京都大学 / 新制・論文博士 / 博士(医学) / 乙第13502号 / 論医博第2261号 / 新制||医||1061(附属図書館) / (主査)教授 村川 泰裕, 教授 柳田 素子, 教授 長船 健二 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Layer 3 (L3) pyramidal neurons are involved in intrinsic and extrinsic corticocortical communications that are integral to area specific cortical functions. The functional and morphological properties of these neurons are altered in the lateral prefrontal cortex (LPFC) of aged rhesus monkeys, changes which parallel the decline of working memory (WM) function. What is not yet understood is the time course of these neuronal alternations during the aging process, or the impact of neuronal changes on the function of local networks that underlie WM. By comparing the properties of L3 pyramidal neurons from the LPFC of behaviorally characterized rhesus monkeys over the adult lifespan using whole cell patch clamp recordings and neuronal reconstructions, the present dissertation demonstrates that WM impairment, neuronal hyperexcitabilty and spine loss begin in middle age. We use bump attractor models to predict how empirically observed changes affect performance on the Delayed Response Task and Delayed Recognition Span Task (spatial). The performance of both models is affected much more by neuronal hyperexcitability than by synapse loss. In a separate study, we examine pathological changes of L3 pyramidal neurons in the perilesional ventral premotor cortex following acute ischemic injury to the primary motor cortex. Neurons from lesioned monkeys exhibit hyperexcitability and changes the excitatory:inhibitory synaptic balance in favor of inhibition. As oxidative stress and inflammation are known to exacerbate both age-related and injury-induced neuronal pathology, we characterize neuronal properties in both conditions after administering therapeutic interventions which target inflammatory pathways and which have previously been shown to ameliorate behavioral deficits. Chronic dietary curcumin treatment dampens neuronal hyperexcitability in middle-aged subjects, but the neuronal changes are not correlated with WM improvements. Treatment with mesenchymal-derived extracellular vesicles lowers firing rates and restores excitatory:inhibitory synaptic balance, and importantly, these changes correlate significantly with motor function.

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) worldwide which is expected to affect 80% of the world’s population. IVD degeneration (IDD) is a key player in the degenerative cascade associated with LBP. Pro-inflammatory cytokines and mediators, such as nitric oxide, have been shown to be triggers and mediators of IDD. Due to the avascular nature of the adult IVD, the disc is unable to heal or regenerate when damaged. The multi-components of the IVD, namely glycosaminoglycan (GAG)-rich nucleus pulposus (NP), a concentric collagen dense annulus fibrosis (AF), and cartilage endplates (CEPs), further complicate possible regenerative solutions. Cell therapies show promise. This is supported by studies that demonstrate the use of mesenchymal stem cells (MSCs) in animal models showing potential in mitigating inflammatory signaling as well as recovering proteoglycan content. Despite these promising findings, several gaps in knowledge remain. While the biochemical and mechanical properties of an injured disc (via physical or chemical stimulation) have been characterized, the resulting inflammatory signaling cascades remain undefined. A growing body of evidence suggests that TLR4 is involved in the pathogenesis of the IVD. However, it is unknown how TLR4 mediates injury responses of the IVD. Second, it is unknown how mechanical loading of IVDs can influence the transcriptome or secretome of the IVD. The IVD is normally exposed to multimodal loading (e.g., compression, tension, shear, hydrostatic pressure, and osmotic pressure). Both frequency and magnitude regulate whether loading is beneficial or detrimental to disc integrity, which will be explored. Furthermore, the secretome of the IVD, especially during loading, may be essential to creating therapies targeted for regeneration of the IVD. There may be key, distinct paracrine factors that are released in IVD conditioned loading media which can influence the regenerative and anti-inflammatory capabilities of cell-based therapies. To address these gaps, this thesis describes a series of experiments employing novel ex vivo organ culture model to study the response of the IVD to various injury modalities (inflammatory stimulation, puncture injury, compressive loading), and resulting changes in inflammatory, biomechanical, and biochemical responses. Through methods such as RNA sequencing and proteomics, we now have expanded the characterization to beyond candidate genes or proteins, and are more informed on (1) the IVD response to injury, (2) the role of TLR4 signaling in this ex vivo organ culture model, in addition to (3) the downstream effects of loading and how paracrine factors can be used to improve and develop potential cell and molecular therapies. Sex-based differences, in male and female rat caudal IVDs, were also identified and are analyzed in the context of response to injury.

