Approaches to developing novel bioengineered skin substitutes

In this body of work, a new method of creating a tunable porous scaffold - emulsion templating - to reconstitute the dermal layer of skin was explored. This method has traditionally been used to create polymeric high internal phase emulsions (polyHIPEs). The challenge of using emulsions for templating protein hydrogel networks is the preservation of native protein conformation in an emulsion system. In this instance, a non-ionic surfactant blend was used to create a metastable biocompatible emulsion system for templating collagen, collagen-fibrin and fibrin polymeric hydrogels. This method of manufacture has the added advantage of ease of control of pore size and formation of nanofibrous filaments within the scaffold itself. Parameters for control of pore size such as hydrophile-lipophile balance (HLB) values of surfactant mixes, surfactant concentration, excipient concentration, temperature and mixing rate were explored. Protein-surfactant interaction was investigated using confocal fluorescence microscopy, tryptophan fluorescence, Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism and enzymatic assays. Using Fluorescein Isothiocyanate Conjugate Bovine Serum Albumin (FITC-BSA) as a model protein, it was demonstrated that nonionic surfactants competitively adsorbed at interfaces, allowing FITC-BSA to remain in the aqueous phase and retaining its native conformation. Using a decane emulsion system, a biocompatible emulsion system was formulated, and emulsion droplets were used to create a hierarchical microporous scaffold (EmDerm). The mechanical properties and degradation profile of EmDerm scaffolds were also characterized. Three different cell types (human dermal fibroblasts, human dermal endothelial cells, mesenchymal stem cells) were seeded onto EmDerm scaffolds and cell proliferation was quantified as well as compared to commercial scaffolds Matriderm and Integra. The EmDerm scaffolds were shown to be highly biocompatible and comparable to commercial scaffolds. Co-cultures of various cell types were also performed to investigate the interaction of different cell types of the emulsion templated scaffolds. The proteome and secretome for both monoculture and co-cultures were also extracted and characterized to investigate effect of EmDerm scaffolds on secretion of degradative enzymes, structural proteins and growth factors as an indicator for in vivo activity of such scaffolds. EmDerm scaffolds implanted in rat wound healing models demonstrated no systemic inflammatory response. However, there was mild to moderate localized inflammatory response at implantation site at Day 7. This was associated with increased vascularization of the wound site at Day 14. Electrospinning of gelatin-polymer blends was also performed to create a pseudo-basement membrane as an epidermal carrier delivery system. The aim is to create an epidermal skin substitute to graft onto the neodermis when the EmDerm scaffolds have integrated into the wounds. Keratinocytes were shown to proliferate well on such membranes and the mechanical properties and degradation of the membranes were also characterized. In conclusion, emulsion-templated scaffolds are biocompatible and promote wound vascularization and integration in murine wound healing models. This work has potentially beneficial implications in the field of tissue engineering and regenerative medicine, particularly for burns and chronic wounds.</p

Manufacture and characterisation of EmDerm—novel hierarchically structured bio-active scaffolds for tissue regeneration

There are significant challenges for using emulsion templating as a method of manufacturing macro-porous protein scaffolds. Issues include protein denaturation by adsorption at hydrophobic interfaces, emulsion instability, oil droplet and surfactant removal after protein gelation, and compatible cross-linking methods. We investigated an oil-in-water macro-emulsion stabilised with a surfactant blend, as a template for manufacturing protein-based nano-structured bio-intelligent scaffolds (EmDerm) with tuneable micro-scale porosity for tissue regeneration. Prototype EmDerm scaffolds were made using either collagen, through thermal gelation, fibrin, through enzymatic coagulation or collagen-fibrin composite. Pore size was controlled via surfactant-to-oil phase ratio. Scaffolds were crosslink-stabilised with EDC/NHS for varying durations. Scaffold micro-architecture and porosity were characterised with SEM, and mechanical properties by tensiometry. Hydrolytic and proteolytic degradation profiles were quantified by mass decrease over time. Human dermal fibroblasts, endothelial cells and bone marrow derived mesenchymal stem cells were used to investigate cytotoxicity and cell proliferation within each scaffold. EmDerm scaffolds showed nano-scale based hierarchical structures, with mean pore diameters ranging from 40–100 microns. The Young’s modulus range was 1.1–2.9 MPa, and ultimate tensile strength was 4–16 MPa. Degradation rate was related to cross-linking duration. Each EmDerm scaffold supported excellent cell ingress and proliferation compared to the reference materials Integra™ and Matriderm™. Emulsion templating is a novel rapid method of fabricating nano-structured fibrous protein scaffolds with micro-scale pore dimensions. These scaffolds hold promising clinical potential for regeneration of the dermis and other soft tissues, e.g., for burns or chronic wound therapies