abstract: Introduction. Intervertebral disc degeneration (DD) is one of the most common diagnoses in patients with neck pain and contributes to worldwide disability. Despite the advances in diagnostic imaging today, little is known about functional status of cervical DD. The purpose of this research was to 1) develop and validate an ovine model of cervical spine DD, 2) to quantify and compare the effect of disc lesions on dynamic spinal stiffness, and 3) study the effect of disc lesions on spinal accelerations and displacements during two types of spinal manipulative therapy (SMT). Methods. Fifteen sheep received surgically induced disc injury to the mid-cervical spine via scalpel wound a minimum of five months earlier and 15 sheep served as controls. All animals were biomechanically assessed at the level of the lesion using swept-sine mechanical loads from 0-20 Hz under load control to quantify dynamic dorsoventral (DV) spine stiffness (load/deformation, N/mm). The effect of disc lesion on stiffness was assessed using a one-factor repeated measures ANOVA comparing 32 mechanical excitation frequencies. Tri-axial accelerometers rigidly attached to adjacent vertebrae across the target level further evaluated the effect of disc lesion on spinal motion response during two types of SMTs. A 2x6x2 repeated measures ANOVA examined the effect of disc lesion and SMT force-time profile on spine motion response. Postmortem histological analysis graded specimens at the target site and comparison was made with descriptive statistics. Results. Annular disc tears were only observed in the disc lesion group and the mild degeneration identified was localized to the injured annular tissue that did not progress to affect other areas of the disc. No difference in overall DD grading was found among the groups. DV stiffness was significantly increased in the disc lesion group by approximately 34% at 31 of 32 frequencies examined (p<.05). SMTs resulted in decreased displacements in the disc lesion group (p<.05), and SMT type significantly influenced spinal accelerations for both the DV and axial planes. Conclusion. Disc lesions in the ovine cervical spine produce localized annular degenerative changes that increase the cervical spine dynamic stiffness and reduce its spinal motion response during manual examination and treatment that is further augmented by the force-time profile administered by the clinician. / Dissertation/Thesis / Doctoral Dissertation Kinesiology 2015

