Development of a Biomimetic Multi-Layered Functionalised Antimicrobial Biomaterial Scaffold for Healing of Complex Wounds

Diabetic foot ulcers (DFUs) are chronic non-healing wounds that constitute one of the most devastating complications of diabetes. Dysfunctional angiogenesis plays a key role in DFU, limiting tissue repair leaving wounds open and prone to infection¹. Biomaterial approaches, and in particular the use of collagen-glycosaminoglycan (CG) scaffolds, have shown promise to promote tissue regeneration and burn wound healing². These biomaterial scaffolds provide a platform for infiltrating cells into the wound environment but do not directly deal with the underlying pathology of diabetic wounds, so further functionalisation is needed for these complex wounds. The aim of this project was to engineer a biomimetic bi-layered biomaterial scaffold designed to replicate the anatomical structure of the native skin with an epidermal antimicrobial collagen/chitosan film to prevent wound infection and a pro-angiogenic, matrix molecule functionalised dermal porous CG scaffold layer to heal complex wounds.<br/><br/>A bi-layered scaffold was developed by combining an antimicrobial collagen/chitosan film (0.5%, 1% / 0.75%) with a type I collagen, chondroitin-6-sulfate slurry by lyophilisation. Mechanical properties, including interlayer adhesion, were optimised through the film’s collagen content and crosslinking treatment (non-crosslinked (NXL)/dehydrothermal/carbodiimide EDAC crosslinking). The CG layer was then functionalised with either fibronectin, collagen IV, or laminin-1 to enhance its angiogenic and regenerative potential. The film’s antimicrobial potential was tested versus <i>staphylococcus aureus, </i>and the ability of the film to support re-epithelialisation was verified through seeding of human epidermal keratinocytes (HaCaTs). The vascularisation potential of the scaffold was investigated through the culture of human umbilical vein endothelial cells (HUVECs), and human dermal fibroblasts (HDFs) on the functionalised CG scaffold layer.<br/><br/>The bi-layered scaffolds had enhanced mechanical properties suitable for implantation following EDAC crosslinking (compressive modulus = 1.83 ± 0.0035 kPa (EDAC), 0.72 ± 0.15 kPa (NXL)) adhesion strength between layers = 32.3 ± 4.5 kPa (EDAC), 22.5 ± 8.8 kPa (NXL)) Biological characterisation showed that all collagen/chitosan films had antimicrobial activity versus s<i>taphylococcus aureus </i>regardless of crosslinking regime and collagen composition and supported the metabolic activity of human epidermal keratinocytes. However, crosslinked scaffolds showed enhanced infiltration and growth of vascular cells³. Functionalisation, of the CG layer with laminin-1 significant increased production of vascular endothelial growth factor (VEGF) by HDFs over 7 days (1044 ± 112 pg/ml). In addition, HUVECs seeded on these scaffolds formed tubular structures.<br/><br/>This study has led to the development of a novel biomimetic multi-layered functionalised antimicrobial biomaterial scaffold for complex wound healing. The collagen/chitosan film layer demonstrated the ability to inhibit growth and infiltration of <i>staphylococcus aureus</i>, the most common bacterial isolate found in infected wounds⁴ and supported metabolic activity of epidermal cells, demonstrating significant potential as a protective barrier to cover the wound and as a surface for re-epithelialisation. Functionalisation of the CG scaffold layer with laminin-1 enhanced the pro-angiogenic regenerative potential of the dermal CG scaffold layer, with increased growth factor production and vascular cell tube formation. Taken together, these results demonstrate the promise of this scaffold as a biomaterial-based solution to treat DFUs.<br/><br/><b>References</b><br/>(1) Lavery et al., Diabetes Care 29:1288-1293, 2006.<br/>(2) Yannas et al., Science 215:174-176, 1982.<br/>(3) McGrath et al. ACS Appl. Mater. Interfaces 2023, 15, 14, 17444–17458<br/>(4) Shettigar et al. Eur J C Micro In 39: 2235-2246, 2020.<br/><br/><b>Acknowledgements</b><br/>Science Foundation Ireland (SFI), AMBER Centre, Grant No.: SFI/12/RC/2278_2