Manuka Honey as a Tissue Engineering Bioactive: Effect on Neutrophil Inflammatory Behavior

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

Honey-Based Templates in Wound Healing and Tissue Engineering

Over the past few decades, there has been a resurgence in the clinical use of honey as a topical wound treatment. A plethora of in vitro and in vivo evidence supports this resurgence, demonstrating that honey debrides wounds, kills bacteria, penetrates biofilm, lowers wound pH, reduces chronic inflammation, and promotes fibroblast infiltration, among other beneficial qualities. Given these results, it is clear that honey has a potential role in the field of tissue engineering and regeneration. Researchers have incorporated honey into tissue engineering templates, including electrospun meshes, cryogels, and hydrogels, with varying degrees of success. This review details the current state of the field, including challenges which have yet to be overcome, and makes recommendations for the direction of future research in order to develop effective tissue regeneration therapies

Application of electrospun fibers in tissue engineering

The process of electrospinning has experienced decades of development for a variety of applications including air filtration and tissue engineering. However, it has not been until the past decade that this process has been a focus in the field of tissue engineering. The unique properties of electrospun fibers have drawn remarkable attention within the field of tissue engineering due to its capacity to mimic the native tissue structural element, the extracellular matrix, and its ability to promote cell proliferation and enhanced tissue regeneration. Herein, we have summarized some different applications of electrospun fibers in tissue engineering while demonstrating the cellular interactions between various cells and electrospun fiber scaffolds. The key aspect is a focus on the potential use of electrospinning in tissue regeneration. The hope is that this book will be beneficial to students and researchers in developing a general knowledge of the tissue engineering and regeneration application of electrospun fibers. We hope it will also inspire their curiosity and creativity to allow for the fabricating of advanced tissue engineering scaffolds for use in developing successful products that will allow for improving the quality of life

Methods for Quantifying Neutrophil Extracellular Traps on Biomaterials

Neutrophils rapidly accumulate at sites of inflammation, including biomaterial implantation sites, where they can modulate the microenvironment toward repair through a variety of functions, including superoxide generation, granule release, and extrusion of neutrophil extracellular traps (NETs). NETs are becoming increasing implicated as a central player in the host response to a biomaterial, and as such, there is a need for reliable in vitro methods to evaluate the relative degree of NETs and quantify NETs on the surface of biomaterials. Such methods should be relatively high throughput and minimize sampling bias. In this chapter, we describe two procedures, (1) fluorescent image analysis and (2) a NETs-based ELISA, both of which have been specifically optimized to quantify NETs generated from human neutrophils on electrospun polydioxanone templates. Both methods are valid and also compatible with tissue culture plastic, but have a variety of advantages and disadvantages. Therefore, both methods can be used to concomitantly study NETs on the surface of a biomaterial. Finally, while these methods were developed for electrospun templates in a 96-well cell culture plate, they may be easily adapted to a large scale and for other biomaterials, including but not limited to metallics, ceramics, and natural and synthetic polymers

Manuka Honey Modulates the Inflammatory Behavior of a dHL-60 Neutrophil Model under the Cytotoxic Limit

Recent work has shown that Manuka honey, an increasingly popular wound additive with potent antibacterial properties, also has anti-inflammatory properties. However, little research has been done examining its effect on neutrophils. This study investigates the hypothesis that Manuka honey reduces neutrophil superoxide release and chemotaxis and reduces the activation of the inflammatory nuclear factor-κB (NF-κB) signaling pathway under honey’s cytotoxic limit. A differentiated HL-60 cell line was used as a neutrophil model and cultured in various concentrations of Manuka honey for 3 and 24 hours to measure cytotoxicity via mitochondrial activity and visual trypan-exclusion count. Cytochrome C and Boyden chamber assays were used to measure the effect of Manuka honey on superoxide release and chemotaxis toward fMLP, respectively. Additionally, a Western blot for NF-κB inhibitor α (IκBα) was performed to measure Manuka honey’s effect on the NF-κB pathway via IκBα phosphorylation. The results indicate a cytotoxic limit of 3-5% v/v. The presence of 1% honey decreased superoxide release at 24 hours. The 0.5, 1, and 3% honey concentrations reduced chemotaxis and IκBα phosphorylation in a dose-dependent fashion. These results suggest that Manuka honey significantly reduces neutrophil recruitment and inflammatory behavior in the wound site in a dose-dependent fashion under the cytotoxic limit