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

Foi avaliada a participação dos corpos vertebrais e discos intervertebrais na lordose lombar e, a contribuição destes nas curvaturas lombares de diferentes magnitudes. Foram avaliadas as radiografias lombares em perfil de 350 adultos assintomáticos (143 homens e 207 mulheres, idade média 29 anos). Foram mensuradas a curvatura lombossacra (L1S1), a curvatura lombolombar (L1L5), a angulação de cada corpo vertebral e cada disco intervertebral por meio de uma variação do método de Cobb. A participação percentual dos corpos vertebrais e dos discos intervertebrais na curvatura lombossacra também foi determinada. Comparações entre os sexos e as faixas etárias foram realizadas. Os indivíduos foram divididos em três subgrupos populacionais, de acordo com a magnitude da lordose lombossacra, de modo a separar os indivíduos pertencentes aos extremos da curva de distribuição. Os componentes da curvatura lombar (corpos vertebrais e discos intervertebrais) foram comparados nestes três subgrupos. A medida da curvatura lombossacra no grupo inicial foi -60,9o (-33o a -89o). Os corpos vertebrais eram cifóticos em L1 (2,15o), tendiam ao neutro em L2 (-0,36o) e eram progressivamente lordóticos de L3 (-1,56o) a L5 (-9,23o). Os discos intervertebrais eram progressivamente lordóticos (variando de -4,99o em L1-L2 a -15,58o em L5-S1). Os corpos vertebrais e discos intervertebrais apresentaram participação progressivamente maior na curvatura lombossacra no sentido crânio-caudal. Os discos intervertebrais participaram com cerca de 80% da curvatura lombossacra, sendo que os elementos mais caudais (corpos vertebrais L4 e L5 e discos intervertebrais L4-L5 e L5-S1) corresponderam a mais de 65% da curvatura lombossacra. Os indivíduos mais velhos apresentaram medidas das curvaturas lombares maiores cerca de 4º em comparação aos indivíduos mais jovens, havendo diferença significante para as medidas dos corpos vertebrais L2 e L5 e o disco intervertebral L3-L4, sendo maiores as medidas nos indivíduos mais velhos. As medidas das curvaturas lombares e dos corpos vertebrais L2 e L4 apresentaram diferença estatisticamente significante entre os sexos, sendo as medidas maiores nos indivíduos do sexo feminino. A curvatura lombossacra apresentou média de -46,9° no subgrupo lordose menor; -61,59° no subgrupo lordose intermediária e; -74,13° no subgrupo lordose maior. A curvatura lombolombar apresentou média de -33,28° no subgrupo lordose menor; -45,34° no subgrupo lordose intermediária e; -56,96° no subgrupo lordose maior. Os corpos vertebrais e os discos intervertebrais apresentaram medidas absolutas menores no subgrupo lordose menor do que as dos subgrupos lordose intermediária e lordose maior, mas a participação dos discos intervertebrais na curvatura lombossacra no subgrupo lordose menor (88%) foi maior que nos subgrupos lordose intermediária (81%) e no subgrupo lordose maior (75%). Complementarmente, os corpos intervertebrais apresentaram maior participação nos subgrupos lordose maior e lordose intermediária. Individualmente, os corpos vertebrais apresentaram maior participação no subgrupo lordose maior, exceto pelo corpo vertebral L5 que apresentou maior participação no subgrupo lordose menor. A maior participação percentual dos discos intervertebrais no subgrupo lordose menor era devida à inclinação cifótica dos corpos vertebrais mais cefálicos (especialmente L1 e L2) no subgrupo lordose menor do que nos demais subgrupos, que, por um efeito compensatório, causava uma maior participação discal nas curvaturas menores. Os demais subgrupos apresentavam os corpos vertebrais cefálicos com inclinação muito mais lordótica do que o observado no subgrupo lordose menor. Concluímos que os discos intervertebrais são os principais responsáveis pela curvatura lombar e que a contribuição dos corpos vertebrais e discos intervertebrais na lordose lombar difere entre indivíduos com curvaturas de diferentes magnitudes. Apesar de ocorrer um aumento gradual do acunhamento lordótico do corpo e disco a cada nível vertebral conforme aumenta a medida da lordose, as vértebras mais cefálicas provocam uma diferença na contribuição percentual entre discos intervertebrais e corpos vertebrais nas curvaturas de tamanhos diferentes. / The vertebral bodies and intervertebral discs participation in lumbar lordosis and their contribution between lumbar curves of different size were studied. 350 lumbar spine radiographs of asymptomatic adults (143 men and 207 women, average age 29 years) were evaluated. Lumbosacral (L1S1) and lumbolumbar (L1L5) curves and the angular inclination of each vertebral boby and intervertebral disc were measured using a Cobb method variant. The percentile participation of each vertebral body and intervertebral disc in the lumbossacal curve was calculated. Sex and age were compared. The subjects were separated in tree subgroups, in acording to lumbosacral curve size. The compounds of lumbar curve (discs and vertebrae) were compared in these tree subgroups. The mean lumbosacral curve was ?60,9º (-33º to ?89º). L1 vertebral body was kyphotic (2,15º), L2 was neutral (-0,36º), and the other ones were progressively lordotic from L3 (-1,56º) to L5 (-9,23º). The intervertebral discs were progressively lordotic from L1-L2 (?4,99º) to L5-S1 (?15,58º). Both vertebrae and discs showed a progressive participation in cephalic-caudal direction. The participation of discs was about 80% of lumbosacral curve, and the caudal elements (L4, L5 vertebrae and L4-L5, L5-S1 discs) contributed far 65% of the curve. The older subjects presented lumbar curves larger than younger 4º average, with significant statistical difference to L2, L5 and L3-L4 measures, with older subjects presenting bigger angular values. There were statistical differences of lumbar curves, L2 and L4 measures between sexes, with females presenting bigger values. The lumbosacral curve presented average -46,9º in minor lordosis subgroup, -64,59º in intermediate lordosis sugbroup, and ?74,13º in major lordosis subgroup. The lumbolumbar curve presented average ?33,28º in minor lordosis subgroup, -45,34º in intermediate lordosis subgroup, and ?56,96º in major lordosis subgroup. The absolut values of vertebrae and discs angles were smaller in minor lordosis subgroup than in major lordosis subgroup, but the intervertebral discs participation of was bigger in minor lordosis subgroup (88%) than intermediate lordosis (81%) and major lordosis (75%) subgroups. Complementarely, the vertebrae had a bigger participation in intermediate and major lordosis subgroups. Individually, the vertebrae presented a larger participation in major lordosis subgroup, excepting L5 that presented bigger participation in minor lordosis subgroup. The discs presented larger participation in minor lordosis subgroup. That is consequence of a more kyphotic inclination of the cephalic vertebrae in minor lordosis subgroup than the other ones, causing a compensating effect, with a larger disc participation in the small curves. The intermediate and major lordosis subgroups had the cephalic vertebrae more lordotic than that of the minor lordosis subgroup. We concluded that the intervertebral discs are the main responsible for the lumbar curve angulation and that the contribution of vertebrae and discs in lumbar curves of different sizes is not equal. In spite of a gradual increase of lordotic wedging while lumbar curve increase, the cephalic vertebrae make the disc and vertebrae participation different between different magnitude lumbar curves.

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 &lt; 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.

No description available.

No description available.

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