Melbourne Dental School - Theses
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ItemThe Role of Hyaluronic Acid in the Prevention of Neoplastic Therapy-induced Oral Mucositis( 2022)ABSTRACT GENERAL SUMMARY Mucositis is a common and most debilitating complication associated with cancer therapy. Approximately 30–40% of cancer patients treated with chemotherapy develop mucositis; this percentage rises to 60–85% for patients receiving conditioning regimens before hematopoietic stem cell transplantation (HSCT) and can increase to almost 100% for head and neck cancer (HNC) patients receiving radiotherapy concomitant with chemotherapy. The condition affects the entire alimentary canal from the mouth to the anus, causing ulceration and severe pain in both the oral cavity and intestinal tract. The consequences of mucositis are far reaching and can lead to a delay or even cessation of otherwise durable cancer treatment and negatively impacting the benefits of chemoradiation. Mucositis is not only an important driver of patients’ symptoms and infection risk, but its onset is also associated with poor health outcomes, the use of health resources, and incremental costs. Given that mucositis is ultimately a predictable and potentially preventable condition, this project aimed at optimising a pre-clinical model of mucositis and testing the protective effect of a natural compound on chemotherapy-induced mucosal injury. Chapter 1 discusses the generalities of mucositis, its causative agents and risk factors, its pathophysiology diagnosis, and management. After recalling basic concepts of cancer treatment (causative factor) and mucosal structure (target tissue), in this chapter I explain the clinical relevance and economic implications of mucositis. A large section is devoted to the pathophysiology of mucositis as this represents the starting point for the development of novel mechanism-based preventative and therapeutic strategies. The chapter then focuses on oral mucositis and, in this context, types of assessment and scoring scales are extensively discussed. Current management strategies of mucositis are scrutinised in the final part of Chapter 1. Despite its clinically devastating consequences, there is currently little to offer patients in the way of effective treatment to prevent or mitigate mucositis, and this provided the rationale for developing the aims of this project. General materials and methods are detailed in Chapter 2. The experimental design included in vitro experiments using an oxidative stress-induced model of human oral mucosal injury and an in vivo dual murine model of 5-FU-induced oral/intestinal mucositis. Formulations of hyaluronic acid (HA) tested in these models included Mucosamin, cross-linked (xl-), and non-crosslinked high molecular weight HA (H-MW-HA). Cell lines, culture conditions, morphological, functional and molecular assays (e.g. cell viability, cytotoxicity, and proliferation; intracellular ROS production, superoxide dismutase enzyme activity) are described. For the animal study, methodology for clinical, histopathological and morphometric assessment of oral and intestinal mucositis is reported in detail. Specific materials and methods are additionally described in the relevant chapters. In the experiments described in Chapter 3, we aimed to develop and characterise an in vitro model of oral mucosal injury that could mimic the initial events that trigger oral mucositis. Since the molecular mechanisms underlying chemotherapy and radiotherapy-induced oral mucositis involve the production of ROS at early stages and consequent activation of oxidative stress pathways, we established an oxidative stress model on human oral keratinocytes cultures whereby were able to identify optimum concentrations of hydrogen peroxide and incubation period for each of the cell line tested. Such a model allowed the screening of potentially xxx drugs reported later in this thesis. In Chapter 4, the biocompatibility of several hyaluronic acid derivatives in our in vitro cell model system was assessed through an extensive series of experiments. We defined the optimal concentrations of the following compounds: Mucosamin (1%, 5%, 7% and 10% v/v), native high molecular weight HA (H-MW-HA; 0.01%, 0.03%, 0.05%, 0.07% and 0.1% w/v), and cross-linked (xl-) HA (0.01% v/v of xl-HA 5/5, xl-HA 30/30, and xl-HA 100/100). Oral keratinocytes were incubated for 24, 48, and 72 hours in the presence of these HA products and a dose-response curve was developed. All HA compounds tested significantly promoted oral epithelial cell proliferation compared to control, except for Mucosamin; this commercial preparation did not affect cell growth at concentrations of up to 5% (v/v) and was cytotoxic at higher concentrations. Drawing on the results of the pilot experiments described in the previous chapters, in Chapter 5 we tested the protective effects of HA in a model of oxidative stress-induced oral mucosal injury. OKF6 cells were incubated with 400 uM (IC50 value) hydrogen peroxide (H2O2) and the effects of Mucosamin, H-MW-HA, Xl-HA 5/5, Xl-HA 30/30, and Xl-HA 100/100 were tested. While all HA compounds could attenuate to some extent the detrimental effects of ROS, the most marked effects were obtained with H-MW-HA. Specifically, pre- and then co-incubation of OKF6 cells with 0.01 % (w/v) H-MW-HA for 24 hours resulted in a sizeable increase in cell viability when compared to H2O2- treated cells. Remarkably, H-MW-HA also reduced the intracellular level of H2O2-induced ROS production, as measured by the ability of cells to oxidize CM-H2DCFDA. Hence, H-MW-HA at 0.01 % (w/v) was selected for the animal study. Chapter 6 describes a detailed set of experiments to show that HA prevents oral and intestinal mucositis induced by chemotherapy in vivo. To overcome the limitations of current models, whereby oral and intestinal mucositis are studied separately using different, organ-specific models, we first characterized a pre-clinical dual (oral and intestinal) murine model of mucositis by using intravenous 5-FU injections (50 mg/kg) every 48 hours for 2 weeks. In the test group, the mice were pre-treated one day prior to the initial 5-FU treatment and then daily thereafter with high molecular weight HA (H-MW-HA) (0.01 % w/v) in drinking water. Mice were monitored clinically for weight loss, diarrhea (as a surrogate of intestinal injury), and incidence and extent of oral mucositis. Microscopically, histomorphometric analyses of the tongue and intestinal tissues were conducted. The results strongly indicated that H-MW-HA prevented 5-FU-induced damage to the intestinal mucosa and tongue epithelium. We also demonstrated that H-MW-HA enhanced the activity of the SOD enzyme in the blood serum of 5-FU treated mice. This thesis comes to a conclusion with Chapter 7, where our results are discussed in light of current literature. These results reported in this thesis provide an experimental rationale to develop a novel HA-derived treatment as a therapeutic agent to protect against oral and intestinal mucositis associated with chemotherapy in cancer patients. We note that systemic administration of HA as a preventive or therapeutic tool in oral and intestinal mucositis may have profound clinical implications in patients, such as potential interference with cancer treatment, that require further elucidation. Future studies will address this important aspect of our research.