## 7 Use of Biomaterials in the Release of Extracellular Vesicles for Treatment of Cutaneous Wounds
### Alginate Hydrogels  
Alginate has high biocompatibility, is inexpensive and easy to gel using divalent cations. In addition, alginate gels have a high capacity to absorb large amounts of wound exudate. These properties have allowed it to be used for drug delivery [105]. Thus, EV derived from cultured rat adipose-tissue-derived stromal cells (ADSC) have been encapsulated in this type of hydrogel at a concentration of 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL. They were applied in a rat skin-wound model, where it significantly improved wound closure, collagen synthesis and vessel formation, with respect to treatment with alginate hydrogel only [106]. In such a study, the MSC-EV used were also found to induce human umbilical-cord-vein endothelial cell (HUVEC) migration in vitro. That could be related to their angiogenic activity observed in vivo [106]. Also, based on alginate, an innovative sponge-like wound dressing has been developed. In this case, under the principles of good manufacturing practices (GMP), it contained the total secretome derived from human AD-MSC, cultured for 48 h, at a concentration of 4 \(\times\) 10\({}^{6}\) equivalent cells per mL. The resulting sponge-like wound dressing was freeze-dried (lyophilized). When applied to wounds in a mouse skin-ulcer model, it was hydrated with phosphate-buffered saline (PBS) buffer. That generated a gelatinous matrix, favoring the release of bioactive compounds from the secretome [107]. In treated wounds, such dressing induced increased levels of proteins involved in complement and coagulation cascades, cytoskeleton and extracellular matrix remodeling. Thus, in relation to the application of only alginate sponge-like wound dressing, incorporation of total secretomes produced an enhanced immune response. Additionally, it produced increased granulation tissue formation, more vascularization and collagen deposition in such in vivo model [107]. This study, therefore, shows the possibility of developing desiccated or lyophilized products based on biomaterials, containing MSC-EV and/or MSC-derived factors. This may facilitate their storage, transportation and clinical applications. Alginate has also been used as a base for the development of more complex hydrogels, with layers of different resistance to degradation. This may allow the release of some particular type(s) of EV in the early stages of healing, and other(s) in later stages. Generally, the application of MSC-EV to wounds decreases inflammation, increases angiogenesis, promotes fibroblast proliferation and increases collagen deposition. These effects are mainly beneficial in the inflammatory and proliferative phases of healing. But high vascularization, fibroblast proliferation and collagen production may also favor the appearance of unwanted hyperplastic scar tissue. Considering this, a bilayered thiolated alginate/PEG diacrylate hydrogel has been developed. Thus, they encapsulated BMMSC-derived EV in the first layer and BMMSC-derived miR-29b-3p-enriched EV, genetically engineered for miRNA overexpression, in the second layer [108]. Interestingly, it has been found that miR-29b mimics (mimicry) inhibited excessive blood vessel formation and collagen deposition, in endothelial cells and fibroblasts [109]. While BMMSC-derived EV promote angiogenesis and collagen formation through activation of PI3K/Akt, Erk1/2 and Smad3/TGF\(\beta\)1 signaling pathways, miR-29b-3p-enriched EV inhibited these pathways. Application of hydrogel containing both (BMMSC-derived EV and miR-29b-3p-enriched EV) types of MSC-EV in different layers provide a temporally separated release. That allowed greater acceleration of healing in a full-thickness skin defect model of rat and rabbit ears. In the early phases, the hydrogel promoted angiogenesis and collagen deposition. In the later ones, as a consequence of the release of miR-29b-3p-enriched EV, the tissue formed was characterized by more uniform vascularization, better rearrangement of collagen fibers, and lower volume of hyperplastic scar tissue [108].