Manuka honey reduces NETosis on an electrospun template within a therapeutic window

Manuka honey, a topical wound treatment used to eradicate bacteria, resolve inflammation, and promote wound healing, is a focus in the tissue engineering community as a tissue template additive. However, its effect on neutrophil extracellular trap formation (NETosis) on a tissue engineering template has yet to be examined. As NETosis has been implicated in chronic inflammation and fibrosis, the reduction in this response within the wound environment is of interest. In this study, Manuka honey was incorporated into electrospun templates with large (1.7-2.2 μm) and small (0.25-0.5 μm) diameter fibers at concentrations of 0.1%, 1%, and 10%. Template pore sizes and honey release profiles were quantified, and the effect on the NETosis response of seeded human neutrophils was examined through fluorescence imaging and myeloperoxidase (MPO) analysis. The incorporation of 0.1% and 1% Manuka honey decreased NETosis on the template surface at both 3 and 6 h, while 10% honey exacerbated the NETosis response. Additionally, 0.1% and 1% Manuka honey reduced the MMP-9 release of the neutrophils at both timepoints. These data indicate a therapeutic window for Manuka honey incorporation into tissue engineering templates for the reduction in NETosis. Future in vivo experimentation should be conducted to translate these results to a physiological wound environment

Manuka honey modulates the release profile of a dHL-60 neutrophil model under anti-inflammatory stimulation: Manuka Honey Modulates Anti-inflammatory dHL-60 Cytokine Output

Manuka honey, a wound treatment used to eradicate bacteria, resolve inflammation, and promote wound healing, is a current focus in the tissue engineering community as a tissue template additive. However, Manuka honey\u27s effect on neutrophils during the inflammation-resolving phase has yet to be examined. This study investigates the effect of 0.5% and 3% Manuka honey on the release of cytokines, chemokines, and matrix-degrading enzymes from a dHL-60 neutrophil model in the presence of anti-inflammatory stimuli (TGF-β, IL-4, IL-4 +IL-13). We hypothesized that Manuka honey would reduce the output of pro-inflammatory signals and increase the release of anti-inflammatory signals. The results of this study indicate that 0.5% honey significantly increases the release of CXCL8/IL-8, CCL2/MCP-1, CCL4/MIP-1β, CCL20/MIP-3α, IL-4, IL-1ra, and FGF-13 while reducing Proteinase 3 release in the anti-inflammatory-stimulated models. However, 3% honey significantly increased the release of TNF-α and CXCL8/IL-8 while reducing the release of all other analytes. We replicated a subset of the most notable findings in primary human neutrophils, and the consistent results indicate that the HL-60 data are relevant to the performance of primary cells. These findings demonstrate the variable effects of Manuka honey on the release of cytokines, chemokines, and matrix-degrading enzymes of this model of neutrophil anti-inflammatory activity. This study reinforces the importance of tailoring the concentration of Manuka honey in a wound or tissue template to elicit the desired effects during the inflammation-resolving phase of wound healing. Future in vivo investigation should be undertaken to translate these results to a physiologically-relevant wound environment

The Effect of Manuka Honey on dHL-60 Cytokine, Chemokine, and Matrix-Degrading Enzyme Release under Inflammatory Conditions

A large body of and evidence indicates that Manuka honey resolves inflammation and promotes healing when applied topically to a wound. In this study, the effect of two different concentrations (0.5% and 3% v/v) of Manuka honey on the release of cytokines, chemokines, and matrix-degrading enzymes from neutrophils was examined using a differentiated HL-60 cell line model in the presence of inflammatory stimuli. The results indicate that 0.5% honey decreased TNF-α, IL-1β, MIP-1α, MIP-1β, IL-12 p70, MMP-9, MMP-1, FGF-13, IL-1ra, and IL-4 release, but increased MIP-3α, Proteinase 3, VEGF, and IL-8 levels. In contrast, 3% honey reduced the release of all analytes except TNF-α, whose release was increased. Together, these results demonstrate a dose-dependent ability of Manuka honey to modify the release of cytokines, chemokines, and matrix-degrading enzymes that promote or inhibit inflammation and/or healing within a wound. The findings of this study provide further guidance for the future use of Manuka honey in wounds or tissue engineering templates. Future investigation is warranted to validate the results and translate these results to physiologically relevant environments