Back pain is one of the most common physical conditions in the United States, for which approximately 15% of the population will visit a doctor every year. The most common type of back pain is low back pain (LBP) and millions of dollars are spent every year healthcare are a due to LBP. Although poorly understood, low back pain has been associated to interveterbral disc (IVD) degeneration. The IVD is an important structure that helps maintaining normal skeletal support. It is composed of three different tissues called the annulus fibrosus (AF), and the nucleus pulposus (NP), attached to a cartilage endplate (CEP) at its top and bottom surfaces. The AF tissue is composed of chondrocyte-like cells, while the NP tissue is composed of notochordal cells at a young age, which are replaced by mature NP cells later in life. Common signs of degeneration are the inability to maintain extracellular matrix integrity and calcification of the cartilage endplate. Extracellular matrix synthesis and cartilage endplate calcification are closely related to production of adenosine triphosphate (ATP) or energy metabolism of the cells. AF and NP tissues are known to be structurally and compositionally different; therefore it is believed that their metabolic pathways are also distinct. The objective of this study was to determine the differences between AF and NP cells, specifically in their energy metabolism with and without dynamic loading.

Lower back disorders due to whole-body vibration (WBV) are the most common injuries reported by professional drivers. Such injuries often have long-term complications leading to significant personal and societal costs. An improved mathematical model of the whole human body would contribute to a better understanding of the mechanisms of lower back injury and be valuable in injury prevention research. Current biodynamic human models reported in the literature lack detailed information for predicting the non-linearity due to vibration amplitude of transmission of vibration from seat to a human. Therefore, one of the primary objectives of this research has been to develop and validate a detailed threedimensional biodynamic human model, with special attention given to the incorporation of active trunk muscles with non-linear stiffness properties. These muscles have been incorporated into an existing spine and neck model of a MADYMO 50th percentile male occupant model. A detailed multi-body human model has been developed, called MODEL ONE. This thesis shows that incorporating non-linear stiffness functions and energy dissipation using hysteresis or damping into a human model is appropriate for predicting non-linear biodynamic responses in arbitrary excitation functions. A major advantage of MODEL ONE compared to other multi-body models and lumped mass models is its ability to predict nonlinear seat-to-human transmissibility. However MADYMO 50th male occupant models use simplified geometry and rigid bodies to represent the lower lumbar spine. These simplified spinal models have no ability to simulate the internal stresses and deformations of soft tissues, even if these are the apparent cause of lower back pain (LBP). Therefore a detailed finite element human lower lumbar spine model - with appropriate material properties and capable of simulating internal stresses⎯is necessary, in order to better understand spinal injuries under WBV. A three-dimensional finite element model of a lower lumbar spine motion segment - called MODEL TWO - has thus been developed for the present study. MODEL TWO comprises a detailed geometric description of vertebrae, nucleus pulposus, endplates, and intervertebral discs. The intervertebral discs lump together the annulus fibrosus, ground substance and ligaments. The vertebrae have been assumed to be rigid. The material properties of the intervertebral discs of MODEL TWO were obtained from test matrices and from various parameter data reported in the literature. MODEL TWO has been validated against cadaveric experiments reported in the literature. The mechanical behaviour and stress distribution within the MODEL TWO intervertebral disc agree reasonably well with the cadaveric experiments. MODEL TWO was integrated into MODEL ONE to form a new human model, called MODEL THREE, which was subsequently dynamically validated against volunteers� responses to WBV reported in the literature. MODEL THREE, as presented in this thesis, consists of a multi-body human model with detailed representation of a finite element (FE) lower lumbar spine. As far as the author is aware, MODEL THREE is the first model with detailed representation of a FE lower lumbar spine to successfully demonstrate that it is capable of simulating the stress profile of the entire intervertebral disc and endplate region due to WBV. The simulated results revealed abnormal stress concentrations in both the posterior and xviii the posterolateral annulus. The stresses increased most in the posterolateral intervertebral discs region during WBV, suggesting a possible mechanism for disc mechanical overload leading to fatigue fracture and degeneration. The results from MODEL THREE are promising and lead to a more comprehensive understanding of the behaviour of the intervertebral disc under WBV. MODEL THREE has also provided a good foundation for the development of a bio-fidelity human model. However, implementation of currently unavailable and/or inadequate in vitro and in vivo experimental studies is needed to further validate and develop MODEL THREE. A better understanding of injury mechanisms and the clinical significance of LBP will ultimately be arrived at using a combination of analytical models with in vitro and in vivo experimental data.

Replacing a diseased disc with a tissue engineered disc has the potential to restore normal spinal biomechanics. However, recreating the interface between annulus fibrosus (AF) and vertebral bone (VB) will be necessary to facilitate proper function of the implant in vivo. This study characterizes the native bovine AF-VB interface and assesses adult human discs. The AF insertion site in humans and cows is uniquely differentiated from other soft tissue-bone interfaces, as AF collagen fibers anchor into the calcified region of vertebral endplate through a zone of hyaline cartilage and have a different organization in inner and outer AF. Mineralization-associated proteins are present in this region and the chondroid tissue undergoes calcification over time. Based on these observations an in vitro AF culture system was developed and demonstrated that AF cells can induce mineralization. Understanding mechanism(s) regulating AF mineralization will help develop conditions to ensure proper integration of bioengineered AF.

