégénérative a pu être étudié dans certains modèles animaux, de tissus post-mortem ou parfois dans des tissus chirurgicaux mais ce phénomène reste encore mal compris. La dégénérescence des disques intervertébraux peuvent être à l’origine de diverses pathologies rachidiennes telles que la hernie discale, la radiculopathie et la myélopathie. Le disque subit un déséquilibre homéostasique, une néoinnervation et une néovascularisation laissant pénétrer les cellules immunitaires au sein du disque. Nous avons analysé 78 échantillons de plasma de patients atteints de pathologie du rachis ayant eu recours à la chirurgie. Les patients ont été stratifié en quatre groupes selon la présence ou l’abscence de myélopathie, hernie discale, radiculopathie, spondylolisthésis, lésions nerveuses et canal étroit. Nous avons identifié un profil de biomarqueurs plasmatiques propre à chacun des groupes. Un profil pro-inflammatoire (augmentation de la protéine C-ractive (CRP), du facteur de nécrose tumorale (TNFα)) semble s’apparié aux patients souffrant de myélopathie, avec une augmentation significative de neurofilament à chaine légère (NfL), confirmant une atteinte de la moelle épinière et une dégradation axonale. Les patients atteints de radiculopathie, ont de faibles niveaux de cytokines pro-inflammatoires. Les patients souffrant de spondylolisthésis semblent être caractérisés par l’augmentation de la chimiokine 22 à motif CC (CCL22) et de la molécule soluble d’adhésion intercellulaire (sICAM-1). La concentration en adiponectine est diminuée chez tous les patients. Des niveaux élevés des biomarqueurs de CD40-ligand soluble (sCD40L), de lipocalin-2 (LCN2) et du sérum amyloïde A (SAA) étaient associés à la dégénérescence des disques intervertébraux. / The role of inflammation in degenerative disc disease has been studied in animal models, in post-mortem tissue and surgical tissue, but the phenomenon remains poorly understood. Degeneration of the intervertebral discs contributes to various spinal pathologies such as disc herniation, radiculopathy, and myelopathy. The disc undergoes homeostatic imbalance, neoinnervation, and neovascularization, allowing immune cells to penetrate the disc tissue. We analyzed blood samples from patients with spinal pathology who had undergone surgery. We stratified patients according to their clinical criteria, and measured inflammatory, antiinflammatory, and trophic biomarkers in samples and compared them with healthy donor samples. We develop a plasma biomarker profile specific to subgroups of patients. A pro-inflammatory profile (increased CRP, TNFα) appears to be associated with patients suffering from myelopathy, with a significant increase in NfL, confirming damage to the spinal cord and axonal degradation compared with other patients and healthy donors. Patients with radiculopathy had low levels of pro-inflammatory cytokines. Patients suffering from spondylolisthesis seem to be characterized by an increase in CCL22 and sICAM-1, which may allow macrophages to penetrate the damaged disc. Adiponectin concentration was lower in all patient subgroups compared to healthy donors. The elevated levels of the biomarkers sCD40L, LCN2 and SAA, were linked to intervertebral degeneration.

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