Diabetes, paralysis, being bed ridden and other conditions can result in chronic wounds that fail to heal, with significant personal consequences for patients and an annual cost of £4.5-5.1 billion annually (Guest et al., 2015) to the National Health Service. Animal models for assessing new wound healing therapies have inherent issues related to animal suffering and in some cases, their relevance to human tissues. ‘Proof of concept’ in vitro testing for new treatments typically uses scratch wounds in monolayer cultures of keratinocytes or fibroblasts but such highly simplified two-dimensional systems fail to account for the complexities of the real wound environment. In particular, the signalling between nerves and epithelial cells, and signalling by the array of immune cells, which collectively promote healing. Additionally, cells behave differently in a 3D environment, changing their sensitivity to the extracellular signals that drive wound closure and interacting with the matrix texture, physical stresses and molecules in their immediate vicinity. A few 3D human skin models have been established recently (using an air water interface to encourage skin-like keratinisation). Some incorporate neuron co-cultures (Lebonvallet et al., 2011; Martorina et al., 2017) but none also incorporate immune cells, which are essential players in the healing process, and these systems have never been used to explore the process of wound closure.
Aim: To develop an improved in vitro 3D skin, sensory neuron, immune cell co-culture model that better recapitulates key aspects of the natural wound environment. To manipulate the environmental conditions to optimise wound closure. To better understand the molecular basis of the interaction and interdependency between skin, neuron and immune cells in the healing process with a view to developing novel clinical therapies.
Experimental plan: We will co-culture human keratinocytes (from human skin or a cell line of human keratinocytes) and human dorsal root ganglion neurons (a cell line) on modified extracellular matrices. In some cases we will use a flexible substratum and a mechanical frame to mimic the physical properties and repetitive stretch they experience in human skin. This aspect is absent in existing in vitro wound healing models and our work will determine whether it contributes to scar formation and how excessive scarring could be prevented. Once the skin/nerve co-cultures are well established we will make full thickness circular punch wounds, seeding the wounds with human immune cells (focussing on monocytes, macrophages proinflammatory/wound healing types and T cells). Wound closure will be assessed by measuring wound area at discrete time points and by using histology, and immunofluorescence, qPCR, and flow cytometry techniques to define the expression and location of wound healing parameters. We will identify the 3D geometry, the molecular properties and the cell type profiles in the wounds over time. The healing of our skin model will be assessed under clinical conditions know to alter the rate of healing including high glucose, inflammation and hypoxia. In some cases we will cover the wound area with dressings shown clinically to improve healing compared to standard gauze dressings, but whose mode of action is not yet clear at the cellular level.
Impact: This model will expedite preclinical testing of new therapies for recalcitrant wounds with the potential to ease patient suffering and reducing the financial burden of care of chronic wounds to the NHS. We envision a particular utility for diabetic ulcers since healing could be compared in cultures derived from diabetic and non-diabetic donors, permitting a better understanding of the molecular basis for the failure of diabetic wound healing and potential innovative treatments.
This project is advertised in relation to the research areas of MEDICAL SCIENCES. Formal applications can be completed online: https://www.abdn.ac.uk/pgap/login.php
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Guest JF, Ayoub N, McIlwraith T, et al (2015) Health economic burden that wounds impose on the National Health Service in the UK. BMJ Open 2015;5:e009283. doi: 10.1136/bmjopen-2015-009283
Lebonvallet N, Boulais N, Le Gall C, et al, (2011) Effects of the re-innervation of organotypic skin explants on the epidermis. Exper Dermatol. 21:154-160 Doi:10.1111/j.1600-0625.2011.01421.x
Martorina F, Casale C, Urciuolo F, et al (2017). In vitro activation of the neuro-transduction mechanism in sensitive organotypic human skin model. Biomaterials 113:217-229.