**4.1. Stem cells**

acid)‐co‐poly‐(e‐caprolactone) (PLLCL) solutions [56]. An initial 44.9% burst release from EIF blended electrospun nanofibers was observed over a period of 15 days. The epidermal differentiation potential of adipose‐derived stem cells (ADSCs) was used to evaluate the scaffolds prepared either by core‐shell spinning or by blend spinning. After 15 days of cell culture, the proliferation of ADSCs on EIF‐encapsulated core‐shell nanofibers was the highest. Moreover, a higher percentage of ADSCs were differentiated to epidermal lineages on EIF‐ encapsulated core‐shell nanofibers compared to the cell differentiation of EIF‐blended nanofibers, and this can be attributed to the sustained release of EIF from the core‐shell nanofibers. A method for coating commercially available nylon wound dressings using the layer‐by‐layer process was utilized to control the release of VEGF165 and PDGF‐BB [57]. Animal evaluation was performed using a db/db mouse model of chronic wound healing. This combination delivery system promotes significant increases in the formation of granulation tissue and/or cellular proliferation when compared to dressings utilizing single growth factor

Current therapeutic regiments of wounded patients are static and mostly rely on matrices, gels, and engineered skin tissue. Accordingly, there is a need to design next‐generation grafting materials to enable biotherapeutic spatiotemporal targeting from clinically approved matrices. Peptides are good candidates for controlling wound infections. A drug carrier system was designed for delivering an insect metalloproteinase inhibitor (IMPI) drug to enable treatment of chronic wound infections [58]. Poly(lactic‐co‐glycolic acid) (PLGA) supplies lactate that accelerates neovascularization and promotes wound healing. Delivery systems of LL37 peptide encapsulated in PLGA nanoparticles (PLGA‐LL37 NP) were evaluated in full‐ thickness excisional wounds. A significantly higher collagen deposition, re‐epithelialized and neovascularized composition were found in PLGA‐LL37 NP‐treated group. *In vitro*, PLGA‐ LL37 NP induced enhanced cell migration but had no effect on the metabolism and prolifer‐ ation of keratinocytes. Interestingly, it displayed antimicrobial activity on *E. coli* [59]. CM11 peptide (WKLFKKILKVL‐NH2) (128 mg/L), a short cecropin‐melittin hybrid peptide, was delivered by an alginate sulfate‐based hydrogel as the antimicrobial wound dressing, and its

healing effects were tested on skin infections caused by MRSA (200 μL, 3 × 108

containing peptide treatment groups showed similar levels of wound healing.

**4. Cell delivery systems in wound healing**

mouse model [60]. During 8‐day period, the 2% mupirocin treatment group and hydrogel

Wound healing involves the coordinated efforts of several cell types, including keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets. The migration, infiltration, prolifer‐ ation, and differentiation of these cells will culminate in an inflammatory response, the formation of new tissue and ultimately wound closure [39]. Cell‐based therapies for wound repair are limited by inefficient delivery systems that fail to protect cells from acute inflam‐ matory environments [61]. Wound dressing of cells laden in biomaterials on wound surfaces

CFU/mL) in a

therapeutics.

**3.2. Delivery of peptides**

80 Wound Healing - New insights into Ancient Challenges

Stem cell therapy offers a promising new technique for aiding in wound healing; however, current findings show that stem cells typically die and/or migrate from the wound site, greatly decreasing the efficacy of the treatment. Most stem cells studied in wound healing delivery systems are mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), adipose‐ derived stem cells (ASCs), umbilical cord perivascular cells (UCPCs), and circulating angio‐ genic cells (CACs). MSCs have been shown to improve tissue regeneration in several preclinical and clinical trials [62]. MSCs from various sources, such as bone marrow and adipose tissue, have been reported in the delivery systems for wound healing [10, 63].

A 3D membrane (FBMSC‐CMM) from a freeze‐dried bone marrow mesenchymal stem cells‐ conditioned medium (FBMSC‐CM) can hold over 80% of the paracrine factors, which could significantly accelerate wound healing and enhance the neovascularization as well as epithe‐ lialization through strengthening the trophic factors in the wound bed [11]. Scaffolds strongly influence key parameters of stem cell delivery, such as seeding efficiency, cellular distribution, attachment, survival, metabolic activity, and paracrine release [64]. Pullulan was used to form a composite with collagen hydrogel for the delivery of MSCs into wounds [65]. Hydrogels induced MSC secretions of angiogenic cytokines and expression of transcription factors associated with the maintenance of pluripotency and self‐renewal (Oct4, Sox2, Klf4) when compared to MSCs grown in standard conditions. Engrafted MSCs were found to differentiate into fibroblasts, pericytes, and endothelial cells but did not contribute to the epidermis. Wounds treated with MSC‐seeded hydrogels demonstrated significantly enhanced angiogen‐ esis, which was associated with increased levels of VEGF.

