Correlations between severity of clinical signs and histopathological changes in 60 dogs with spinal cord injury associated with acute thoracolumbar intervertebral disc disease

The outcome of spinal surgery in dogs with absent voluntary motor function and nociception following intervertebral disc (IVD) herniation is highly variable, which likely attests to differences in the severity of spinal cord damage. This retrospective study evaluated the extent to which neurological signs correlated with histologically detected spinal cord damage in 60 dogs that were euthanased because of thoracolumbar IVD herniation. Clinical neurological grades correlated significantly with the extent of white matter damage (P<0.001). However, loss of nociception also occurred in 6/31 (19%) dogs with relatively mild histological changes. The duration of clinical signs, Schiff-Sherrington posture, loss of reflexes and pain on spinal palpation were not significantly associated with the severity of spinal cord damage. Although clinical-pathological correlation was generally good, some clinical signs frequently thought to indicate severe cord injury did not always correlate with the degree of cord damage, suggesting functional rather than structural impairment in some cases.

Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

The transpedicular approach as an alternative route for intervertebral disc regeneration

STUDY DESIGN Descriptive anatomical study on ovine and human cadaveric lumbar spinal segments. OBJECTIVE To describe the alternative transpedicular approach to deliver therapeutic agents into intervertebral disc (IVD). SUMMARY OF BACKGROUND DATA The present delivery approach of therapeutic agents (growth factors/cells/hydrogels) within the IVD is through injection, via the annulus fibrosus (AF). However, it has recently been demonstrated that small needle puncture of the AF leads to further degeneration and disc herniation. In addition, the injected material has a high chance to be extruded through the AF injury. METHODS Lumbar ovine and human spinal segments were used. Under fluoroscopy, a 2-mm Kirschner wire was introduced in the caudal vertebra through the pedicle and the inferior endplate to the nucleus pulposus. Gross anatomy analysis and high-resolution peripheral quantitative computed tomography (HR-pQCT) were performed to assess the right position of the wire in pedicles. Discography and nucleotomy were performed using a 14G cannula insertion or a 2-mm arthroscopic shaver blade, respectively. Nucleoplasty was also performed with agarose gel/contrast agent and imaged with HR-pQCT. RESULTS Gross anatomy, fluoroscopy, and HR-pQCT images showed that the nucleus pulposus could be approached through the endplate via the pedicle without affecting the spinal canal and the neural foramina. The contrast agent was delivered into the IVD and nucleus pulposus was removed from the disc and filled with agarose gel. CONCLUSION This study describes how a transpedicular approach can be used as an alternative route to deliver therapeutic agents to the disc without disruption of the AF showing the potential use of this technique in preclinical research and highlighting its clinical relevance for IVD regeneration.

Region Specific Response of Intervertebral Disc Cells to Complex Dynamic Loading: An Organ Culture Study Using a Dynamic Torsion-Compression Bioreactor

The spine is routinely subjected to repetitive complex loading consisting of axial compression, torsion, flexion and extension. Mechanical loading is one of the important causes of spinal diseases, including disc herniation and disc degeneration. It is known that static and dynamic compression can lead to progressive disc degeneration, but little is known about the mechanobiology of the disc subjected to combined dynamic compression and torsion. Therefore, the purpose of this study was to compare the mechanobiology of the intervertebral disc when subjected to combined dynamic compression and axial torsion or pure dynamic compression or axial torsion using organ culture. We applied four different loading modalities 1. control: no loading (NL), 2. cyclic compression (CC), 3. cyclic torsion (CT), and 4. combined cyclic compression and torsion (CCT) on bovine caudal disc explants using our custom made dynamic loading bioreactor for disc organ culture. Loads were applied for 8 h/day and continued for 14 days, all at a physiological magnitude and frequency. Our results provided strong evidence that complex loading induced a stronger degree of disc degeneration compared to one degree of freedom loading. In the CCT group, less than 10\% nucleus pulposus (NP) cells survived the 14 days of loading, while cell viabilities were maintained above 70\% in the NP of all the other three groups and in the annulus fibrosus (AF) of all the groups. Gene expression analysis revealed a strong up-regulation in matrix genes and matrix remodeling genes in the AF of the CCT group. Cell apoptotic activity and glycosaminoglycan content were also quantified but there were no statistically significant differences found. Cell morphology in the NP of the CCT was changed, as shown by histological evaluation. Our results stress the importance of complex loading on the initiation and progression of disc degeneration.

