abstract: Intervertebral Disc Degeneration (IVDD) is a complex phenomenon characterizing the desiccation and structural compromise of the primary joint in the human spine. The intervertebral disc (IVD) serves to connect vertebral bodies, cushion shock, and allow for flexion and extension of the vertebral column. Often presenting in the 4th or 5th decades of life as low back pain, this disease was originally believed to be the result of natural “wear and tear” coupled with repetitive mechanical insult, and as such most studies focus on patients between 40 and 50 years of age. Research over the past two decades, however, has demonstrated that environmental factors have only a modest effect on disc degeneration, with genetic influences playing a much more substantial role. Extensive research has focused on this process, though definitive risk factors and a clear pathophysiology have proven elusive. The aim of this study was to assemble a cohort of patients exhibiting definitive signs of degeneration who were well below the average age of presentation, with minimal or no exposure to suspected environmental risk factors and to conduct a targeted genome analysis in an attempt to elucidate a common genetic component. Through whole genome sequencing and analysis, the results corroborated findings in a previous study, as well as demonstrated a potential connection and influence between mutations found in IVD structural or functional genes, and the provocation of IVDD. Though the sample size was limited in scale and age, these findings suggest that further IVDD research into the association of variants in collagen, aggrecan and the insulin-like growth factor receptor genes of young patients with an early presentation of disc degeneration and minimal exposure to suspected risk factors is merited. / Dissertation/Thesis / Masters Thesis Biology 2016

El documento digital no refiere asesor / Describe las alteraciones discales en resonancias magnéticas de columna lumbosacra en postulantes asintomáticos a una empresa productora de sanitarios. Desarrolla un estudio descriptivo, transversal, desde enero del 2013 a noviembre del 2015. Evalúa 559 resonancias magnéticas de columna lumbosacra de postulantes asintomaticos a una empresa productora de sanitarios. Encuentra que la prevalencia de postulantes con lesiones asintomáticas fue de 44.9% (n=251). Del total de postulantes asintomáticos, el 18% tuvo prominencia discal en al menos un nivel, el 20% protrusión discal, el 4% extrusión discal, sin observarse casos de secuestro. Adicionalmente, el 3% presentó hernia intracorporal de Schmorl. Según la regresión de Poisson ajustado, las asociaciones evidenciadas en el modelo crudo se mantuvieron estadísticamente significativas, por cada incremento en 5 años de la edad la posibilidad de presentar lesiones asintomáticas aumenta en 17%, PR=1.17 IC 95% (1.07 a 1.28); mientras que por cada incremento en 5 Kg/m2 de índice de masa corporal, la posibilidad de tener lesiones asintomáticas se incrementa en 15%; PR=1.15 IC 95% (1.00 a 1.33). Del mismo modo, los participantes con educación universitaria tenían 55% mayor posibilidad de tener lesiones asintomáticas comparado con el grupo de participantes con educación secundaria; PR=1.55 IC 95% (1.15 a 2.11). Concluye que en los exámenes de resonancia magnética de columna lumbosacra, hasta cinco de diez personas asintomáticas tendrán alteraciones discales. Debido a esto, las prominencias, protrusiones y hernias intracorpóreas pueden ser una coincidencia. Los resultados de resonancias magnéticas de columna lumbosacra deben ser cuidadosamente analizados, conjuntamente con la evaluación clínica y el criterio médico al momento de tomar una conducta médica. El término "hernia" puede ser demasiado genérico por tener gran relevancia clínica. La clasificación de prominencias, protrusiones, extrusiones y secuestros resulta ser más útil en la tipificación de los hallazgos. / Tesis

