Abstract
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.
Highlights
Hierarchical interconnected porous architecture and nanoscale structure are fundamental requirements of threedimensional protein-based bio-active scaffolds, which are essential for the functions of cell conductivity, nutrient perfusion, angiogenesis and vasculogenic differentiation [1,2,3]
The use of decane oil-in-water (o/w) emulsions was successful in forming scaffolds from each type of scaffold protein, neutralised type I collagen acetic acid extract, which gels spontaneously on warming from a 4 °C solution to 37 °C; and fibrinogen, enzymatically coagulated with thrombin 37°C
For scaffolds made from emulsion with 0.1% surfactant, a mean pore size of around 100 μm was obtained, and this decreased to approximately 40 μm with 0.7% surfactant
Summary
Hierarchical interconnected porous architecture and nanoscale structure are fundamental requirements of threedimensional protein-based bio-active scaffolds, which are essential for the functions of cell conductivity, nutrient perfusion, angiogenesis and vasculogenic differentiation [1,2,3]. One method for achieving controlled porosity in protein hydrogels is controlled freezing and lyophilisation, in which pores are formed by material exclusion from the ice crystal porogen. This typically results in dense lamellar structured material, largely devoid of nano-scale structure. This is shown by many of the current scaffolds, notably acellular collagen scaffolds [4]. This method is still in widespread use despite the long processing times required to make such scaffolds [5]. Foam formation by aeration is another methodology [7,8,9,10], and has some limitations, due to a large exposed air interface for protein denaturation, intrinsic bubble instability and foam drainage during gelation and cross-linking, which cause difficulty in achieving a biologically acceptable degree of homogeneity
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