Assessment of the Matrix Degenerative Effects of MMP-3, ADAMTS-4 and HTRA1 injected into a bovine Intervertebral Disc Organ Culture Model

Study Design. In vitro study to develop an intervertebral disc degeneration (IDD) organ culture model, using coccygeal bovine intervertebral discs (IVDs) and injection of proteolytic enzymes MMP-3, ADAMTS-4 and HTRA1.Objective. This study aimed to develop an in-vitro model of enzyme-mediated IDD to mimic the clinical outcome in humans for investigation of therapeutic treatment options.Summary of Background Data. Bovine IVDs are comparable to human IVDs in terms of cell composition and biomechanical behavior. Researchers injected papain and trypsin into them to create an IDD model with a degenerated nucleus pulposus (NP) area. They achieved macroscopic cavities as well as a loss of glycosaminoglycans (GAGs). However, none of these enzymes are clinically relevant.Methods. Bovine IVDs were harvested maintaining the endplates. Active forms of MMP-3, ADAMTS-4 and HTRA1 were injected at a dose of 10μg/ml each. Phosphate buffered saline (PBS) was injected as a control. Discs were cultured for 8 days and loaded diurnally (day 1 to day 4 with 0.4 MPa for 16 h) and left under free swelling condition from day 4 to day 8 to avoid expected artifacts due to dehydration of the NP. Outcome parameters included disc height, metabolic cell activity, DNA content, glycosaminoglycan (GAG) content, total collagen content, relative gene expression and histological investigation.Results. The mean metabolic cell activity was significantly lower in the NP area of discs injected with ADAMTS-4 compared to the day 0 control discs. Disc height was decreased following injection with HTRA1, and was significantly correlated with changes in GAG/DNA of the NP tissue. Total collagen content tended to be lower in groups injected with ADAMTS4 and MMP-3.Conclusion. MMP-3, ADAMTS-4 and HTRA1 neither provoked visible matrix degradation nor major shifts in gene expression. However, cell activity was significantly reduced and HTRA1 induced loss of disc height which positively correlated with changes in GAG/DNA content. The use of higher doses of these enzymes or a combination thereof may therefore be necessary to induce disc degeneration

Human intervertebral disc stiffness correlates better with the Otsu threshold computed from axial T2 map of its posterior annulus fibrosus than with clinical classifications

Degeneration of the intervertebral disc, sometimes associated with low back pain and abnormal spinal motions, represents a major health issue with high costs. A non-invasive degeneration assessment via qualitative or quantitative MRI (magnetic resonance imaging) is possible, yet, no relation between mechanical properties and T2 maps of the intervertebral disc (IVD) has been considered, albeit T2 relaxation time values quantify the degree of degeneration. Therefore, MRI scans and mechanical tests were performed on 14 human lumbar intervertebral segments freed from posterior elements and all soft tissues excluding the IVD. Degeneration was evaluated in each specimen using morphological criteria, qualitative T2 weighted images and quantitative axial T2 map data and stiffness was calculated from the load-deflection curves of in vitro compression, torsion, lateral bending and flexion/extension tests. In addition to mean T2, the OTSU threshold of T2 (TOTSU), a robust and automatic histogram-based method that computes the optimal threshold maximizing the distinction of two classes of values, was calculated for anterior, posterior, left and right regions of each annulus fibrosus (AF). While mean T2 and degeneration schemes were not related to the IVDs' mechanical properties, TOTSU computed in the posterior AF correlated significantly with those classifications as well as with all stiffness values. TOTSU should therefore be included in future degeneration grading schemes.

Finite element analyses of human vertebral bodies embedded in polymethylmethalcrylate or loaded via the hyperelastic intervertebral disc models provide equivalent predictions of experimental strength