Abstract The purpose of the current study was to evaluate the intercorrelations of magnetic resonance imaging (MRI) findings and clinical symptoms and signs in sciatic patients. Furthermore, determinants of spontaneous HNP resorption and the effect of anti-inflammatory treatments (periradicular methylprednisolone injection and intravenous infliximab) on spontaneous HNP resorption were evaluated. MRI follow-up was performed at baseline, after two months, after six months and after one-year for patients with unilateral sciatica to evaluate determinants of spontaneous HNP resorption and the effect of periradicular methylprednisolone injection on spontaneous HNP resorption. At baseline the study population consisted of 160 patients (group A). MRI follow-up for 21 patients with unilateral sciatica was performed at baseline and after two weeks, after three months and after six months to evaluate the effect of infliximab, a monoclonal TNFα antagonist, infusion on spontaneous HNP resorption (group B). Patients in group A were randomized to receive either periradicular saline or methylprednisolone. Volume of HNP, extent and thickness of enhancement (in Gd-DTPA MRI) and degree of disc displacement were measured and the symptoms and signs were followed repeatedly. The extent of rim enhancement correlated significantly with the degree of disc displacement. The duration of sciatic symptoms correlated negatively with enhancement parameters. The clinical symptoms did not correlate significantly with the different enhancement parameters or disc herniation volume. Achilles reflex abnormality correlated significantly with all enhancement parameters for lesions at L5-S1. Significant decrease in HNP volume occurred from baseline to two moths, and even more so during the whole one year follow-up period. Higher baseline scores of rim enhancement thickness, higher degree of HNP displacement in the Komori classification and age category of 41–50 years were associated with a higher resorption rate. Clinical symptoms alleviation occurred concordantly with a faster resorption rate. No significant difference was noted in the decrease of HNP volume in the saline and methylprednisolone injection groups in follow-up imaging during one year. The enhancement parameters (thickness and extent of rim enhancement) did not differ significantly in the different treatment groups. In group B, 11 patients received intravenous infliximab and 10 saline. Baseline demographic data, pain scores, and clinical status, did not differ between the treatment groups. HNP volume decreased significantly in both groups (P < 0.01). There was no significant difference in HNP volume changes between the treatment groups. By two weeks, enhancement thickness increased significantly in the infliximab compared placebo group (p=0.003). Two patients in each group required back surgery prior to the 6-month assessment.

Back pain is a highly prevalent condition whose pathogenesis is associated with intervertebral disc (IVD) degeneration. Degeneration is driven by abnormal cell biology, particularly within the IVD’s inner core, the nucleus pulposus (NP). In recent years, there has been an ever-increasing search for cell-based therapies aimed at correcting the cell biology and thus repairing/regenerating the degenerate IVD. The success of these novel therapies, however, requires a thorough understanding of IVD development and of the phenotype of its cells. The embryonic, foetal and juvenile NP is populated by large vacuolated notochordal cells that with skeletal maturity are replaced by smaller NP cells. Since notochordal cells have been shown to display protective and anabolic roles in the IVD their loss in humans has often been suggested to initiate the degenerative process. As such, a detailed understanding of notochordal cells and their regulatory pathways may help identify factors involved in IVD homeostasis and aid the development of novel cell-based therapies targeting IVD degeneration. The study of human notochordal cells has, however, been hindered by ethical, logistical and technical difficulties in obtaining suitable samples and, as such, the human notochordal cell phenotype is, to date, unknown, constituting a major limitation in the field. The work presented here was conducted with the objective of developing a methodology to isolate human developing notochordal cells (NP progenitors) from adjacent sclerotomal cells (annulus fibrosus and vertebral body progenitors), to characterise the notochordal cell phenotype and identify potential factors involved in notochordal cell biology. Initially, human embryonic and foetal spines were characterised to assess their suitability as a source of notochordal cells and to identify a notochord-specific marker that could be used to isolate notochordal cells for microarray studies. The human developing spine contained large vacuolated notochordal cells in all stages analysed (3.5-18 weeks post-conception (WPC)) that specifically expressed KRT8, KRT18 and KRT19 at all stages and CD24 between 5.5-18 WPC. KRT18 and CD24 were independently used to label notochordal cells (7.5-14 weeks post-conception) and separate them from sclerotomal cells. Methodologies were developed to allow extraction of RNA of sufficient quality for microarray analysis from fixed, permeabilised (in the case of KRT18) and/or, labelled and sorted cells (CD24). Microarray analysis identified and real-time qPCR and, for some markers, immunohistochemistry, validated GRB14, SLC19A1, FGF10, ADORA3, TBXA2R, CDH6, ANPEP, CD69, CD24, RTN1, PRPH, MAP1B, ISL1 and CLDN1 as human notochordal cell markers. Ingenuity pathway analysis was performed to investigate the pathways/networks and upstream regulators and downstream effectors of notochordal cells. Inhibition of inflammation and angiogenesis were identified as relevant to notochordal cell biology, function and, possibly, to the known protective and anabolic role notochordal cells display in the IVD. Notochordal marker gene expression was identified in adult NP tissue, and negatively correlated with degeneration. Proteins encoded by ADORA3 and MAP1B were expressed by a proportion of adult NP cells, suggesting the presence of notochord-derived cells in the adult NP.Importantly, this is the first study to detail a methodology and successfully isolate human notochordal cells. Such methodology has the potential to be used to culture and investigate the biology of viable human notochordal cells (CD24+ve). Future studies aimed at developing cell-based therapies for IVD degeneration could also use these identified markers to assess appropriate stem cell differentiation to notochordal cells.

