Mielografia lombar no diagnóstico de extrusão do disco intervertebral toracolombar em cães: estudo prospectivo

Thoracolumbar disk extrusion is the most common cause of extradural compression of the spinal cord in dogs. Myelography is one of the most commonly performed techniques for the diagnosis of this affection. This study aimed to evaluate the applicability and effectiveness of lumbar myelography in the diagnosis of thoracolumbar intervertebral disk extrusion in dogs, as well as its major complications. Twenty dogs were used in this study. Animals were included when neurological examination suggested thoracolumbar spine lesion, myelography was used as a complementary diagnostic method, and diagnosis of disk extrusion was surgically confirmed. The accuracy of the exam to predict location and lateralization of extruded disk material were evaluated, as well as complications associated to the procedure. Lumbar myelography exhibited 95% and 60% accuracy for location and lateralization of the lesion, respectively, with minimal complications.

Intervertebral disc regeneration or repair with biomaterials and stem cell therapy--feasible or fiction?

The "gold standard" for treatment of intervertebral disc herniations and degenerated discs is still spinal fusion, corresponding to the saying "no disc - no pain". Mechanical prostheses, which are currently implanted, do only have medium outcome success and have relatively high re-operation rates. Here, we discuss some of the biological intervertebral disc replacement approaches, which can be subdivided into at least two classes in accordance to the two different tissue types, the nucleus pulposus (NP) and the annulus fibrosus (AF). On the side of NP replacement hydrogels have been extensively tested in vitro and in vivo. However, these gels are usually a trade-off between cell biocompatibility and load-bearing capacity, hydrogels which fulfill both are still lacking. On the side of AF repair much less is known and the question of the anchoring of implants is still to be addressed. New hope for cell therapy comes from developmental biology investigations on the existence of intervertebral disc progenitor cells, which would be an ideal cell source for cell therapy. Also notochordal cells (remnants of the embryonic notochord) have been recently pushed back into focus since these cells have regenerative potential and can activate disc cells. Growth factor treatment and molecular therapies could be less problematic. The biological solutions for NP and AF replacement are still more fiction than fact. However, tissue engineering just scratched the tip of the iceberg, more satisfying solutions are yet to be added to the biomedical pipeline.

Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system

STUDY DESIGN: Ex vivo in vitro study evaluating a novel intervertebral disc/endplate culture system. OBJECTIVES: To establish a whole-organ intervertebral disc culture model for the study of disc degeneration in vitro, including the characterization of basic cell and organ function. SUMMARY OF BACKGROUND DATA: With current in vivo models for the study of disc and endplate degeneration, it remains difficult to investigate the complex disc metabolism and signaling cascades. In contrast, more controlled but simplified in vitro systems using isolated cells or disc fragments are difficult to culture due to the unconstrained conditions, with often-observed cell death or cell dedifferentiation. Therefore, there is a demand for a controlled culture model with preserved cell function that offers the possibility to investigate disc and endplate pathologies in a structurally intact organ. METHODS: Naturally constrained intervertebral disc/endplate units from rabbits were cultured in multi-well plates. Cell viability, metabolic activity, matrix composition, and matrix gene expression profile were monitored using the Live/Dead cell viability test (Invitrogen, Basel, Switzerland), tetrazolium salt reduction (WST-8), proteoglycan and deoxyribonucleic acid quantification assays, and quantitative polymerase chain reaction. RESULTS: Viability and organ integrity were preserved for at least 4 weeks, while proteoglycan and deoxyribonucleic acid content decreased slightly, and matrix genes exhibited a degenerative profile with up-regulation of type I collagen and suppression of collagen type II and aggrecan genes. Additionally, cell metabolic activity was reduced to one third of the initial value. CONCLUSIONS: Naturally constrained intervertebral rabbit discs could be cultured for several weeks without losing cell viability. Structural integrity and matrix composition were retained. However, the organ responded to the artificial environment with a degenerative gene expression pattern and decreased metabolic rate. Therefore, the described system serves as a promising in vitro model to study disc degeneration in a whole organ.