Quantitative computer tomography (QCT)-based finite element (FE) models of vertebral body provide better prediction of vertebral strength than dual energy X-ray absorptiometry. However, most models were validated against compression of vertebral bodies with endplates embedded in polymethylmethalcrylate (PMMA). Yet, loading being as important as bone density, the absence of intervertebral disc (IVD) affects the strength. Accordingly, the aim was to assess the strength predictions of the classic FE models (vertebral body embedded) against the in vitro and in silico strengths of vertebral bodies loaded via IVDs. High resolution peripheral QCT (HR-pQCT) were performed on 13 segments (T11/T12/L1). T11 and L1 were augmented with PMMA and the samples were tested under a 4° wedge compression until failure of T12. Specimen-specific model was generated for each T12 from the HR-pQCT data. Two FE sets were created: FE-PMMA refers to the classical vertebral body embedded model under axial compression; FE-IVD to their loading via hyperelastic IVD model under the wedge compression as conducted experimentally. Results showed that FE-PMMA models overestimated the experimental strength and their strength prediction was satisfactory considering the different experimental set-up. On the other hand, the FE-IVD models did not prove significantly better (Exp/FE-PMMA: R²=0.68; Exp/FE-IVD: R²=0.71, p=0.84). In conclusion, FE-PMMA correlates well with in vitro strength of human vertebral bodies loaded via real IVDs and FE-IVD with hyperelastic IVDs do not significantly improve this correlation. Therefore, it seems not worth adding the IVDs to vertebral body models until fully validated patient-specific IVD models become available.

Finite Element Based Nonlinear Normalization of Human Lumbar Intervertebral Disc Stiffness to Account for Its Morphology

Disc degeneration, usually associated with low back pain and changes of intervertebral stiffness, represents a major health issue. As the intervertebral disc (IVD) morphology influences its stiffness, the link between mechanical properties and degenerative grade is partially lost without an efficient normalization of the stiffness with respect to the morphology. Moreover, although the behavior of soft tissues is highly nonlinear, only linear normalization protocols have been defined so far for the disc stiffness. Thus, the aim of this work is to propose a nonlinear normalization based on finite elements (FE) simulations and evaluate its impact on the stiffness of human anatomical specimens of lumbar IVD. First, a parameter study involving simulations of biomechanical tests (compression, flexion/extension, bilateral torsion and bending) on 20 FE models of IVDs with various dimensions was carried out to evaluate the effect of the disc's geometry on its compliance and establish stiffness/morphology relations necessary to the nonlinear normalization. The computed stiffness was then normalized by height (H), cross-sectional area (CSA), polar moment of inertia (J) or moments of inertia (Ixx, Iyy) to quantify the effect of both linear and nonlinear normalizations. In the second part of the study, T1-weighted MRI images were acquired to determine H, CSA, J, Ixx and Iyy of 14 human lumbar IVDs. Based on the measured morphology and pre-established relation with stiffness, linear and nonlinear normalization routines were then applied to the compliance of the specimens for each quasi-static biomechanical test. The variability of the stiffness prior to and after normalization was assessed via coefficient of variation (CV). The FE study confirmed that larger and thinner IVDs were stiffer while the normalization strongly attenuated the effect of the disc geometry on its stiffness. Yet, notwithstanding the results of the FE study, the experimental stiffness showed consistently higher CV after normalization. Assuming that geometry and material properties affect the mechanical response, they can also compensate for one another. Therefore, the larger CV after normalization can be interpreted as a strong variability of the material properties, previously hidden by the geometry's own influence. In conclusion, a new normalization protocol for the intervertebral disc stiffness in compression, flexion, extension, bilateral torsion and bending was proposed, with the possible use of MRI and FE to acquire the discs' anatomy and determine the nonlinear relations between stiffness and morphology. Such protocol may be useful to relate the disc's mechanical properties to its degree of degeneration.

Immunohistochemical demonstration of lumbar intervertebral disc innervation in the dog.

Low back pain is a common ailment in dogs, particularly in specific breeds such as the German shepherd dog. A number of structures such as facet joint capsules, ligaments, dorsal root ganglia, periosteum, vertebral endplates and meninges have been associated with this condition. Yet, in spite of all diagnostic efforts, the origin of pain remains obscure in a substantial proportion of all cases. A further structure often being involved in vertebral column disorders is the intervertebral disc. The presence of nerves, however, is a precondition for pain sensation and, consequently, structures lacking innervation can be left out of consideration as a cause for low back pain. Nerve fibres have been demonstrated at the periphery of the intervertebral disc in man, rabbit and rat. With regard to the dog, however, the extent of intervertebral disc innervation is still being disputed. The goal of the present study, therefore, was to substantiate and expand current knowledge of intervertebral disc innervation. Protein gene product (PGP) 9.5 was used for immunohistochemical examination of serial transversal and sagittal paraffin sections of lumbar discs from adult dogs. This general marker revealed nerve fibres to be confined to the periphery of the intervertebral discs. These results indicate that even limited pathological processes affecting the outer layers of the intervertebral disc are prone to cause low back pain.