Intervertebral disc (IVD) degeneration is associated with low back pain (LBP). It has been suggested that changes in the IVD physio-chemical microenvironment (i.e. hypoxia, reduced nutrient and acidic conditions) may lead to disc degeneration. Studying the response of human nucleus pulposus (NP) cells to these conditions could establish the causal relationship between IVD microenvironment and aberrant cellular behaviour, characteristic of disc degeneration. Human bone marrow mesenchymal stem cells (BM-MSCs) are a promising cell population for disc regeneration. However, knowledge of their survival and functioning in the microenvironment of the IVD is still lacking. Moreover, in vitro co-culture model studies that are used to study MSC/disc cell interaction, also need to consider the effect of the microenvironment on cellular responses. BM-MSCs and degenerate NP cells were cultured alone or co-cultured in monolayer under hypoxia (2%O2), reduced nutritional (2% serum or/and 5mM glucose) and acidic (moderate pH 6.8 or severe pH 6.5) conditions alone or in combination for 7 days. Cell viability, proliferation, gene and protein expression was assessed. Degenerate NP cells and BM-MSCs maintained good cell viability under all conditions. Both cell types demonstrated overall similar proliferation and gene and protein responses under the majority of the conditions and combinations studied. Hypoxia promoted aggrecan and versican matrix biosynthesis in both cell types. Nutrient deprived and moderate acidic conditions (pH 6.8) inhibited proliferation of both cell types. Interestingly the combination of hypoxia with these conditions showed a protective effect in modulating cell proliferation. These results imply that hypoxia may be beneficial in some instances. Nutrient deprived conditions had a relatively minor effect on degenerate NP cell gene and protein expression but these conditions specifically inhibited VCAN expression in BM-MSCs. The combination of hypoxia with these conditions increased or restored VCAN expression. Interestingly the combination of hypoxia with reduced glucose conditions increased aggrecan and versican matrix biosynthesis in both NP cells and BM-MSCs. The combination of hypoxia and complete nutrient deprived conditions (both reduced serum and reduced glucose) impaired ACAN, VCAN and PAX-1 gene and aggrecan and versican protein expression in degenerate NP cells implicating disc hypoxia and complete nutrient deprived combined microenvironment in accelerating degenerate changes in NP cells. In contrast, these conditions showed no such detrimental effects on BM-MSC gene and protein expression. pH 6.5 was critical for both cell types proliferation and ACAN and VCAN gene expression suggesting that severe acidic conditions may exacerbate degenerative changes and be inhibitory for implanted MSCs. Finally, a combination of hypoxia, complete nutrient deprived and moderate acidic conditions, reduced cell proliferation without affecting the gene expression profile of both cell types. IVD-like physio-chemical microenvironmental conditions also appeared to influence differentiation of BM-MSC and modulation of degenerate NP cell phenotype observed during co-culture. Noticeably hypoxia, reduced serum or reduced glucose conditions stimulated BM-MSC differentiation and modulation of degenerate NP cell phenotype. Hypoxia also increased or recovered changes at gene expression level in both BM-MSCs and degenerate NP cells under nutrient deprived (reduced serum or/and reduced glucose) conditions during co-culture. Degenerate NP cell and BM-MSC co-culture also showed noticeable increase in aggrecan and versican biosynthesis under hypoxia and reduced glucose combine conditions, implicating these in improving the co-culture responses. Severe pH condition alone, pH 6.8 in combination with hypoxia and finally all IVD-like physio-chemical conditions together compromised co-culture responses. Such results imply that IVD-like physio-chemical microenvironmental conditions may influence MSC based regenerative outcomes. This work has increased our understanding about the influence of disc harsh microenvironment on degeneration and regeneration processes.

