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

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Degenerative disc disease (DDD) is a medical condition whereby the intervertebral discs (IVD) of the human spine degenerates and may cause pain which significantly affects the quality of one掇 life. Its prevalence has sparked off much research in deciphering its causes and developing new treatments. Recently, attempts to treat this degenerative problem have turned to seeking answers from regenerative medicine. One approach is to deliver mesenchymal stem cells (MSCs) with or without carriers to the nucleus pulposus (NP) in degenerative disc to restore both its structural and functional properties. However, the optimal conditions and signals for inducing MSCs differentiation toward NP-like phenotype have not been identified. This work aimed to develop injectable microspheres with matrix microenvironment mimicking that of native NP tissue for MSCs delivery. Firstly, it was aimed to establish a collagen microencapsulation based 3D culture system for maintenance of the phenotype of nucleus pulposus cells (NPCs) and remodeling of the collagen matrix to one that was similar to the native NP. Secondly, it was aimed to optimize a decellularization protocol for complete removal of the encapsulated NPCs with minimal loss of remodeled extracellular matrix. Thirdly, it was aimed to investigate whether this acellular matrix produced by NPCs was inductive for MSCs discogenic differentiation. Finally, it was aimed to evaluate the efficacy of the MSC-seeded acellular matrix microspheres in a pilot rabbit disc degeneration model. It demonstrated that NPCs maintained their phenotype, survived within the collagen microspheres and produced NP-like ECM such as glycosaminoglycan (GAG) and collagen type II. GAG production of NPCs was found to positively correlate with the dosage of TGF-? within a short period, initial collagen concentration and cell seeding density. An optimized decellularization protocol with 50mM SB-10, 0.6mM SB-16 and 0.14% Triton X-200 was established to completely remove the encapsulated NPCs with partial retention of the GAG-rich matrix. The decellularized microspheres were able to be repopulated with human MSCs (hMSCs) or rabbit MSCs (rMSCs). Within the NPC-derived acellular matrix, the repopulated hMSCs were able to partially exhibit NPC phenotype with upregulated expression of a few NPC markers and NP-like ECM according to histological, biochemical, immunohistological and real-time PCR results. In the pilot in vivo evaluation study, preliminary results showed that intra-discal injection of MSCs reseeded acellular NPC-matrix microspheres maintained a better water content than the control MSC-microspheres without the NPC-derived acellular matrix. This work reconstituted in vitro a NP-like 3D matrix and provided preliminary evidence on discogenic differentiation of MSCs in such a matrix environment. This work laid down the foundation to future development of stem cell-based therapies for DDD. Further studies should focus on deciphering the soluble and insoluble composition of such a NP-like matrix environment and understanding the molecular mechanism of the cell-matrix interactions involved. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

Degeneration of the lumbar intervertebral disc, a condition widely implicated in the cause of low back pain among adult humans, is typically characterised by progressive biochemical and structural changes to the extracellular matrix. Comprehensive descriptions of the structural and functional inter-relationships within the extracellular matrix are therefore critical to understanding the degenerative process and developing effective treatments. In the anulus fibrosus, this matrix has a complex, hierarchical architecture comprised of collagens, proteoglycans, and elastic fibres. Elastic fibres are critical constituents of dynamic biological structures that functionally require elasticity and resilience. Studies to date of elastic fibre network structure in the anulus fibrosus have been qualitative and limited in scope. Additionally, there is poor understanding of the structural and functional associations between elastic fibres and other matrix constituents such as collagen, and, critically, there have been no studies directly examining the nature and magnitude of the contribution made by elastic fibres to anulus fibrosus mechanical behaviour. In this thesis, multiple experimental studies are described that specifically examine each of these areas. Novel imaging techniques were developed and combined with histochemistry and light microscopy to facilitate the visualisation of elastic fibres at a level of detail not previously achieved. Examination of elastic fibre network structure revealed architectural differences between the intralamellar and interlamellar regions, suggesting that elastic fibres perform functional roles at distinct levels of the anulus fibrosus structural hierarchy. The density of elastic fibres within lamellae was found to be significantly higher in the lamellae of the posterolateral region of the anulus than the anterolateral, and significantly higher in the outer regions than the inner, suggesting it may be commensurate with the magnitude of the tensile strains experienced by each region of the disc in bending and torsion. The nature of the structure-function associations between elastic fibres and collagen was then examined with respect to the reported structural mechanisms of collagen matrix tensile deformation. Histological assessment of collagen crimp morphology in specimens from which elastic fibres had been enzymatically removed revealed no observable differences when compared with controls, suggesting that any contribution made by elastic fibres to maintaining crimp is minimal. Elastic fibres in anulus fibrosus specimens subjected to radial tensile deformations exhibited complex patterns of re-arrangement, suggesting that they maintain cross-collagen fibre connectivity. Elastic fibres were also observed to maintain physical connections between consecutive lamellae undergoing relative separation. Finally, the nature and magnitude of the contribution made by elastic fibres to anulus fibrosus mechanical properties at the tissue level was investigated using a combination of biochemically verified enzymatic treatments and biomechanical tests. Targeted degradation of elastic fibres resulted in a significant reduction in both the initial modulus and the ultimate modulus, and a significant increase in the extensibility, of radially oriented anulus fibrosus specimens. Separate treatments and mechanical tests were used to account for any changes attributable to non-specific degradation of glycosaminoglycans. These results suggest that elastic fibres enhance the mechanical integrity of the anulus fibrosus extracellular matrix in the direction perpendicular to the plane containing the collagen fibres. In summary, the results of the studies presented in this thesis provide important new insights into the structure and function of the anulus fibrosus elastic fibre network, and highlight its potential importance as a contributing or ameliorating factor in the progression of the structural and mechanical changes associated with intervertebral disc degeneration. Additionally, these results establish an improved framework for the development of more accurate analytical and finite element models to describe disc behaviour. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317006 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2008

Bibliography: leaves 148-160. / xix, 160, [40] leaves, [19] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the process of neovascularization of intervertebral disc components, with ageing and in disease. Establishes the extent of end plate vascularization in the lumbar discs of young normal sheep, and demonstrates that some components of the disc undergo neovascularization in response to physical trauma, possibly as a part of tissue repair response. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 1996?

Bibliography: leaves 102-116. / 116, [43] leaves, [51] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Analyses the characteristics and relative incidence of annular defects in the human lumbar spine and investigates their role in the pathogenesis of invertebral disc degeneration. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 1992

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.