published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Background: An association between cartilaginous endplate (CEP) defects and intervertebral disc (IVD) degeneration has been previously suggested in animal and cadaveric studies. CEP defects may also be involved in Schmorl’s nodes (SN). There have been no previous reports in the literature that describe the use of ultrashort time-to-echo (UTE) MRI to assess the CEP in humans in vivo. In chapter 5 of this thesis, a retrospective study of paediatric spines and the neurocentral synchondrosis (NCS) was singled out to report the incidence of NCS and to raise the hypothesis of NCS as a precursor of SN. Purpose: To assess the feasibility of detecting CEP defects in live humans using UTE MRI, and to assess their relationship with IVD degeneration and SN. Subjects and Methods: A total number of 22 subjects underwent T2-weighted (T2W) and UTE MRI to assess for the presence and severity of IVD degeneration, the presence of SN and for the presence of CEP defects. SN and IVD degeneration were confirmed by assessing T2W images and IVD degeneration was graded according to the Schneiderman classification. CEP defects were defined as discontinuity of high signal over 4 consecutive images and were independently assessed by two raters. Results: Analyses of CEP defects between IVD degeneration and SN were performed separately. For the study of CEP defects and IVD degeneration, subjects with SN were excluded. 37 out of 108 (34.3%) CEPs had defects, which mainly occurred at T12/L1, L1/L2 and L4/L5 (p=0.008). Inter-rater reliability was substantial (Kappa statistic= 0.67, p<0.001). Multivariate logistic regression revealed that lower BMI (p=0.009) and younger (p=0.034) individuals had a decreased likelihood of having CEP defects. A statistically significant association was found to exist between the presence of cartilaginous endplate defects and intervertebral disc degeneration (p=0.036). Degenerated discs with CEP defects were found in L4/5 and L5/S1, while degenerated discs with no CEP defects were found throughout the whole lumbar region. Mean degeneration scores of L4/5 and L5/S1 levels with CEP defects were higher than that of L4/5 and L5/S1 levels without. For the study of CEP defects and SN, with all 22 subjects assessed, 125 out of 264 (47.3%) CEPs had defects. 40 SN were found, and among those, 35 SN had CEP defects (87.5%). 125 CEPs had the presence of CEP defects; among them, a large number of CEP defects did not have SN underneath (92 out of 125, 73.2%). Conclusion: The studies demonstrate the feasibility of using UTE MRI in live humans to assess the integrity of the CEP. Longitudinal studies may reveal the diagnosis of CEP defects to be clinically beneficial for assessment of IVD degeneration and SN. / published_or_final_version / Diagnostic Radiology / Master / Master of Philosophy

