abstract / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Back pain is a prevalent musculoskeletal disorder affecting populations worldwide. Degeneration of the intervertebral discs (IVD) is indicated to be one of the main etiologic factors of back pain. The loss of proteoglycan and consequently dehydration and reduction of swelling of the intervertebral disc core, the nucleus pulposus (NP), is one of the earliest degenerative events. The replacement of large vacuolated notochordal cells by the smaller chondrocyte-like cells in the NP coincide with the onset of IVD degeneration, the loss of the notochordal cells was therefore postulated to be a cause of the degeneration and associated with ageing . To date, the true origin of the smaller chondrocyte-like cells was still under dispute. In this study, Egfp reporter was constitutively and permanently activated in the notochord via specific Cre expression by the Foxa2mNE enhancer under the recombination system in the Foxa2mNE-cre; Z/EG double transgenic mice. The notochord descendent cells were therefore labeled with green fluorescent protein (GFP) and their localization and expression characteristic were tracked during development, maturation, ageing of mouse tail IVD. To further investigate the cellular changes during degeneration, disc degeneration in the murine disc was induced by puncture. The GFP labeled notochord cells were demonstrated to be entrapped and remained in the NP of the murine disc. With the validation of degeneration by the multichromatic FAST staining, a grading system based on altered sulfated glycosaminoglycan content and cellular organization specialized to murine disc was proposed. The segregation of the notochordal NP cell mass segregation was found to be a common pathway of both age-related and puncture-induced degeneration. While apoptosis and loss of NP cells was presented in puncture-induced disc degeneration, invasion of lamellae cells was only found in severely degenerated discs. Consistent with the findings of the murine puncture model, increased expression of Col2a1 and Col1a1 were demonstrated in punctured discs by ISH, which the notochordal NP cells were showing a remarkable elevation in expression. The data supports a chondrogenic differentiation of the endogenous notochordal cells instead of invasion of chondrocyte-like cells into the NP. Three NP markers identified as a common genes from microarray of rat and human NP, (Carbonic anhydrase3 (Car3), UDP-N-acetyl-alpha-D-galactosamine:polypeptide; N-acetylgalactosaminyltransferase 3 (Galnt3) and HOP homeobox (Obl)), were found specifically co-localized with the notochord descendent cells in all ages and different time-points after puncture, suggesting them as novel but reliable markers for notochordal NP cells. This study provides direct evidence of the origin of NP cells in aged and degenerated IVDs using transgenic mice, and thus enhances the understanding of the role of notochordal cells in the progression of IVD degeneration. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

abstract / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Intervertebral disc (IVD) degeneration could lead to many serious complications including low back pain and disc herniation. However, the mechanism of disc degeneration is not fully understood, hindering the development of the therapeutics to cure this disease. The integrity of the nucleus pulposus (NP), which is derived from the notochord and situated in the core of the IVD, has long been implicated in the function and homeostasis of the IVD. Previous puncture-induced disc degeneration mouse model showed segregation of NP cell mass during the early stage of disc degeneration, indicating that an alteration in the cell adhesion molecule activities is involved in this process. By microarray analysis, our group have revealed specific expression of Cdh2 gene, encoding cadherin 2/N-cadherin, a subtype of cadherins in the NP cells, suggesting a regulatory role of cadherin 2 in the IVD. Cadherins are single transmembrane glycoproteins mediating calcium-dependent intercellular adhesions. Cadherin 2 is involved in chondrogenesis and skeletogenesis, suggesting that it is important in skeletal development and function. This study hypothesized that cadherin 2 is required in the normal IVD development and homeostasis. The purposes of this project is firstly to fully characterize changes in cadherin 2 expression in the normal and degenerative discs in rodent and human, and secondly to examine the effect of loss of function of cadherin 2 on IVD homeostasis by functional blocking of the protein in the rodent NP and conditional knock out of cadherin 2 from the murine NP. The rodent adult NP is similar to human fetal NP, where cadherin 2 is homogeneously expressed in the cell membranes of the notochordal (NC) cells, suggesting that cadherin 2 is a potential NC cell marker. The rodent degenerative NP is similar to human adult NP, where down-regulation of cadherin 2 is observed, the NC cells are replaced by small round cells, and the cell-cell contact is lost. Blocking cadherin 2 function in the rodent NP and conditional knock out of cadherin 2 in the notochord and consequently the NP will lead to transformation of NC cells into small cells, loss of cell-cell contact and a change in the extracellular matrix (ECM), suggesting that cadherin 2 is important in the maintenance of the phenotype and intercellular adhesion of the NC cells. In conclusion, this study indicates that cadherin 2 is mainly expressed in the NC cells of the NP and serves as a potential NC cell marker. It plays a regulatory role in the IVD homeostasis through the maintenance of the NC cell phenotype by intercellular adhesions. This study contributes to the knowledge about the role of cadherin 2 in the disc homeostasis and the early mechanism of disc degeneration, and this would help in developing a therapeutic method to intervene or even reverse the disease process of disc degeneration. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 278-292). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 269-295). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 208-243). Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 208-243). Also available online.

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