Abstract Background: The process that involves the migration of the nucleus pulposus from the innermost annular layers and culminates with the final extrusion of the nucleus has been limited to a few studies. This investigation was directed towards a better understanding of the herniation process. The architecture of the annulus fibrosus and the mechanism of progressive disc herniation were analyzed, using a controlled porcine model. Microscopic and histochemical techniques were employed. <br /><br /> Methodology: Two studies were performed. In the first stage, the macroscopic and microscopic structures of twelve cervical intervertebral discs were compared with young human disc data from studies reported in the literature. Important structural features were studied such as annulus fibrosus thickness, number of lamellae, lamellae thickness, orientation of the lamellae fibers and blood supply. In the second study, sixteen fresh-frozen functional spine units were submitted to repetitive flexion?extension motions combined with a low compressive load in a servo-hydraulic dynamic testing system. Discograms, dissections and histochemical techniques were applied to characterize the cumulative damage. The experiment produced eight complete herniations, four partial herniations and four specimens without any microscopic detectable annular damage. <br /><br /> Results and Discussion: The structure of the cervical porcine disc resembles the lumbar human disc. Some differences are evident. The size of the annulus is smaller, the thickness of the lamellae is narrower and the number of layers is fewer in the pig. It is hypothesized that the flexion-extension motion combined with a low-level load produced an increased hydraulic pressure in the inner wall of the posterior annulus. This pressure and repetitive motion first produced a small cleft, spreading the collagen bundles inside the first layer. The nuclear material was "pumped" through the small cleft to the first layer filling the layer creating a fluid-filled pocket between the collagen fibers. Once the "pocket" acquired enough pressure a new cleft was produced in the weakest part of the layer allowing the nuclear material to create a new "pocket" in the second layer. This was the first stage of damage and disc herniation production. This mechanism was repeated until the nucleus traveled along the annulus reaching the posterior longitudinal ligament. At this point a complete extrusion herniation was produced. <br /><br /> Conclusion: The porcine model appears to be suitable as a model to understand the mechanism of disc herniation when the spine is subjected to flexion-extension motions combined with a low-level load. The first cumulative injury appears to be a cleft between the lamellae bundles produced by the nuclear hydraulic pressure. A cumulative load/cumulative injury model approach was used to create the damage that was quantified in the study.

This study investigates the changes due to crosslinking treatment in stiffness, permeability, and glycosaminoglycan (GAG) content of bovine intervertebral discs. The objective of this study was to determine the mechanical and biochemical effects of crosslinking treatment on lumbar bovine tissue. Previous studies have found that crosslinking can increase stiffness and permeability in the intervertebral disc. These changes have not yet been investigated by confined compression, stress-relaxation tests of young bovine tissue. Eleven lumbar motion segments were harvested from calf spines and soaked in a saline solution or one of four crosslinking treatments (genipin, methylglyoxal, proanthrocyanidin, and EDC). Five mm diameter samples were removed from the midannulus region at anterior / anterior-lateral locations, confined in a saline bath, swelled to equilibrium, and tested in confined compression stress-relaxation to 15% strain in 5% increments. Radial samples were also harvested, treated with saline solution and EDC, and tested in the same manner. The aggregate modulus and hydraulic permeability were calculated using the nonlinear biphasic theory. Swelling pressure was calculated as the load at swelling equilibrium. GAG content was measured using the dimethylmethylene blue assay. Differences with P value < 0.05 were considered significant. In the axial orientation, all crosslinking treatments except methyglyoxal at least doubled the aggregate modulus relative to soaked controls (P less than 0.05). Genipin treatment resulted in 78% lower axial permeability, proanthrocyanidin (PA) 50% lower, and EDC treatment 84% lower relative to soaked controls (P &lt; 0.05). GAG content measured in the methyglyoxal treatment group was 25% lower than in soaked control group. Genipin (G), proanthrocyanidin (PA), and EDC treatment increased the swelling pressure by at least 65% (P less than 0.05). In the radial orientation, EDC treatment increased the stiffness by 75%, and did not significantly affect the permeability or swelling pressure. Some crosslinking treatments proved effective in increasing the stiffness and swelling pressure of the disc. The increased swelling pressure in G, PA, and EDC treatment groups relative to soaked controls suggests reduced GAG leaching during soaking treatment, further confirmed by the reduction in permeability in these groups.

