A developmental glycobiology approach to formulate an enhanced hyaluronic acid-based hydrogel for intervertebral disc regeneration

Intervertebral disc degeneration (IDD) is the leading cause of lower back pain, which affects the majority of the world population at some point in their lives. IDD is a progressive event marked by inflammation, loss of cellularity, extracellular matrix degradation, loss of water, and structural deterioration. Standard care includes symptom management, pain relief, and invasive surgeries in advanced cases. Addressing the need for regenerative therapeutic approaches, scientists have shifted their focus on the properties of a unique population of cells in the nucleus pulposus (NP) region of the intervertebral disc (IVD). Notochordal cells (NC) developmentally descend from the notochord before transitioning into what is identified as NP cells after maturity and age. Some animals, such as pigs and non-chondrodystrophic dogs, retain this population throughout their lives, while humans lose it after NP maturation. The changes that occur in the disc, including the loss of NCs, coincide with the pattern leading to degeneration, which suggests that the notochordal cells and their niche have a role in keeping IVD healthy. To understand these mechanisms better, molecular aspects of degeneration and maturation must be studied. One overlooked subject in this regard is glycosylation. It is known that heavily glycosylated molecules are involved in disc degeneration. Considering the loss of NCs and the inflammatory changes that occur during the degeneration of the disc, it is hypothesised that introducing the healthy and young NP glycome within a supportive anti-inflammatory hydrogel network could induce changes in the disc that are indicative of tissue remodelling and regeneration. This study identified the glycosylation patterns in young and mature NPs that are rich in notochordal cells. A thorough analysis of the ultra-performance liquid chromatography (UPLC) and mass spectrometry (MS) data revealed over 300 N-glycan species in the NP, more than 45% of which are sialylated and/or fucosylated. In addition, 5% increase α(1,2/3) fucosylation and 9% decrease in α(2,3) sialylation was detected at maturation. With these properties in mind, a hyaluronic acid (HA) hydrogel network was formulated by crosslinking the carboxylic groups of HA with 8-arm polyethylene glycol (PEG), which was enhanced with the notochordal cell-derived matrix (NCM) to mimic the notochordal niche. This HA-NCM formulation formed a hydrogel within 15 minutes. The fabricated hydrogel showed high swelling capacity (up to 150% in weight), and supported cells such as NPs, NCs and stem cells for at least 14 days. The hydrogel also showed signs of mitigating the pro-inflammatory cytokines in the long-term. To test the regenerative potential of the HA-NCM hydrogel system, the last phase of the project investigated an in vivo study utilising a rat-rail model after introducing an acute injury to the NP. Even though the injury created is more than what is expected in the natural course of IDD, the HA-NCM hydrogel can improve the disc height by 36% after injury. The work described in this thesis introduces a significant potential of a glycomic approach to a clinical problem. Future studies include more robust and efficient methods to analyse the glycosylation patterns and further charcaterisation of NCM to validate its potential while focusing on the mechanistic approaches to induce regeneration and remodelling of the IVD.

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University of Galway

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Attribution-NonCommercial-NoDerivatives 4.0 International

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University of Galway Theses (PhD Theses)