Periodic Reporting for period 1 - SKIN SUBSTITUTE (Development of hybrid artificial skin substitute for chronic diabetic wounds) Reporting period: 2018-08-01 to 2020-07-31 Summary of the context and overall objectives of the project In recent years, diabetes has become a global disease with accelerating incidence in newly industrialized countries whose societies have become increasingly westernized. In Ireland, 1 in 10 people is currently diagnosed as having diabetes, which is a disease that occurs when the sugar (glucose) level in the blood is too high. A common side effect of poorly controlled diabetes is damage to nerves and blood vessels in the legs and feet, which can result in the breakdown of skin, and the development of non-healing open wounds called diabetic foot ulcers. The annual cost of treating patients with this condition in Ireland is estimated at around €1 billion. Typical therapies for diabetic foot ulcers are daily wound dressing and application of ointments or antibiotics. However, most of the time these treatments do not completely cure the problem of slow wound healing that is associated with diabetic foot ulcers, and in many cases, these wounds can become infected and it is necessary to amputate the lower leg. In Europe, it is estimated that the number of adults diagnosed as diabetic will increase to 43 million by 2030. Around 15% of diabetes-affected patients develop a diabetic foot ulcer, and of these patients, approximately 20% will require amputation of the lower leg. This project aims to develop multicomponent-based artificial skin. The use of an artificial skin substitute (referred to as an artificial extracellular matrix scaffold (aECM) for diabetic wound healing would be of great help in the treatment of diabetic foot ulcers, promoting healing of the wound by providing all of the necessary components for the skin to regenerate. The overall research aim of the project was to develop for the first time a 3-dimensional aECM scaffold incorporating chemically engineered chondroitin sulfate (CS) and anti-inflammatory cytokine to resolve the cytokine imbalance in the chronic diabetic wound (Figure 1). The native ECM mimicking scaffold will act as a customized platform for technologies that deliver anti-inflammatory cytokine and chemically engineered CS to resolve the multi-faceted nature of the pathology of chronic diabetic wounds. The proposed aECM scaffold combined with anti-inflammatory cytokine and chemically engineered CS is designed based on the limitation of marketed skin grafts. The hypothesis underlying the project is that an aECM scaffold incorporating anti-inflammatory cytokine and chemically engineered CS will reduce the chronic inflammation and promote angiogenesis and re-epithelization in a diabetic mice ulcer model. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Chemically engineered CS was synthesized using acidic methanol treatment. The product was characterized using characterized Attenuated total reflection (ATR) - Fourier-transform infrared spectroscopy (FTIR), CHNS elemental analysis, Dimethylmethylene blue (DMMB )assay, Gel permeation chromatography (GPC), and High-performance liquid chromatography (HPLC). The molecular interaction between chemically engineered CS and pro/anti-inflammatory cytokines and chemokines was examined via surface plasmon resonance analysis. Additionally, molecular modeling analysis will also be performed in collaboration with Dr Damien Thompson, UL, Ireland (delayed due to COVID-19 lockdown/restrictions). The aECM scaffold was fabricated by crosslinking the polymers blends of chemically engineered CS and 4-arm polyethylene glycol (PEG) using 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). The characterization of the hydrogel was performed in terms of surface morphology (SEM), swelling, and rheological properties. SEM micrographs revealed the micro-porous and filamentous nature of the scaffold. No significant difference in surface morphology was observed by varying the concentration of the polymers. The degradation behavior of the scaffold was examined using gravimetric analysis on phosphate buffer saline pH 7.4 supplemented with chondroitinase ABC and it was observed that the scaffolds were stable for more than 2 weeks. Cytocompatibility of the scaffolds was accessed by cultivating human dermal fibroblasts (HDFs) and human epidermal keratinocytes (HEKs) on the scaffold for 3 and 7 days, respectively. Alamar blue and DNA quantification were performed on HDFs and HEKs cultivated scaffold to analyzed initial cellular adhesion and proliferation. It was found that all types of scaffold support cellular migration and proliferation. In vitro scratch assay was performed by injuring on the monolayer of HDFs and HEKs to examine the wound healing efficacy of the biomaterials. Cellular behavior was examined using immunofluorescence imaging (n-cadherin, e-cadherin, CD44, vimentin, and keratin 14). Besides, the optimized scaffolds showed no significant effect on cytokine/chemokines expression on THP1. The cellular influence of the degradation products of the optimized scaffolds was examined on THP1 cells and no changes in morphology or cytokine/chemokine expression profile were observed. Based on these findings, we can suggest that aECM scaffold loaded with anti-inflammatory cytokine is a promising candidate as a biomaterial for dermal wound healing. The optimized scaffolds have been selected to examine the in-vivo wound healing efficacy in db/db genetically diabetic mice model. However, due to the COVID-19 lockdown/restriction, the tasks related to this particular work package (WP4) are still ongoing. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) After completion of in vivo experiments, this project will be a significant advancement in the medical field by improving patients’ lives, reducing pain caused by slow-healing ulcers, and making the chances of amputation less likely. This could result in an improvement in the quality of life of many patients, who currently need daily wound monitoring and dressing changes. Patients may be required to attend appointments only once weekly with the use of this artificial skin substitute. Additionally, pain caused by slow healing wounds could be reduced, and the chances of infection could be minimised, meaning it would be less likely that amputation would be required. Medical professionals, caregivers, and medical health institutions would significantly benefit from the advancement of research and therapies in this area. Scientists and engineers would also benefit from the findings of this study as it will add to the overall knowledge in this field.Based on the findings of my MSCA-IF at NUI Galway, I have published one article in Advanced Functional Materials, 2020 (DOI: 10.1002/adfm.201910031) and will submit three more manuscripts in the upcoming months in open-access peer-reviewed journals, in line with the Horizon 2020 guidelines of making research outputs from the EU research programs accessible to the general population.