Date of Award
Doctor of Philosophy
Marie van der Merwe
The overall goal of tissue engineering research is to develop resorbable templates that induce functional regeneration in damaged tissues within the body. The insertion of these templates into the body requires the creation of a wound, triggering the tissue response continuum that occurs with any injury to vascularized tissue. Integral to this tissue response is the activity of neutrophils, the predominant immune cells which flood any wound site soon after injury and power the initial inflammatory response. While neutrophil inflammatory behavior is key to creating the acute inflammation response necessary to begin the healing process, excessive neutrophil inflammatory activity has been implicated in creating a state of chronic inflammation which impairs wound healing. In such environments, the neutrophil release of anti-bacterial superoxide and proteases causes excessive tissue degradation which prevents the wound from closing. Excessive neutrophil NETosis, a response in which a mixture of DNA and degradative proteases is ejected to trap and kill bacteria, can also lead to fibrotic capsule formation surrounding a tissue engineering template which prevents tissue-template integration. Discovering ways to mitigate this neutrophil inflammatory response will enable the design of more effective tissue engineering templates and treatments for chronic wounds and other inflammatory diseases. Manuka honey is a honey variety produced by bees from the nectar of the Leptospermum scoparium shrub of New Zealand which has potent wound healing properties. When applied to a wound, Manuka honeys high solute concentration creates an osmotic gradient which draws fluid and nutrients up from the subcutaneous tissue into the wound site and pulls debris and bacteria out of the wound. The low pH of the honey creates a favorable environment for fibroblast and macrophage activity, while floral-derived flavonoids scavenge reactive oxygen species to reduce tissue damage. Manuka honeys unique methylglyoxal content is a potent weapon against bacterial infection, including antibiotic-resistant bacteria. For these reasons, Manuka honey has become an increasingly predominant wound treatment, and has also become the subject of research as a tissue engineering bioactive additive to reduce inflammation and eliminate bacterial infection. However, the effect of Manuka honey on neutrophil inflammatory behavior has yet to be examined. As this phenomenon is a crucial determinant of the success or failure of tissue engineering templates, it is imperative that the response of neutrophils to Manuka honey be observed and characterized. The work contained in this dissertation characterizes the effect of Manuka honey on a variety of neutrophil activities. Chapter 1 contains an introduction into the role of neutrophils in inflammation and wound healing, and Chapter 2 gives a background explanation of the various mechanisms of Manuka honey in wound healing and a literature review of honey in tissue engineering research. Chapters 3, 4, and 5 utilize a dHL-60 cell line model of a neutrophil, which allows for more experimental reproducibility than primary human neutrophils. Chapter 3 examines the cytotoxic limit of Manuka honey on a dHL-60 neutrophil model, which was found to be in the range of 3-5% v/v, and investigates the honeys effect on several neutrophil inflammatory behaviors. A cytochrome C assay was used to measure Manuka honeys effect on superoxide release, and it was found that concentrations of 1% v/v honey and above decrease superoxide release after 24 hours. A Boyden chamber assay was used to measure Manuka honeys effect on dHL-60 chemotaxis towards fMLP, and a Western blot for the NF-B inhibitor (IB) measured the honeys effect on the activation of the NF-B pathway. These experiments demonstrated that 0.5-3% v/v honey reduce chemotaxis and IB phosphorylation in a dose-dependent fashion. Together, the work contained in Chapter 3 indicates that Manuka honey reduces several neutrophil inflammatory behaviors.Chapter 4 contains an in-depth examination of how Manuka honey affects dHL-60 cytokine, chemokine, and matrix-degrading enzyme release in the presence of various inflammatory stimuli. The results indicated that 0.5% honey decreased the release of the inflammatory signals TNF-, IL-1, MIP-1, MIP-1, and IL-12 p70, the matrix-degrading enzymes MMP-9 and MMP-1, the angiogenic growth factor FGF-13, and the anti-inflammatory signals IL-1ra and IL-4, but increased the pro-inflammatory signals MIP-3 and IL-8, the matrix-degrading enzyme Proteinase 3, and the angiogenic growth factor VEGF. However, 3% honey reduced the release of all measured analytes except TNF-, which was increased. Similarly, the work described in Chapter 5 tests the response of dHL-60 cytokine, chemokine, and matrix-degrading enzyme release to Manuka honey when in an anti-inflammatory stimulation environment. The results of this work indicate that when under anti-inflammatory stimulation, 0.5% honey increases the release of the pro-inflammatory signals IL-8, MCP-1, MIP-1, and MIP-3, the anti-inflammatory signals IL-4 and IL-1ra, and the angiogenic growth factor FGF-13 while reducing the release of the matrix-degrading enzyme Proteinase 3. However, 3% honey reduced the release of all analytes except the inflammatory signals TNF- and IL-8, which were increased. The results of these two chapters indicate the dramatic difference that a slight change in the dose of Manuka honey, from 0.5% to 3%, can elicit a completely different cytokine response in the inflammatory environment. In Chapter 6, Manuka honey is incorporated into electrospun templates with small-diameter (SD) and large-diameter (LD) fibers and its effect on porosity, the honey release rate, and the effect on the NETosis response of primary human neutrophils are examined. Honey incorporation was found to create more restrictive pore sizes within both SD and LD templates. SD templates were found to release honey at a higher rate compared to LD templates with equivalent honey loads, as expected from their higher surface-area-to-volume ratio. Fluorescence imaging and an MPO assay indicated that 0.1%-1% Manuka honey reduced neutrophil NETosis, on the surface of both SD and LD templates while also reducing MMP-9 output. Together, these results indicate a role for Manuka honey in the reduction of neutrophil inflammatory activity in the area surrounding an electrospun tissue engineering template.
Dissertation or thesis originally submitted to ProQuest
Minden-Birkenmaier, Benjamin Alexander, "Manuka Honey as a Tissue Engineering Bioactive: Effect on Neutrophil Inflammatory Behavior" (2020). Electronic Theses and Dissertations. 2678.