Replacing a diseased disc with a tissue engineered disc has the potential to restore normal spinal biomechanics. However, recreating the interface between annulus fibrosus (AF) and vertebral bone (VB) will be necessary to facilitate proper function of the implant in vivo. This study characterizes the native bovine AF-VB interface and assesses adult human discs. The AF insertion site in humans and cows is uniquely differentiated from other soft tissue-bone interfaces, as AF collagen fibers anchor into the calcified region of vertebral endplate through a zone of hyaline cartilage and have a different organization in inner and outer AF. Mineralization-associated proteins are present in this region and the chondroid tissue undergoes calcification over time. Based on these observations an in vitro AF culture system was developed and demonstrated that AF cells can induce mineralization. Understanding mechanism(s) regulating AF mineralization will help develop conditions to ensure proper integration of bioengineered AF.

No description available.

The muscle fibre cytoskeleton of skeletal and heart muscle cells is composed mainly of intermediate filaments (IFs), that surround the myofibrils and connect the peripheral myofibrils with the sarcolemma and the nuclear membrane. Desmin is the first muscle specific IF protein to be produced in developing muscles and is the main IF protein in mature muscles. In skeletal muscle, desmin is particularly abundant at myotendinous and neuromuscular junctions. In the heart an increased amount of desmin is found at intercalated discs and in Purkinje fibres of the conduction system. Interactions between the IFs themselves, and between IFs and other structures such as Z-discs and the sarcolemma, are mediated by intermediate filament associated proteins (IFAPs). A transgenic mice model, which lacks the desmin gene have been developed to study the function of desmin. In these mice, morphological abnormalities are observed in both heart and skeletal muscles. Similar defects have been observed in human myopathies, caused by different mutations in the desmin gene. In the present thesis, skeletal and heart muscles of both wild type and desmin knock-out (K/O) mice have been investigated. Furthermore the cytoskeletal organisation in skeletal muscles from human controls and from a patient with desmin myopathy was examined. In the desmin K/O mice, no morphological alterations were observed during embryogenesis. These mice postnatally developed a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. Ruptures of the sarcolemma appear to be the primary event leading to muscle degeneration and fibrosis both in cardiac and affected skeletal muscles. In the heart the muscle degeneration gave rise to calcifications, whereas in skeletal muscles regeneration of affected muscle was seen. In mature wild type mice, the IF proteins synemin and paranemin, and the IFAP plectin were present together with desmin at the myofibrillar Z-discs, the sarcolemma, the neuromuscular junctions and the myotendinous junctions. Nestin was only found in these junctional regions. In desmin K/O mice, all four proteins were detected at neuromuscular and myotendinous junctions. The normal network of synemin and paranemin were not observed, whereas the distribution of plectin was preserved. In normal human muscles, synemin, paranemin, plectin and αB-crystallin were colocalised with desmin in between the myofibrils, at the sarcolemma and at myotendinous and neuromuscular junctions. In the human desmin myopathy, the distribution of desmin varied considerably. A normal pattern was seen in some fibres areas, whereas other regions either contained large subsarcolemmal and intermyofibrillar accumulations of desmin or totally lacked desmin. Nestin, synemin, paranemin, plectin and αB-crystallin also exhibited an abnormal distribution. They were often aggregated in the areas that contained accumulations of desmin. In cultured satellite cells from the patient, a normal network of desmin was present in early passages, whereas aggragates of desmin occurred upon further culturing. In the latter, also the nestin network was disrupted, whereas vimentin showed a normal pattern. αB-crystallin was only present in cells with a disrupted desmin network. Plectin was present in a subset of cells, irrespective of whether desmin was aggregated or showed a normal network. From the present study it can be concluded that an intact desmin network is needed to maintain the integrity of muscle fibres. Desmin may be an important component in the assembly of proteins, which connect the extrasarcomeric cytoskeleton with the extracellular matrix.

The effect of 1,2,3,4-tetrahydro-9-aminoacridine (THA) on quantal transmitter release was examined at the frog neuromuscular junction. THA (3 μM) caused an increase in m (no. of quanta released) as measured by K+-evoked miniature endplate potential (MEPP) frequency. This was due to an increase in p (probability of release), as n (no. of functional release sites) was unchanged. The increase in p was dose-dependent over a range of 0.3-10 μM. By contrast, physostigmine (3 μM) caused a decrease in p, and neostigmine, which does not cross the nerve membrane, had no consistent effect on p. At the postsynaptic site, neostigmine produced the largest increase in MEPP size (79.2%), and THA produced the smallest (17.5%). The divergent effects of THA and physostigmine on p indicate a fundamental difference in their actions at the nerve terminal.

Intracellular recordings were made from isolated frog sciatic-sartorius nerve-muscle preparations, and the effects of sphingosine 1-phosphate (S1-P) on miniature endplate potentials (MEPPs) were studied. Extracellular application of S1-P (1 and 30 μM) had no significant effects on the frequency and amplitude of MEPPs. Delivery into nerve terminals by liposomes containing 10-5, 10-4 or 10-3 M S1-P was associated with a concentration-dependent increase in MEPP frequency of 37, 63 and 86%. The per cent of median MEPP amplitude was not significantly changed, but there was an increase in the number of 'giant' MEPPs. Pre-exposure of the preparations to S1-P 10-5 but not 10-8 M entrapped in liposomes for 15 min blocked the effects of subsequent superfusion of S1-P (10-4 M)-filled liposomes on MEPP frequency. Thus, intracellular S1-P receptors seem to undergo 'desensitization' to higher concentrations of S1-P. The result provides the first evidence that S1-P acting intracellularly but not extracellularly enhances spontaneous transmitter release at the frog neuromuscular junction.

No description available.

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