Σκοπός: Ο νεοεννευρωμένος μεσοσπονδύλιος δίσκος (ΜΔ), κλινικά θεωρείται μια σημαντική πηγή πόνου στους ασθενείς με οσφυαλγία. Η ταυτοποίηση των νεοσχηματιζόμενων νεύρων, που εκφράζουν τους υποδοχείς των νευροτροφινών, στην εκφύλιση του ΜΔ, σπονδυλικών επιπέδων που αναφέρονται ως πηγή δισκογενή πόνου και η συσχέτισή τους με το ενδοκανναβινοειδές σύστημα αναλγησίας, αποτελεί το αντικείμενο αυτής της μελέτης. Υλικό και μέθοδος: Συλλέχθηκαν 50 ανθρώπινοι ΜΔ ή τμήματα αυτών, μετά από δισκεκτομή σε χειρουργεία κήλης και εκφυλιστικών παθήσεων της Οσφυϊκής Μοίρας της Σπονδυλικής Στήλης (ΟΜΣΣ). Εφαρμόσθηκε ανοσοϊστοχημεία για την ανίχνευση νευρικών ινών, στους ΜΔ, αφού διαπιστώθηκε η εκφύλισή τους. Συσχετίσθηκε η ανάπτυξη νευρικών ινών των εκφυλισμένων ΜΔ με την έκφραση των υψηλής και χαμηλής συγγένειας υποδοχέων των νευροτροφινών, TrkA, TrkB, TrkC και p75, αντίστοιχα. Στα περιστατικά με ανάπτυξη νευρικών ινών ανιχνεύθηκε ανοσοϊστοχημικά η έκφραση του υποδοχέα CB1 των ενδοκανναβινοειδών. Αποτελέσματα: Η εκτεταμένη νεύρωση στον ΜΔ που φέρεται στον έσω ινώδη δακτύλιο (ΙΔ) και μπορεί να φτάνει μέχρι τον πηκτοειδή πυρήνα (ΠΠ), φαίνεται να είναι εξαρτώμενη από τους υποδοχείς των νευροτροφινών TrkA, TrkB, TrkC και p75. Μικρά αιμοφόρα αγγεία συνοδεύουν τις νευρικές ίνες στα σπονδυλικά επίπεδα του ΜΔ που δίνουν δισκογενή πόνο και εκφράζουν επίσης τους υποδοχείς των νευροτροφινών. Ο CB1 υποδοχέας των ενδοκανναβινοειδών ταυτοποιήθηκε σε όλα τα περιστατικά που εμφάνιζαν ανάπτυξη νευρικών ινών. Βρέθηκε να εκφράζεται στα αγγεία που συνοδεύουν τις νευρικές ίνες και σε μερικά νεύρα. Δεν ταυτοποιήθηκε ανάπτυξη νευρικών ινών σε εκφυλισμένους ΜΔ σπονδυλικών επιπέδων που δεν αποτελούσαν αιτία δισκογενούς οσφυαλγίας. Συμπέρασμα: Η αλγοϊδιοδεκτική ανάπτυξη νευρικών ινών στον ΜΔ συνδέεται με την έκφραση των υποδοχέων των νευροτροφινών στις νεοσχηματιζόμενες νευρικές ίνες, δείχνοντας ότι όχι μόνο ο NGF, αλλά και άλλες νευροτροφίνες εμπλέκονται στην ανάπτυξη νευρικών ινών. Η έκφραση του CB1 υποδοχέα των ενδοκανναβινοειδών αποτελεί πεδίο έρευνας για τη θεραπεία του δισκογενή πόνου. / The current study was designed in order to detect immunohistochemically the expression of cannabinoid CB1 receptor and high and low affinity neurotrophin receptors TrkA, TrkB, TrkC and p75 respectively in human painful intervertebral disc (IVD). Previous data have shown an association between nerve ingrowth in human degenerated IVD and patient’s experience of pain. The nociceptive nerve ingrowth into painful IVD is causally linked with Nerve Growth Factor (NGF) production by blood vessels growing into the IVD. NGF causes further innervation and is involved in processes that result in an inflammatory hyperalgesia. There is evidence that cannabinoids show a neuronal CB1 receptor-mediated antihyperalgesic action and a separate inhibition of proinflammatory neuroimmune processes. A total of 50 IVD specimens after lumbar discectomy were included in the present study. The degeneration of IVD was identified and connected with clinical background of patients. An immunoperoxidase method on formalin-fixed, paraffin-embedded tissue sections and specific antibodies against CB1 and neurotrophin receptors (TrkA, TrkB, TrkC and p75) were used. Immunohistochemistry was also performed to detect neural and vascular markers: tubulin (βIII) and CD31. Neurotrophin receptors (TrkA, TrkB, TrkC and p75) expression was detected only in innervated degenerated IVD. Blood microvessels accompanied nerve fibers growing into IVD expressed also neurotrophin receptors. Furthermore, in some nerve fibers CB1 cannabinoid receptor expression was identified. Interestingly, CB1 receptor expression was detected in the majority of vessels accompanying nerve fibers. Nociceptive nerve ingrowth into degenerated IVD was strongly associated with all neurotrophin receptors expression indicating that not only NGF but also and others neurotrophins are involved in nerve ingrowth. Expression of CB1 cannabinoid receptor in innervated IVD suggests a possible therapeutic site of analgesic action. Additional studies would be necessary to clarify the functional role of CB1 cannabinoid receptor in degenerated intervertebral disc.

