## 8 Functional Aspects of Honey-Loaded Scaffolds for Wound Healing
Increasing honey concentrations affected hydrogel's absorption capacity from 512 \(\pm\) 21% for the blank film down to 197 \(\pm\) 9% for the 10% (\(w/v\)) honey-loaded sample [169]. This is in line with the results by [PERSON], which described a fivefold reduction of the percentage of swelling (from 520% to 100%) when honey concentration was increased from 10% to 30% (at a fixed crosslinking level) in honey (H)/polyvinyl alcohol (P)/chitosan (CS) electrospun nanofibers. Honey addition also impacted on the structural features of the fibres, with an increase of almost 100 nm in the mean diameter for any 10% increase in honey concentration [167]. It has been hypothesised that the frequently observed reduction in swelling ability of hydrogels upon honey addition might be ascribed to honey high water solubility, causing a higher rate of degradation when the scaffold is immersed in water [167]. Another explanation might be that honey occupies the polymer's sites for hydrogen bonding, which are otherwise available for interactions with water molecules [169]. Conversely, honey addition to double cross-linked alginate hydrogels led to a significant improvement in fluid uptake capacity up to 700% (measured in 10 mL deionised water), with an optimum observed with the 4% honey structure, which showed pronounced water absorption for 40 h followed by a plateau. This is in line with the controlled degradation kinetics, which involved non-crosslinked residuals first [170]. Analogously, the swelling ratio of honey-silk fibroin scaffolds significantly increased with higher honey concentrations, which was possibly due to honey hygroscopicity generating an osmotic pressure that directed the medium influx. However, the degradation rate also increased in the fibrous mats with higher honey loading, together with a reduction in compressive strength. These effects on the mechanical features could be explained by the enhanced pore size and porosity of the tridimensional structures upon the incorporation of increasing honey quantities [171]. Similarly, when incorporated as an additive during the electrospinning process of silk fibroin (SF) solutions, Manuka honey has been found to function as an effective hydrophilicity-enhancer, significantly increasing the water retention capacity of the control (pristine silk fibroin scaffolds) at both the honey concentrations tested (1% and 5%), up to a maximum swelling ratio of almost 400% of the initial dry weight [172]. Although a dose-dependency of honey antibacterial and antioxidant properties has been described in the literature [79; 109], when it is incorporated into a scaffold, simply increasing its concentration does not necessarily guarantee that the beneficial effects are maximised. For example, among the formulations developed by [PERSON] et al. for silk fibroin-based skin substitutes (loaded with multifocal honey up to 10% of the final concentration), as imaged in Figure 4, the best pro-healing performance with reduced scar formation was obtained with the 4% honey scaffold. Figure 2: Schematic representation illustrating critical aspects for the optimisation and characterisation of a honey-loaded scaffold for wound repair, with examples of polymers successfully demonstrated in published literature, as discussed in Section 8. This sample had an optimal microstructure, which allowed a flux of nutritious substances to the wound site, with enhanced fibroblast proliferation and migration. In particular, a full skin regeneration occurred both at the epidermal and dermal level, with the prevalence of collagen I over collagen III, angiogenesis, and formation of skin appendages such as hair follicles and sebaceous glands after 2 weeks of treatment [171].