Intervertebral disc (IVD) degeneration is associated with low back pain (LBP), which affects approximately 80% of the global population and is a huge socioeconomic burden. Presently, conservative and surgical therapies are inadequate and have poor long term outcomes; hence there is a necessity for alternative options, such as cell-based therapies, that will address the underlying pathogenesis.Mesenchymal stem cells (MSCs), specifically bone marrow (BM-MSCs) and adipose derived (AD-MSCs) have been shown to differentiate to a nucleus pulposus (NP) like cell (discogenic differentiation); although to date optimum discogenic differentiation protocols have not been defined. The most common method is the use of a transforming growth factor (TGF-β) however, this growth factor is commonly used to induce chondrogenesis. Thus, there is a need to identify growth factors or small molecules that can induce discogenic differentiation and appropriate matrix formation, particularly when exposed to IVD microenvironmental factors. The initial objective of the study was to investigate the discogenic potential of BM-MSCs and AD-MSCs when cultured with members of the TGF-β superfamily. Results showed growth differentiation factor 6 (GDF6) induced an improved discogenic phenotype in human AD-MSCs (aNPCs) resulting in a proteoglycan rich matrix, compared to growth differentiation factor 5 (GDF5) or TGF-β treatment or BM-MSCs exposed to the same growth factors. AD-MSCs supplemented with GDF6 were also exposed to hypoxia and load to mimic the IVD microenvironment. Whilst hypoxia and load increased discogenic differentiation, matrix synthesis was aberrantly altered resulting in a stiffer matrix than that produced in standard conditions. Another feature of the degenerate IVD environment, to which any implanted cells will be subjected to, is the milieu of cytokines specifically interleukin-1β (IL-1β). Thus, the response of aNPCs to IL-1β was compared to native degenerate NP cells. Interestingly, IL-1β had no detrimental catabolic effect on aNPCs suggesting that these cells may be able to withstand the effects of the IL-1β milieu of the degenerate IVD niche. The effect of GDF6 on native NP cells was also investigated and found to restore a nondegenerate NP phenotype by upregulating NP marker gene expression and increasing ECM synthesis. Furthermore, when NP cells were treated with GDF6 and exposed to IL-1β there was still an upregulation of ECM and suppression of catabolic genes, suggesting GDF6 may have a protective role. Having established the efficacy of GDF6, preliminary experiments were undertaken to assess the feasibility of using GDF6 loaded microparticles to provide sustained delivery of the growth factor; results were comparable to exogenous delivery of GDF6. In addition, initial studies were undertaken to develop an ex vivo IVD model in order to test the efficacy of regenerative therapies such as those described throughout the thesis. Taken together,this study shows that GDF6 is a promising biological therapy for IVD regeneration strategies for the treatment of IVD degeneration.
|Date of Award||1 Aug 2016|
- The University of Manchester
|Supervisor||Judith Hoyland (Supervisor) & Stephen Richardson (Supervisor)|