There are other kinds of stem cells that have been used in combination with 3D scaffolds as a promising approach in the field of regenerative medicine. For instance, human umbilical cord perivascular cells (HUCPVC) [66], amniotic fluid‐derived stem cells (AFSs) [67], EPCs [68], and circulating angiogenic cells (CACs). CACs are known as early EPCs and are isolated from the mononuclear cell fraction of peripheral blood, and provide a potential topical treatment for nonhealing diabetic foot ulcers. A scaffold fabricated from type 1 collagen facilitates topical cell delivery of CACs to a diabetic rabbit ear wound (alloxan‐induced ulcer). Increased angiogenesis and increased percentage wound closure were observed with the treatment of collagen and collagen seeded with CSCs [69].

Compared to MSCs and EPCs, adipose‐derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility. ASCs are autologous, non‐immunogenic, plentiful, and easily obtained [70]. An acellular dermal matrix (ADM) scaffold made from cadaveric skins of human donors (AlloDerm, LifeCell Corp., Branchburg, NJ, USA) was served as a carrier for the delivery of ASCs [12]. ASCs‐ADM grafts secreted various cytokines, including VEGF, HGF, TGFβ, and bFGF. Novel technology and biocompatible biomaterials have been applied for stem cell delivery. A silk fibroin‐chitosan (SFCS) scaffold serving as a delivery vehicle for human adipose‐derived stem cells (ASCs) was evaluated in a murine soft tissue injury model [71]. Microvessel density at wound bed biopsy sites at 2 weeks postoperative was significantly higher in the ASC‐SFCS group vs. SFCS alone (7.5 ± 1.1 vs. 5.1 ± 1.0 blood vessels per high‐power field). A newly developed thermorespon‐ sive poly(ethylene) glycol (PEG)‐hyaluronic acid (HA) hybrid hydrogel with multiple acrylate functional groups provides an efficient delivery dressing system for human adipose‐derived stem cells (hADSCs) [72]. Although cellular proliferation was inhibited, cellular secretion of growth factors, such as VEGF and PDGF production, increased over 7 days, whereas IL‐2 and IFNγ release were unaffected. Injectable gelatin microcryogels (GMs) were used to load human ASCs [73]. The results demonstrated the priming effects of GMs on the upregulation of stemness genes and improved secretion of growth factors of hASCs for potential augmented wound healing. In a full‐thickness skin wound model in nude mice, multisite injections and dressings of hASC‐laden GMs significantly accelerated the healing compared to free stem cell injection.

### **4.2. Other cells**

Endothelial cells (ECs), keratinocytes, and fibroblasts are the most studied cells in terms of accelerated wound healing and improved skin tissue regeneration. A growing number of studies indicate that endothelial cells (ECs) and endothelial progenitor cells (EPCs) may regulate vascular repair in wound healing via paracrine mechanisms [61]. Using dried reagent patches that incorporate dextran (DEX) and a bulk aqueous phase comprising a cell culture medium containing poly(ethylene) glycol (PEG), Bathany et al. made a micro‐patterned localized delivery of fluorescent molecules and enzymes for cell detachment [74]. Keratino‐ cytes were delivered to dermal wounds in mice via cell‐adhesive peptides attached to chitosan membranes [75]. Two peptides of 12 or 13 amino acids each that bind to cell surface heparin‐ like receptors (A5G27 and A5G33) were found to promote strong keratinocyte attachment, whereas the one that binds to integrin (A99) was inactive. Recombinant human collagen III (rhCol‐III) gel was used as a delivery vehicle for cultured autologous skin cells (keratinocytes only or keratinocyte‐fibroblast mixtures) [76]. Its effect on the healing of full‐thickness wounds in a porcine wound‐healing model was examined. Two Landrace pigs were used for the study. Fourteen deep dermal wounds were created on the back of each pig with an 8‐mm biopsy punch. The scaffold enhanced early granulation tissue formation. Interestingly, fibroblast‐ containing gel was effectively removed from the wound, whereas gels without cells or with keratinocytes only remained intact.