Introduction: Intervertebral disc (IVD) degeneration is suggested to begin from the nucleus pulposus (NP). Evidence from various studies highlights mesenchymal stem cells (MSC), in most cases using bone marrow derived MSC, as a potential stem cell source for NP regeneration. However MSC can be isolated from many sources with various characteristics. There are indications that fetal or close to fetal tissue sources contain MSC with relatively undifferentiated phenotype with respect to MSC from adult sources. Moreover, umbilical cord (C)-MSC may have better chondrogenic differentiation potential than bone marrow (B)-MSC. We hypothesize CMSC are different from BMSC, and more efficient than BMSC in stimulating NP regeneration. Methods: MSC were isolated from human bone marrow and umbilical cord with corresponding ethical approval. BMSC and CMSC were characterized for cell surface marker expression profile and differentiation potential.. RT-PCR of interest genes in NP cells isolated from scoliosis and degenerate discs was performed to search for NP degeneration indicators. Conditioned media (CM) was collected from confluent MSC monolayer, and used for stimulation of four batches of degenerated NP cells isolated from human degenerative intervertebral discs. Cell proliferation and cytotoxicity were assessed by MTT assay. Proteoglycan content were measured by DMMB assay. Gene expression of a series of degeneration related molecules including ACAN, SOX9, CDH2, CD55, KRT19, KRT18, FBLN1 and MGP, and fibrosis related molecules, including MMP12, HSP47, COL1A1, COL3A1 and FN1, of NP cells in MSC-CM were determined by real- time RT-PCR. All results were normalized to the control cells in basal medium. The expression of discogenic, chondrogenic and osteogenic markers on BMSC and CMSC were compared by RT-PCR. Results and Conclusion: CMSC were similar to BMSC and fulfilled the minimum criteria of MSC, however the expression of CD146, CD106 and Stro-1 was different, and BMSC had a spontaneous osteogenesis tendency while CMSC expressed chondrogenic marker even without TGF-beta stimulation. BMSC demonstrated a paracrine effect on modulating human degenerated NP cells towards a non-degenerative phenotype in stimulating cell proliferation, slightly enhancing proteoglycan production, upregulating KRT19 while downregulating MMP12. Compared with BMSC, a higher paracrine effect of CMSC was disclosed in modulating the phenotype of NP cells in all aspects tested, and an intrinsic higher expression on CMSC of ‘potential NP markers’, including KRT19, KRT18 and CD55, but lower expression of osteogenic markers, including RUNX2 and ALPL, was revealed, which indicate a higher potential of CMSC for future clinical application to treat IVD degeneration diseases. KRT19 and MMP12 were also confirmed to be the highest differentially expressed candidate genes between cultured scoliosis and degenerated human NP cells, indicating a high indicator potential of NP degeneration. Furthermore, a subpopulation was detected in the degenerated NP cells that possessed macrophage-like phenotype and activities, which may play a role in the pathogenesis of IVD degeneration. In conclusion, studies in this thesis highlighted CMSC as a superior source than BMSC for IVD repair. Further investigations into the active agents in the conditioned media and the signalling pathway may help to elucidate the mechanism of the effect. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Intervertebral disc tissue engineering is challenging because it involves the integration of multiple tissues with distinct structures and compositions such as lamellar annulus fibrosus, gel?like nucleus pulposus and cartilage endplate. Each of them has different compositions and different structures. It is hypothesized that integration of tissues can be enhanced with appropriate mechanical and biological stimuli. Meanwhile, effect of torsional stimulus on cell re?orientation in mesenchymal stem cell?collagen tubular constructs is investigated in this study. Furthermore, it is proposed that these findings can be used to fabricate a multicomponent unit for intervertebral disc tissue engineering. It has been demonstrated that mechanical and biological stimuli can stabilize the interface between osteogenic and chondrogenic differentiated constructs with enhanced ultimate tensile stress while the phenotype of osteogenic and chondrogenic differentiated constructs were maintained. Scanning electronic microscopic images have shown aligned collagen fibrils and presence of calcium at the interface, indicating the possibility of the formation of a calcified zone. In addition, it is proven that torsional stimulus triggered re?orientation of mesenchymal stem cells in collagen lamellae towards a preferred angle. Cell alignments were confirmed by using a MatLab?based program to analyze the actin filament and the cell alignment via Phalloidin and Hematoxylin staining, respectively. Cells and actin filaments were inclined around 30o from the vertical axis, while cells and filaments in the control group (static loading) aligned along the vertical axis. Furthermore, a double?layers bioengineered unit was fabricated, with intact osteogenic differentiated parts at both ends. Comparatively higher cell density was observed at the interface between layers, demonstrating the interactions between layers, while the phenotype of each part was maintained in 14 days culture. This study concludes that a multi?components bioengineered unit with preferred cell alignments can be fabricated. This provides new insights to future development of bioengineered spinal motion segment for treating late stage disc degeneration. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Low back pain (LBP) is associated with intervertebral disc (IVD) degeneration and exerts enormous socioeconomic burdens on the society. The nucleus pulposus (NP) is the structural and functional core of the IVD, and plays vital roles in its homeostasis. Although the etiology of IVD degeneration is not fully understood, the cellular changes of the NP have been proposed to be associated with degeneration. Conventional management for IVD degeneration primarily targets to relieve LBP and other symptoms without restoring or preserving disc function. Novel therapeutic strategies have emerged with an aim to retard or even reverse disc degeneration. In particular, the use of growth factors, such as the bone morphogenetic proteins (BMP), has received considerable attention due to their anabolic effects on extracellular matrix (ECM) synthesis by NP cells. BMP-2 and BMP-7 are of great interest for their involvement in osteogenesis, chondrogenesis, and development and maintenance of the IVD. To date, the benefits of BMP-2 on disc degeneration are controversial, given the inconsistent findings from animal model studies. The effectiveness of BMP-7 in disc repair, however, has been well demonstrated both in vitro and in vivo. A better understanding of the differences between BMP-2 and BMP-7 regulatory action on NP cells may facilitate future applications of BMP in disc repair/regeneration. This study hypothesized that BMP-2 and BMP-7 act differentially on human NP cells via different signal transduction processes. The differential effect of BMP-2 and BMP-7 was first tested in bovine NP cells using a three-dimensional culture system (alginate beads). Both BMP-2 and BMP-7 enhanced ECM production and phenotypic characteristics of bovine NP cells. Notably, BMP-7 was significantly more potent than BMP-2 in this regard. The effects of BMPs were further tested on non-degenerated (ND-NP) and degenerated (D-NP) human NP cells. The DMMB assay revealed that BMP-7 exerted a superior up-regulatory action on GAG production of D-NP cells compared to BMP-2. Furthermore, the overall response of D-NP cells to BMP-2 and BMP-7 was significantly lower than ND-NP cells. Immunohistochemical staining and quantitative RT-PCR assays demonstrated that D-NP cells possess a more fibroblastic and less chondrocyte-like phenotype than ND-NP cells. At the mRNA level, the BMP receptor BMPR1A was not expressed in D-NP cells. BMP-7, but not BMP-2, induced expression of BMPR1A in D-NP cells. On the other hand, gene expression of selected TGF-β pathway components and hypoxia pathway components were significantly up-regulated by BMP-2 but down-regulated by BMP-7. These findings suggest that D-NP cells can activate differential molecular cascades in response to BMP-2 and BMP-7. In conclusion, this study showed a superior effect of BMP7 in up-regulation of classical BMP signaling components including BMP receptor BMPR1A. The reduced responsiveness of D-NP cells to BMP-2 and BMP-7 stimulation may be related to a different expression pattern of BMP receptors. This study provides insights into the differential regulatory actions of BMP-2 and BMP-7 on human NP cells and facilitates the future application of BMPs in managing disc degeneration. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

No description available.

Thesis (M.D.)--University of Adelaide, 1989. / Includes bibliographical references (leaves 107-109).

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 154-162). Also available in print.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 96-103).

Thesis (Ph.D.)--Drexel University, 2009. / Includes abstract and vita. Includes bibliographical references (leaves 145-161).