Orientador: José Elias Tomazini / Banca: Mauro Hugo Mathias / Banca: Luiz Carlos de Queiroz / Resumo: Para melhor entender os mecanismos degenerativos da coluna vertebral e avaliar o melhor método para seu tratamento é necessário que se conheça o comportamento dos diversos componentes das articulações intervertebrais. Com isso, torna-se essencial o desenvolvimento de dispositivos capazes de simular as condições fisiológicas de movimentos e cargas a fim de realizar testes in vitro que fornecerão dados para posteriormente serem testados in vivo. O objetivo deste trabalho é o desenvolvimento de dispositivos para a realização de testes in vitro com coluna vertebral. Estes dispositivos constam de uma máquina capaz de simular o movimento de flexão-extensão, pré-cargas axiais e a angulação pélvica, e uma lâmina de extensometria para captar as deformações dos tecidos testados. Foram realizados experimentos com discos invertebrais suínos a fim de avaliar a eficiência dos dispositivos. Realizaram-se dois experimentos usando os dispositivos desenvolvidos e um experimento na máquina universal de ensaios. Como resultado obteve-se valores de propriedades mecânicas coerentes com a literatura e o disco intervertebral comportando-se como um material viscoelástico. Outro ponto importante foi a obtenção da pressão intradiscal aproximada relacionando-a com o deslocamento angular da coluna. Conclui-se que os dispositivos apresentaram funcionamento satisfatório, abrindo perspectivas para outros estudos. / Abstract: The best way to understand the degenerative mechanisms of the vertebral column and to evaluate appropriated methods for its treatment it is necessary to know the behavior of the diverse components of the intervertebral joints. So, it becomes essential to simulate the physiological conditions of movements and loads in order to carry through test in vitro that they will supply datas to be tested in vivo. The objective of this study is the development of devices of low cont for the accomplishment of tests in vitro with spine. These devices consist of a machine capable to simulate the movement of flexion-extension, daily pay-loads and the pelvic inclination, and an extensometry blade to catch the deformations of tested structures. POrcine had been carried through experiments with intervertebral discs in order to evaluate the efficiency od the devices. Two experiments had been become fullfilled using the developed devices and an experiment in the universal test machine. As result we got values of coherent mechanical properties with literature and the intervertebral disc behaving as a viscoelastic material. Another important point was the attainment of the intradiscal pressure approached relating it with the angular displacement of the spine. It is concluded that the devices had presented satisfactory functioning, opening perspective for other studies. / Mestre

Resumo: A desidratação do núcleo pulposo que, juntamente com o anulus fibroso, compõe o disco intervertebral, participa do processo de degeneração da coluna vertebral. Torna-se importante entender como este processo de desidratação e a consequente alteração das propriedades mecânicas influenciam na biomecânica dessa articulação. O principal objetivo deste estudo é analisar a influência da desidratação no coeficiente de amortecimento viscoso e como esta variação pode alterar o comportamento mecânico do disco intervertebral lombar. Este estudo é composto de duas etapas: na primeira, o coeficiente de amortecimento viscoso foi obtido experimentalmente de unidades funcionais de colunas lombares suínas antes e após o processo de desidratação das mesmas; na segunda, foi simulado em modelo computacional a resposta harmônica, utilizando-se dados da literatura e do coeficiente de amortecimento viscoso, analisando as diferenças entre uma simulação com o disco intervertebral hidratado e desidratado. O coeficiente de amortecimento viscoso hidratado (4,7) e desidratado (2,7) apresentou diferença estatisticamente significante (p<0,001). Na simulação computacional podese evidenciar uma menor influência da variação do coeficiente de amortecimento viscoso quando analisado de forma isolada do que quando analisado em conjunto com variações nos dados referentes à rigidez, principalmente nas frequências de ressonância e nas amplitudes de pressão intradiscal e deformação do disco intervertebral. Concluiu-se que discos intervertebrais desidratados respondem de forma diferente à vibração, o que pode contribuir para os processos lesivos da coluna vertebral / Abstract: The dehydration of the nucleus pulposus which, with the