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

published_or_final_version / abstract / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Intervertebral disc(IVD) degeneration is associated with low back pain (LBP).Despite the endplates are located adjacent to the IVD, the phenotypes of endplate and vertebral bone marrow changes remain unclear. The objective of this study is to assess the prevalence and the associated determinants of these phenotypes of the lumbar spine. 2449 Southern Chinese subjects (aged 10 –88 years) undergone sagittal T2-weighted magnetic resonance imaging (MRI)examination to assess the presence of endplate and vertebral bone marrow changes including Schmorl’s nodes (SN) and Modic changes (MC),respectively, and scored for additional radiographic features over the lumbar spine. Subjects’ demographics, clinical profile, and functional status were assessed by means of standardized questionnaires. Anovel6-domain SN morphological classification based on MRI was developed to further analyze the characteristics of SN and its association with disc degeneration (DD). The prevalence of SN was 16.4%. Males, taller and heavier individuals had a significantly increased likelihood of SN. Overall presence of SN was age-independent, but was significantly associated with DD, and linearly correlated with increase in severity of DD. SN were particularly associated with severe DD at the upper two lumbar levels (L1/2 – L2/3). Based on the SN classification system, specific SN characteristics and endplate linkage patterns were found. Of these, two SN types were identified:“Typical SN” and “Atypical SN”. “Typical SN” were those smaller size SN with various shapes that were frequently located at caudal endplates of L1/2–L3/4disc levels. One variant of “Atypical SN” were those rectangular shape SN, which predominantly located at the posterior region of the rostral endplates of L1/2–L2/3disc levels. The other variant of “Atypical SN” was larger size SN with irregular shape that frequently presented at the L4/5endplates, they were also likely to be associated with marrow changes. Despite “Atypical SN” only entailed 8.3% of all identified SN, they were associated with increased severity of DD than “Typical SN”. Although the overall prevalence of MC among Southern Chinese was low (5.8%), after adjustments for other confounding factors, the presence of MC was associated with the presence and severity of LBP. Interestingly, the determinants of MC at upper (L1/2 – L3/4) and lower (L4/5 – L5/S1) lumbar showed distinct difference. The presence of MC at upper lumbar levels was only associated with the disc integrity only (i.e. the presence of disc displacement, and DD score), while the presence of MC at the lower levels, apart from the disc integrity, was also associated with increasing age, the presence of SN, smoking and obesity status. This study is the largest MRI study assessing the phenotypes of endplate and vertebral bone marrow changes in-vivo. The prevalence and distribution of SN and MC vary throughout the lumbar spine, and there are level and region-specific variations regarding these phenotypes. Determinants of SN and MC may be similar but do vary, suggesting distinct etiological factors. Findings of this study broaden the understanding of the various phenotypes of the lumbar spine and its association with DD. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Orthopaedics and Traumatology / Master / Master of Philosophy

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