Presented work deals with the strain-stress analysis of a spinal segment while monitoring the influence of total disk replacement Maverick on the global behaviour of the spinal segment. It also assesses the influence of this implant on the nearest upper intervertebral disk. On the basis of CT images was in the program Rhinoceros created the geometry model of spinal segment Th12-L2. The geometry model of implant was applied between vertebra L1 and L2 instead of intervertebral disk from which the whole core was removed, while in the immediate surrounding of implant the part of disk was left, which is important from the view of stability. Subsequently were in the program Ansys Workbench created finite element models for two states: - healthy (unbroken) status - status with applied implant Then the calculations for movements were realized: lateral flexion, flexion, extension, rotation. For the same movements were realized calculations of the model simulated spinal fusion of the vertebras L1-L2. Finally an influence both of methods (total disk replacement, spinal fusion) on the intervertebral disk Th12-L1 was compared.

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Propósito: Analizar si la adición de un espaciador interespinoso o la fusión intersomática ofrece ventajas en relación con la microdiscetomía aislada en el tratamiento de la hernia discal lumbar. Métodos: Pacientes con hernia de disco lumbar que iban a someterse a cirugía fueron elegidos para participar. En este estudio de cohorte los pacientes se dividieron en 3 grupos; Microdiscectomía sola (MD), microdiscectomía más espaciador interespinoso (IS) y fusión intersomática lumbar posterior (PLIF). La medida de resultado primaria fue la eficacia clínica mediante el índice de discapacidad de Oswestry (ODI). También evaluamos varios otros parámetros de resultado, entre los que se incluyeron: escala analógica visual para el dolor (EVA) de espalda y piernas, duración de la estancia, coste desde el ingreso hasta el alta hospitalaria, tasa de complicaciones de 90 días y tasa de reoperación tras 1 año. Resultados: Se incluyeron un total de 103 pacientes cuya edad media fue de 39,1 (± 8,5) años. En los 3 grupos se detectó una mejora significativa de la puntuación inicial del dolor de espalda y piernas con ODI y EVA. Las puntuaciones del ODI cambiaron de 62.66 a 13.77 en el grupo MD, 62.93 a 13.50 en el grupo IS, y 59.62 a 17.62 en el grupo PLIF (p <0.001). Después de 1 año, no se encontraron diferencias significativas en el ODI, ni en la EVA de espalda y piernas entre los 3 grupos. Hubo un aumento del 169% en el coste hospitalario en el grupo IS y del 287% en el grupo PLIF, en relación con la MD (p <0,001). La duración de la estancia fue un 86% mayor en el grupo IS y un 384% más en el grupo PLIF en comparación con MD (p <0,001). Las tasas de reoperación a 1 año fueron de 5,6%, 10% y 16,2% (p = 0,33) en los grupos MD, IS y PLIF respectivamente. Conclusión: La mejoría clínica parece deberse a la microdiscectomía, sin que el implante (interespinoso o fusión) agregue ningún beneficio. La adición de espaciador interespinoso o fusión no protegió contra la reoperación, y aumentó la duración de la estancia hospitalaria y los gastos quirúrgicos.

椎間板変性を引き起こす要因として、骨性終板内の栄養管狭小および軟骨終板の石灰化による椎間板への栄養供給の低下が推察されているが、椎体終板内栄養管の3次元微細構造は未だ明確にされていない。本論文は高解像度マイクロCTを用いて家兎腰椎骨性終板内栄養管の3次元微細構造を明らかにする事を目的とし、各栄養管の長さ,直径,配向及び表面からの深さを定量的に解析することでその多層構造を定量的に評価することができた。 / Insufficient nutrient supply through vertebral canal structures to the intervertebral disc (IVD) has been considered as an important contributor for disc degeneration. In spite of this, three-dimensional (3D) topology inside the vertebral endplate remains poorly understood. This study aims to characterize the 3D canal structure in the rabbit lumbar vertebral endplate using micro computed tomography (µCT), and revealed a distinct depth-dependent structure of the canal in the rabbit vertebral endplate characterized by length, diameter and orientation of the individual canals. / 博士(工学) / Doctor of Philosophy in Engineering / 同志社大学 / Doshisha University