Effects of anatomic conformation on three-dimensional motion of the caudal lumbar and lumbosacral portions of the vertebral column of dogs

OBJECTIVE: To determine the association between the 3-dimensional (3-D) motion pattern of the caudal lumbar and lumbosacral portions of the canine vertebral column and the morphology of vertebrae, facet joints, and intervertebral disks. SAMPLE POPULATION: Vertebral columns of 9 German Shepherd Dogs and 16 dogs of other breeds with similar body weights and body conditions. PROCEDURE: Different morphometric parameters of the vertebral column were assessed by computed tomography (CT) and magnetic resonance imaging. Anatomic conformation and the 3-D motion pattern were compared, and correlation coefficients were calculated. RESULTS: Total range of motion for flexion and extension was mainly associated with the facet joint angle, the facet joint angle difference between levels of the vertebral column in the transverse plane on CT images, disk height, and lever arm length. CONCLUSIONS AND CLINICAL RELEVANCE: Motion is a complex process that is influenced by the entire 3-D conformation of the lumbar portion of the vertebral column. In vivo dynamic measurements of the 3-D motion pattern of the lumbar and lumbosacral portions of the vertebral column will be necessary to further assess biomechanics that could lead to disk degeneration in dogs.

Influence of surgical approach on the efficacy of the intervertebral disk fenestration: a cadaveric study

OBJECTIVES: To investigate the influence of different approach angles on the amount of nucleus pulposus removed during intervertebral disc fenestration in dogs. METHODS: Twenty cadavers of beagle dogs were randomly divided into four groups: a control group and three treatment groups in which intervertebral fenestration was performed using either a dorsal, dorsolateral or lateral approach between the 12th thoracic and second lumbar spaces. The volume of nucleus pulposus, the weight of the residual nucleus pulposus and the angle of the working sector were measured. The ratio of the residual nucleus pulposus weight to the nucleus pulposus volume was used to evaluate the efficacy of the performed fenestration. Data were analysed with Kruskal-Wallis analysis of variance between groups on ranks with correction for ties and Bonferroni correction for multiple comparisons. Correlation between ratio and working angle was calculated using a Spearman's rank test (P<0.05). RESULTS: The calculated ratio of nuclear weight to volume was significantly less in the lateral approach group than that in the other groups. The working sector was widest in the dorsolateral approach group, but this did not correlate with efficient fenestration. CLINICAL SIGNIFICANCE: Using the lateral approach for intervertebral disc fenestration may increase the efficiency of the fenestration procedure.

Lumbar spinal 'juxtaarticular' cyst in a Gordon setter

An 11-year-old Gordon setter bitch was presented with a history of progressive weakness in the right hind limb associated with pain in the lumbar spine. Neurological deficits consisted of ataxia, monoparesis, muscle atrophy and spontaneous over-knuckling of the affected limb. A large 'juxtaarticular' cyst located in a right dorsolateral position of the intervertebral foramen at L3-L4 was diagnosed by magnetic resonance imaging. The cyst was removed through a modified laminectomy. The dog recovered quickly and returned to the owners 4 days after surgery with slight neurological symptoms. During the follow-up examination 2 and 6 months later, the Setter showed normal gait and neurological examination.

Little-known Swiss contributions to the description, diagnosis, and surgery of lumbar disc disease before the Mixter and Barr era

The understanding of lumbar spine pathologies made substantial progress at the turn of the twentieth century. The authors review the original publication of Otto Veraguth in 1929 reporting on the successful resection of a herniated lumbar disc, published exclusively in the German language. His early report is put into the historical context, and its impact on the understanding of pathologies of the intervertebral disc (IVD) is estimated. The Swiss surgeon and Nobel Prize laureate Emil Theodor Kocher was among the first physicians to describe the traumatic rupture of the IVD in 1896. As early as 1909 Oppenheim and Krause published 2 case reports on surgery for a herniated lumbar disc. Goldthwait was the first physician to delineate the etiopathogenes is between annulus rupture, symptoms of sciatica, and neurological signs in his publication of 1911. Further publications by Middleton and Teacher in 1911 and Schmorl in 1929 added to the understanding of lumbar spinal pathologies. In 1929, the Swiss neurologist Veraguth (surgery performed by Hans Brun) and the American neurosurgeon Walter Edward Dandy both published their early experiences with the surgical therapy of a herniated lumbar disc. Veraguth's contribution, however, has not been appreciated internationally to date. The causal relationship between lumbar disc pathology and sciatica remained uncertain for some years to come. The causal relationship was not confirmed until Mixter and Barr's landmark paper in 1934 describing the association of sciatica and lumbar disc herniation, after which the surgical treatment became increasingly popular. Veraguth was among the first physicians to report on the clinical course of a patient with successful resection of a herniated lumbar disc. His observations should be acknowledged in view of the limited experience and literature on this ailment at that time.

