published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Nursing Studies / Master / Master of Nursing in Advanced Practice

published_or_final_version / Nursing Studies / Master / Master of Nursing in Advanced Practice

published_or_final_version / Endodontics / Master / Master of Dental Surgery

published_or_final_version / Endodontics / Master / Master of Dental Surgery

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Medical Sciences

published_or_final_version / Public Health / Master / Master of Public Health

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

Acute glaucoma is an ocular emergency and sight -threatening disease which is caused by a sudden increase in intraocular ocular pressure (IOP) due to blockage of aqueous humor outflow. Acute glaucoma can result in permanent loss of visual acuity and visual field (VF). Prophylactic or therapeutic medicine is rare for acute glaucoma. In animal studies, a well-established model to investigate this acute IOP spike is by fluid infusion and adjustment of the fluid level to induce high IOP within a few seconds. However, there is no blockage of aqueous outflow and the increase in intraocular pressure is unrealistically rapid. To mimic the IOP profile in human acute glaucoma attack, we propose the use of an ophthalmic viscosurgical device (OVD), Healon 5 (AMO, Santa Ana, CA, USA) which is injected intracamerally to block aqueous outflow. The IOP is allowed to increase naturally inside the globe. We found that Healon 5 can induce an acute elevation in IOP with very similar characteristics to those observed in humans. For example, the IOP profile during the attack, changes in the anterior segment and retinal nerve fibre layer (RNFL) thinning are all consistent with findings in human acute angle closure glaucoma (AACG). We believed that our new model can more accurately reflect acute glaucoma than other animal models. Based on these findings we further tested the neuroprotective effect of Lycium barbarum polysaccharides (LBP) on retinal neurons against an acute rise in IOP (attack) with the new model. L. barbarum is an herb that has been used in Chinese medicine for thousands of years. The fruit of this plant is believed to be good for the health of the eyes. In our study we found that oral administration of LBP preceding an acute glaucoma attack can preserve the visual function of the animals despite the loss of neurons in the retinal ganglion cell layer (RGCL). L. barbarum intake seems to inhibit secondary cell death and progression of the disease. In conclusion, we had successfully established a new acute glaucoma attack animal model by intracameral injection of Healon 5. This model more closely resembles the condition observed in human acute glaucoma. We also found that LBP has a prophylactic neuroprotective effect against an acute glaucoma attack in animals. It can protect the visual function and possibly inhibit secondary cell death. Oral consumption of LBP as a health supplement may provide extra benefit to people who are at high risk of developing acute glaucoma, in addition to the protective effects of LBP against other diseases. / published_or_final_version / Anatomy / Master / Master of Philosophy