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Tissue engineering is a promising approach that combines the interactions of biomaterials, cells, and growth factors to stimulate tissue growth and regeneration. As such, selecting a suitable biomaterial is vital to the success of the procedure. Ideally, the material should show similarity to the extracellular matrix in the structure and relative stiffness, and biofunctionality beside others to provide a comfortable environment for the cells. Additionally, the biomaterial properties should allow for the effective diffusion of relevant growth factors and nutrients throughout the material to enable cell growth. Because peptides are composed of amino acids found naturally within the human body, they are considered non-toxic and biocompatible. Ultrashort peptides are peptides with three to seven amino acids that can be self-assembled into helical fibers forming scaffolds of supramolecular structures. These peptide hydrogels formed a highly porous network of nanofibers which can quickly solidify into nanofibrous hydrogels that resemble the extracellular matrix (ECM) and provide a 3D environment for cells with suitable mechanical properties. Furthermore, we can easily tune the stiffness of these peptide hydrogels by just increasing peptide concentration, thus providing a wide range of peptide hydrogels with different stiffness for 3D cell culture applications. Herein we describe the use of ultrashort peptide hydrogels for the maintenance and the differentiation of human mesenchymal stem cells into the osteogenic lineage. Furthermore, we develop a three dimensional (3D) biomimicry acute myeloid leukemia (AML) disease model using biomaterial from a tetramer ultrashort self-assembling peptide. In addition, we evaluate the potential application of peptide hydrogels as a hemostatic agent. The results presented in this study suggest that our biomimetic ultrashort tetrapeptide hydrogels are an excellent candidate for tissue engineering and biomedical applications.

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This study was motivated by a theoretical formulation on mechanobiology of soft and hard skeletal tissue differentiation. To prove this formulation experimentally, I hypothesized that cartilaginous phenotype can be induced in vitro in a seemingly non-cartilaginous cell source from fibrous tissue. In testing this hypothesis, I have focused on cartilage as a target and fibrous tissue as an origin or the source of cell. Four different trials were pursued with one supposition in common, i.e. hydrostatic pressure is one of the main driving forces for chondroinduction in vitro. The first and second trials pertained to the influence of a relatively short and long duration cyclic hydrostatic compression on rat Achilles tendon fibroblasts. The third trial was to examine the effect of two different drugs on cytoskeletal elements of mesenchymal stem cells or mouse embryonic fibroblast lines in pellet cultures combined with the similar duration and/or frequency of cyclic hydrostatic pressure adopted in the aforesaid trials with no pharmacological agents added. Last, attempts were made to implement an advanced technique in molecular biology called 'PCR array' to further quantify expression levels of eighty four pathway-specific genes in mouse TGFbeta/BMP signaling traffic under the same physiological regimen of hydrostatic compression. Results demonstrated that transdifferentation in phenotype from tendon to fibrocartilage may have occurred in vitro in tendon fibroblasts in pellet cultures exposed to hydrostatic pressure. Experiments on the role of the cytoskeleton in mechanotransduction of the applied level of hydrostatic pressure demonstrated that disruption of microfilaments in the presence of cytochalasin-D did not significantly interfere with the anabolic effect of cyclic pressure. However, disruption of microtubule assembly by nocodazole abolished the pressure-induced stimulation in cartilage marker genes. These findings suggest that microtubules, but not microfilaments, are involved in mechanotransduction of hydrostatic pressure by mesenchymal stem cells.

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