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.

Sensory innervation of the dorsal longitudinal ligament and the meninges in the lumbar spine of the dog

Although intervertebral disc herniation is a well-known disease in dogs, pain management for this condition has remained a challenge. The goal of the present study is to address the lack of information regarding the innervation of anatomical structures within the canine vertebral canal. Immunolabeling was performed with antibodies against protein gene product 9.5, Tuj-1 (neuron-specific class III β-tubulin), calcitonin gene-related peptide, and neuropeptide Y in combination with the lectin from Lycopersicon esculentum as a marker for blood vessels. Staining was indicative of both sensory and sympathetic fibers. Innervation density was the highest in lateral areas, intermediate in dorsal areas, and the lowest in ventral areas. In the dorsal longitudinal ligament (DLL), the highest innervation density was observed in the lateral regions. Innervation was lower at mid-vertebral levels than at intervertebral levels. The presence of sensory and sympathetic fibers in the canine dura and DLL suggests that pain may originate from both these structures. Due to these regional differences in sensory innervation patterns, trauma to intervertebral DLL and lateral dura is expected to be particularly painful. The results ought to provide a better basis for the assessment of medicinal and surgical procedures.

Extended duration of torsional loading reduced the survival of the NP cells of the intervertebral disc

Introduction Previous studies on the influence of torsion and combined torsion-compression loading revealed a positive effect on the cell viability when a repetitive short-term torsion was applied at a physiological magnitude to intervertebral disc organ culture.1 However, after an extended period (8 hours) of combined torsion-compression loading, substantial cell death was detected in the nucleus pulposus (NP).2 In this follow-up study, we aimed to investigate the relationship, if any, between the duration of torsion applied to the intervertebral disc (IVD) and the level of NP cell viability. Materials and Methods Bovine caudal discs were harvested and cultured in a custom-built multiaxis dynamic loading bioreactor.2 Torsion (± 2 degrees) was applied to the samples at a frequency of 0.2 Hz. Torsion was applied for durations of 0, 1, 4, and 8 h/d, repeated over 7 days. After the last day of loading, disc tissue was dissected for analysis of cell viability and gene expression. Results Disc NP cell viability remained above 85% after torsional loading for 0, 1, or 4 h/d. Viability was statistical significantly reduced to below 70% when torsion was applied for 8 h/d (p = 0.03) (Table 1). The daily duration of torsional loading did not affect the AF cell viability (> 80% for all loading durations). The trend of collagen 2 gene upregulation and matrix metalloproteases 13 downregulation with an increasing duration of torsion was observed in both NP and AF (Fig. 1).Conclusion We have demonstrated that an extended duration of torsion could inhibit the survival of NP cells within the IVD in organ culture. Acknowledgments Funds from the Orthopedic Department of the Insel University Hospital of Bern and a private donation from Prof. Dr. Paul Heini, Spine Surgeon, Sonnenhof Clinic Bern were received to support this work. Disclosure of Interest None declared References References 1 Chan SC, Ferguson SJ, Wuertz K, Gantenbein-Ritter B. Biological response of the intervertebral disc to repetitive short-term cyclic torsion. Spine 2011;36(24):2021–2030 2 Chan SC, Walser J, Käppeli P, Shamsollahi MJ, Ferguson SJ, Gantenbein-Ritter B. Region specific response of intervertebral disc cells to complex dynamic loading: an organ culture study using a dynamic torsion-compression bioreactor. PLoS ONE 2013;8(8):e72489

Cell therapy for intervertebral disc repair: advancing cell therapy from bench to clinics

Intervertebral disc (IVD) degeneration is a major cause of pain and disability; yet therapeutic options are limited and treatment often remains unsatisfactory. In recent years, research activities have intensified in tissue engineering and regenerative medicine, and pre-clinical studies have demonstrated encouraging results. Nonetheless, the translation of new biological therapies into clinical practice faces substantial barriers. During the symposium "Where Science meets Clinics", sponsored by the AO Foundation and held in Davos, Switzerland, from September 5-7, 2013, hurdles for translation were outlined, and ways to overcome them were discussed. With respect to cell therapy for IVD repair, it is obvious that regenerative treatment is indicated at early stages of disc degeneration, before structural changes have occurred. It is envisaged that in the near future, screening techniques and non-invasive imaging methods will be available to detect early degenerative changes. The promises of cell therapy include a sustained effect on matrix synthesis, inflammation control, and prevention of angio- and neuro-genesis. Discogenic pain, originating from "black discs" or annular injury, prevention of adjacent segment disease, and prevention of post-discectomy syndrome were identified as prospective indications for cell therapy. Before such therapy can safely and effectively be introduced into clinics, the identification of the patient population and proper standardisation of diagnostic parameters and outcome measurements are indispensable. Furthermore, open questions regarding the optimal cell type and delivery method need to be resolved in order to overcome the safety concerns implied with certain procedures. Finally, appropriate large animal models and well-designed clinical studies will be required, particularly addressing safety aspects.

