Throughout the last decade, extracellular vesicles (EVs) have become increasingly popular in several areas of regenerative medicine. Recently, Apis mellifera royal jelly EVs (RJ EVs) were shown to display favorable wound healing properties such as stimulation of mesenchymal stem cell migration and inhibition of staphylococcal biofilms. However, the sustained and effective local delivery of EVs in nonsystemic approachessuch as patches for chronic cutaneous woundsremains an important challenge for the development of novel EV-based wound healing therapies. Therefore, the present study aimed to assess the suitability of type I collagen-a well-established biomaterial for wound healingas a continuous delivery matrix. RJ EVs were integrated into collagen gels at different concentrations, where gels containing 2 mg/ml collagen were found to display the most stable release kinetics. Functionality of released RJ EVs was confirmed by assessing fibroblast EV uptake and migration in a wound healing assay. We could demonstrate reliable EV uptake into fibroblasts with a sustained promigratory effect for up to 7 d. Integrating fibroblasts into the RJ EV-containing collagen gel increased the contractile capacity of these cells, confirming availability of RJ EVs to fibroblasts within the collagen gel. Furthermore, EVs released from collagen gels were found to inhibit Staphylococcus aureus ATCC 29213 biofilm formation. Overall, our results suggest that type I collagen could be utilized as a reliable, reproducible release system to deliver functional RJ EVs for wound healing therapies.

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Apis mellifera Royal Jelly (RJ) is a well-known remedy in traditional medicine around the world and its versatile effects range from antibacterial to anti-inflammatory properties and pro-regenerative properties. Several active compounds have been identified, however, the mechanisms of action still remain widely unknown. As a glandular product, RJ has been shown to contain a substantial number of extracellular vesicles (EVs) and in this study, we aimed to investigate the extent of involvement of RJEVs in wound healing associated effects. Molecular analysis of RJEVs verified the presence of important conserved exosomal markers such as CD63 and syntenin, as well as cargo molecules MRJP1, defensin-1 and jellein-3. RJEV internalization analysis demonstrated the involvement of membrane fusion as well as macropinocytosis or clathrin-dependent endocytosis into mammalian cells. Furthermore, RJEVs have demonstrated to modulate MSCs differentiation and secretome, as well as decrease LPS-induced inflammation in RAW 264.7 macrophages by blocking the MAPK pathway. In vivo studies confirmed anti-bacterial effects of RJEVs, and demonstrated an acceleration of wound healing in a splinted mouse model. Summarizing, this study suggests that RJEVs of potentially exosomal origin play a crucial role in the known effects of RJ by modulating the inflammatory phase and cellular response in wound healing.