anulus fibrosus, composes the intervertebral disc, participates in the process of degeneration of the spine. It is important to understand how this process and the consequent alteration of dehydration of the mechanical properties influence the biomechanics of this joint. The aim of this study is to analyze the influence of dehydration in the viscous damping coefficient and how this variation can change the mechanical behavior of lumbar intervertebral disc. This study consists of two stages: first, the viscous damping coefficient was obtained experimentally in functional units of porcine lumbar spine before and after the dehydration process of them; second, an harmonic response was simulated in a computer model, using literature data and the viscous damping coefficient, analyzing the differences between a simulation with the hydrated and dehydrated intervertebral disc. The hydrated (4.7) and dehydrated (2.7) viscous damping coefficient showed a statistically significant difference (p <0.001). In the computer simulation, a lower influence of variation of the viscous damping coefficient can be detected when analyzed separately than when analyzed together with variation on stiffness data, especially in the resonance frequencies and in the amplitudes of the intradiscal pressure and deformation of the intervertebral disc. We conclude that dehydrated intervertebral discs respond differently to vibration, what can contribute to the damaging processes of the spine / Orientador: José Elias Tomazini / Coorientador: Mauro Hugo Mathias / Banca: Fernando de Azevedo Silva / Banca: José Geraldo Trani Brandão / Banca: Valdeci Donizete Gonçalves / Banca: Magda Francisca Gonçalves Rocha / Doutor

Abstract Magnetic resonance imaging (MRI) provides important information about structural and biochemical changes in organs. MRI is also an effective imaging method for the evaluation of spinal disorders. However, many of its potential applications - particularly diffusion imaging - have not yet been thoroughly explored. The purpose of this study was to determine the MRI-detectable changes in the intervertebral disc after trauma and to test the feasibility of diffusion-weighted MR imaging of the intervertebral discs. A minipig model was used in the experimental study to determine the MRI changes in the intervertebral disc after peripheral annular lesions in different time frames. Three of eight discs with experimental annular lesions had a normal annular appearance in MRI. Annular lesions, when detectable, were manifested as a bulging of the disc or as a high-intensity zone (HIZ) inside the annulus. Either the signal intensity or the area of bright signal intensity in the nucleus had nearly always decreased after one month, but they were still detectable even in cases where no signs of annular trauma could be seen in the MR images. The histology of HIZ is presented for the first time: clusters of nuclear cells and disorganized granulation tissue with capillaries were detected in the HIZ area. Fourteen patients 8 to 21 years of age with histories of vertebral fracture at least one year previously and 14 asymptomatic healthy control subjects 8 to 22 years of age were studied by MRI. In these young people a vertebral fracture, especially with end-plate injury, proved to be a notable risk factor for initiating disc degeneration. The apparent diffusion coefficients (ADCs) of the thoracolumbar intervertebral discs were determined in three orthogonal directions in 18 healthy young volunteers aged 8-22 years. The ADCs were also determined in 10 young patients with previous vertebral fractures, and clear decreases were found in the ADCx and ADCy directions, but in the ADCz direction values had not changed significantly as compared to the values in the controls. The most marked changes were observed in the degenerated discs, followed by those in the discs with a normal signal intensity adjacent to the primary trauma area. Diffusion-weighted MR imaging affords a useful tool for evaluating disc diseases in the early phases. Additionally, 37 adult volunteers without back symptoms were studied by MRI and by magnetic resonance angiography (MRA) and it was found that the status of the lumbar arteries significantly explained the diffusion values in the lumbar intervertebral discs. The correlation between disc degeneration and diffusion was mostly linear, but not significant.