Organ Culture Bioreactors - Platforms to Study Human Intervertebral Disc Degeneration and Regenerative Therapy.

In recent decades the application of bioreactors has revolutionized the concept of culturing tissues and organs that require mechanical loading. In intervertebral disc (IVD) research, collaborative efforts of biomedical engineering, biology and mechatronics have led to the innovation of new loading devices that can maintain viable IVD organ explants from large animals and human cadavers in precisely defined nutritional and mechanical environments over extended culture periods. Particularly in spine and IVD research, these organ culture models offer appealing alternatives, as large bipedal animal models with naturally occurring IVD degeneration and a genetic background similar to the human condition do not exist. Latest research has demonstrated important concepts including the potential of homing of mesenchymal stem cells to nutritionally or mechanically stressed IVDs, and the regenerative potential of "smart" biomaterials for nucleus pulposus or annulus fibrosus repair. In this review, we summarize the current knowledge about cell therapy, injection of cytokines and short peptides to rescue the degenerating IVD. We further stress that most bioreactor systems simplify the real in vivo conditions providing a useful proof of concept. Limitations are that certain aspects of the immune host response and pain assessments cannot be addressed with ex vivo systems. Coccygeal animal disc models are commonly used because of their availability and similarity to human IVDs. Although in vitro loading environments are not identical to the human in vivo situation, 3D ex vivo organ culture models of large animal coccygeal and human lumbar IVDs should be seen as valid alternatives for screening and feasibility testing to augment existing small animal, large animal, and human clinical trial experiments.

NonViral Gene Delivery of Growth and Differentiation Factor 6 (GDF6) to whole bovine Intervertebral Disc

Question: Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and de- generation. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM) [1]. Further studies showed that growth factors from the transforming growth factor (TGF) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC) [2]. Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods: Bovine IVD cells were isolated by pronase/collagenase II overnight digestion. After monolayer expansion up to passage 3, cells were transfected with the plasmid pGDF6 (RG211366, Origene, SF) or with green fluorescence protein (GFP) control using the NeonÒ transfection system (Invitrogen, Basel), both equipped with a Cy- tomegalovirus (CMV) promotor to induce over-expression. We tested a range of yet unpublished parameters for each of the primary disc cells to optimize efficiency. To test a non-viral gene therapy applied directly to 3D whole organ culture, bovine IVDs were harvested from fresh tails obtained from the abattoir within 5 h post-mortem [3]. Discs were then pre-incubated for 24 h in high glucose Dulbecco’s Modified Eagle Medium and 5 % fetal calf serum. Each disc was transfected by injection of 5 lg of plasmid GDF6 (Origene, RG211366) into the center by 25G needle and using Hamilton sy- ringe. Electroporation was performed using 2-needle array electrode or tweezertrodes; 8 pulses at 200mv/cm with an interval of 10 ms were applied using ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) (Fig. 1). After transfection discs were cultured for 72 h to allow expression of GFP or GDF6. Discs were then fixed, cryosectioned and analysed by immunofluorescence against GDF6. Results: We successfully transfected bovine NP and AF cells in monolayer culture with the two plasmids using a 1,400 V, 20 ms and 2 pulses with a *25 % efficiency using 0.15 M cells and 3 lg DNA (Fig. 1). Organ IVD culture transfection revealed GFP6 positive staining in the centre of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GFP posi- tive cells. Conclusions: We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgments: This study was supported by the Lindenhof Foundation ‘‘Forschung und Lehre’’ (Project no. 13-02-F). References 1. Roughly PJ (2004) Spine (Phila) 29:2691–2699 2. 3. Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014) Arthritis Res Ther 16:R67 Chan SC, Gantenbein-Ritter B (2012) J Vis Exp 60(60):e3490