## 7 Use of Biomaterials in the Release of Extracellular Vesicles for Treatment of Cutaneous Wounds
### Chitosan Hydrogels  
Chitosan is one of the most widely used polymers, as a base for manufacturing hydrogels with healing capacity. It is a poly-cationic biopolymer derived from a thermochemical deacetylation of chitin (a polymer found in the exoskeleton of arthropods). Chitosan has antimicrobial properties, promotes blood clotting, and is biocompatible and biodegradable. Therefore, it is a suitable substrate for the development of dressings, for the treatment of skin wounds [95]. In this regard, it has also served as a scaffold to include MSC and MSC-EV. Interestingly, a collagen- and chitosan-based hydrogel enriched in bone marrow-derived MSC or their EV, significantly improved wound healing in a rat model over hydrogel alone. This improvement was greater in wounds treated with a hydrogel containing MSC-EV. This was due to an increase in the deposition and organization of collagen in the extracellular matrix. That shows the therapeutic potential of cell-free therapies, based on hydrogels and VE, for the treatment of skin ulcers [96]. In this sense, the use of EV has the advantage that they can be enriched in different factors, through modifications such as genetic engineering. Thus, for example, in synovium mesenchymal stem cells (SMSC), miR-126-3p has been overexpressed, giving rise to EV enriched in such miRNA. Encapsulation of these EV in a chitosan-based hydrogel and their application to wounds in a diabetic rat model improved healing, by increasing granulation tissue formation and angiogenesis [97]. The rationale is that miR-126-3p induces angiogenesis, through phosphatidylcholine3-kinase (PI3K)/AKT, as well as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways [98]. A hydrogel based on chitosan and glycerol was enriched in EV, derived from human endometrial stem cells. It was used in an in vivo mouse model of skin healing, being compared against treatment with hydrogel without extracellular vesicles. Treatments were performed every three days, showing that hydrogel with MSC-EV had a greater capacity to promote wound closure (83.6% compared to 51.5% in the control), with a high degree of re-epithelialization and greater induction of angiogenesis and granulation tissue formation [99]. On the other hand, carboxymethyl chitosan is a derivative, that has been used together with poloxamer 407 (P407), for the fabrication of a thermo- and pH-sensitive hydrogel, by crosslinking reactions with the natural nontoxic crosslinker genipin (GP) [100]. This hydrogel showed an adequate protective and moisturizing effect on wounds, without stimulating proinflammatory processes; in addition to being biodegradable. The incorporation of EV derived from hUCMSC provides a high healing capacity to the hydrogel, in a cutaneous wound rat model, when applied every three days. Thus, the hydrogel loaded with MSC-EV significantly improved wound closure, re-epithelialization, collagen deposition and the number of skin appendages with respect to hydrogel without MSC-EV, indicating a higher maturation of the regenerated tissue [100]. VE-loaded chitosan-based hydrogels can also exert their positive effect on healing, through the inhibition of apoptosis. Thus, a methylcellulose-chitosan hydrogel loaded with EV derived from placental-mesenchymal stem cells produced a positive effect on skin-wound healing in the db/db diabetic mouse model. That was partly due to its ability to promote angiogenesis and inhibit apoptosis. Indeed, this hydrogel increased the expression of VEGF and BCL-2 (anti-apoptotic) proteins, while _BAX_ (pro-apoptotic) expression decreased, with respect to treatment with hydrogel or EV, separately [101]. Chitosan has also been used as a base for the manufacture of dressings with antimicrobial capacity. For instance, a chitosan-based dressing was developed, including silver nanoparticles (AgNP), attached to EV derived from hUCMSC. AgNP exhibits a broad antimicrobial spectrum, and is being widely used for the treatment of skin infections [102].