As alterações de sinal no osso subcondral dos corpos vertebrais foram descritas primeiramente por Modic, correlacionando o espectro das alterações da medula óssea no corpo vertebral relacionadas a discopatias degenerativas. O Modic tipo I representa a alteração de sinal do tipo edema, já o Modic II representa a alteração de sinal com padrão de lipossubstituição e o Modic III representa alteração de sinal relacionada a esclerose óssea. O objetivo primário deste estudo foi avaliar se há alteração na detecção dos padrões da classificação Modic na coluna lombar comparando a técnica Dixon com o protocolo tradicional. Como objetivo secundário avaliamos a correlação intra-observador e interobservador. O estudo foi realizado de forma retrospectiva com a inclusão de pacientes cujas ressonâncias magnéticas (RM) foram realizadas na Central de Diagnóstico Ribeirão Preto (CEDIRP). Dois médicos radiologistas de forma independente classificaram as alterações do tipo Modic permitindo uma avaliação interobservador e um dos radiologistas realizou uma segunda análise dos exames possibilitando uma avaliação intra-observador. Os resultados evidenciaram que a técnica Dixon apresentou superioridade na detecção do Modic I em todas as análises e também apresentou médias mais altas da espessura das alterações no osso subcondral. Houve correlações intra-observador moderadas e substanciais no protocolo tradicional e substanciais a altas com a técnica Dixon. A correlação interobservador demonstrou uma correlação moderada na avaliação deste tipo de alteração nos platôs superior e inferior de L3 e correlação inversa e não significativa para a o platô superior de L5 utilizando o protocolo tradicional. Já a correlação interobservador com a técnica Dixon evidenciou concordância perfeita entre os observadores na avaliação de tipo de alteração de sinal no osso subcondral do platô inferior de L1, correlação inversa para o platô inferior de L3 e superior de L4, correlação substancial entre o platô inferior de L2, platô inferior de L5 e superior de L5. Concluímos que a técnica Dixon apresentou superioridade na detecção do Modic I em relação ao protocolo tradicional, assim como permitiu detectar médias mais altas da espessura das alterações no osso subcondral. Também conclui-se que houve boa reprodutibilidade intra-observador e correlação interobservador variável entre os platôs vertebrais analisados / Signal changes in the subchondral bone of the vertebral bodies were first described by Modic, correlating the spectrum of bone marrow changes in the vertebral body to degenerative disc diseases. Modic type I represents the change of edema type signal, while Modic II represents the change of signal with liposubstitution pattern and Modic III represents signal change related to bone sclerosis. The main objective of this study was to evaluate if there is difference in the detection of Modic classification in the lumbar spine comparing the Dixon technique with the traditional protocol. We evaluated the intraobserver and interobserver correlation as a secondary objective. The study was performed retrospectively, including patients whose magnetic resonance imaging (MRI) was performed at the Ribeirão Preto Diagnostic Center (CEDIRP). Two radiologist physicians independently classified the Modic type endplate changes allowing an interobserver evaluation and one of the radiologists performed a second analysis of the exams making possible an intraobserver evaluation. The results showed that the Dixon technique presented superiority in the detection of Modic I in all the analyzes and also presented higher averages of the thickness of the alterations in the subchondral bone. There were moderate and substantial intraobserver correlations in the traditional protocol and substantial to high Dixon technique. The interobserver correlation was moderate in the evaluation of this type of alteration for the upper and lower plateau of L3 and inverse and nonsignificant for the upper plateau of L5 using the traditional protocol. On the other hand, the interobserver correlation with the Dixon technique showed perfect agreement among the observers in the evaluation of type of signal change in the subchondral bone of the lower plateau of L1, an inverse correlation for the lower plateau of L3 and higher of L4, a substantial correlation between the lower plateau of L2, lower of L5 and higher of L5. We conclude that the Dixon technique presented superiority in the detection of Modic I compared with the traditional protocol, as well as allowed the detection higher averages of the thickness of alterations in the subchondral bone. It was also concluded that there was good intraobserver reproducibility and a variable interobserver correlation between the vertebral plateaus analyzed

No description available.

Intervertebral disc (IVD) degeneration is a prevalent health problem that is highly linked to back pain. To understand the disease and tissue response to therapies, ex-vivo whole IVD organ culture systems have recently been introduced. The goal of this study was to develop and validate a whole spinal segment culturing system that loads the disc in complex loading similar to the in-vivo condition, while preserving the adjacent endplates and vertebral bodies. The complex loading applied to the spinal segment was achieved with three pneumatic cylinders. The pneumatic cylinders were rigidly attached to two triangular alumni plates at each corner, comprising the loading mechanism. By extending or compressing the pneumatic cylinders, three modes of loading were achieved: flexion-extension, bi-lateral bending, and cyclic compression. The cylinders were controlled via microcontroller, and the entire system was fully automated. The culture container, which housed the spinal segment during culturing, was a flexible silicone container with an aluminum base and lid. The culture container attached to the loading mechanism allows for loading of the spinal segment. It had a vent attached to the aluminum lid that allowed for gas exchange in the system. The dynamic bioreactor was able to achieve physiologic loading conditions with 100 N of applied compression and approximately 2-4 N-m of applied torque. The function of the bioreactor was validated through testing of bovine caudal IVDs with intact endplates and vertebral bodies that were isolated within 2 hours of death and cultured for 14 days under a diurnal cycle. The resulting IVD cell viability following 14 days of loading was approximately 43% and 20% for the nucleus pulposus and annulus fibrosus respectively, which was significantly higher than the unloaded controls. The loading system accurately mimicked flexion-extension, bi-lateral bending, and compression motions seen during daily activities. Results indicate that this complex dynamic bioreactor may be appropriate for extended pre-clinical testing of vertebral mounted spinal devices and therapies.

Purpose. The present work hypothesized that proteolytic dissolution of intervertebral discs could induce biomechanical change comparable to the change observed in natural disc degeneration. A method to do such could be utilized for in vitro research where intersample differences in geometry and chemical makeup render it difficult to compare and aggregate results into generalized conclusions. Methods. Forty-one bovine coccygeal intervertebral discs were isolated with individual functional spinal units. Samples were loaded in three modes: compression/tension, flexion/extension, and axial rotation. The anulus fibrosus of each disc was injected with 200µl trypsin or fetal bovine serum (control) and incubated for an allotted period: 30 minute, 60 minutes, or 180 minutes. Mechanical loading was repeated and the load-displacement responses before and after treatment were compared as were the differences between each time group. Results. Significant change was observed in the discs' total range (stiffness), low range (laxity), and hysteresis. Changes in load-displacement response were observed to be correlated with both treatment and time. Conclusions. Enzymatic degeneration of intervertebral discs shows promise as a means to further understanding of disc mechanics in varying levels of degeneration. In virtually all cases, the trypsinized discs exhibited the increased joint laxity and decreased stiffness that is associated with early stage, natural disc degeneration.