Foreskin‐isolated keratinocytes provide successful extemporaneous autologous paediatric skin grafts

Severe burns in children are conventionally treated with split‐thickness skin autografts or epidermal sheets. However, neither early complete healing nor quality of epithelialization is satisfactory. An alternative approach is to graft isolated keratinocytes. We evaluated paediatric foreskin and auricular skin as donor sources, autologous keratinocyte transplantation, and compared the graft efficiency to the in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. Keratinocytes were isolated from surgical samples by enzymatic digestion. Living cell recovery, in vitro proliferation and epidermal reconstruction capacities were evaluated. Differentiation status was analysed, using qRT–PCR and immunolabelling. Eleven children were grafted with foreskin‐derived (boys) or auricular (girls) keratinocyte suspensions dripped onto deep severe burns. The aesthetic and functional quality of epithelialization was monitored in a standardized way. Foreskin keratinocyte graft in male children provides for the re‐epithelialization of partial deep severe burns and accelerates wound healing, thus allowing successful wound closure, and improves the quality of scars. In accordance, in vitro studies have revealed a high yield of living keratinocyte recovery from foreskin and their potential in terms of regeneration and differentiation. We report a successful method for grafting paediatric males presenting large severe burns through direct spreading of autologous foreskin keratinocytes. This alternative method is easy to implement, improves the quality of skin and minimizes associated donor site morbidity. In vitro studies have highlighted the potential of foreskin tissue for graft applications and could help in tissue selection with the prospect of grafting burns for girls. Copyright © 2013 John Wiley & Sons, Ltd.     

A novel system for expansion and delivery of human keratinocytes for the treatment of severe cutaneous injuries using microcarriers and compressed collagen

Cell therapy with autologous or allogeneic keratinocytes applied as a single‐cell suspension is well established in clinical practice in the treatment of severe burn injuries to augment epithelial barrier restoration. Yet, the application of cell sprays can lead to significant cell loss owing to lack of adhesion of cell suspension to the wound bed. The development of a robust and controllable method of transplanting cells onto the wound bed is yet to be established. The ability to control adhesion and distribution of cells by using a cell carrier embedded in a biodegradable scaffold could significantly improve the treatment of cutaneous wounds with keratinocyte cell therapy. Several microcarrier‐based systems for expanding keratinocytes already exist. A new method for expansion of human keratinocytes in a feeder‐free, defined medium system on microcarriers has been developed. The cells retained their basal, proliferative phenotype after rapid expansion in a clinically relevant time‐frame. The cell‐laden microcarriers were further incorporated into collagen scaffolds fabricated by plastic compression. When cultured in vitro, cells continued to proliferate and migrate along the surface of the collagen scaffold. Using an in vitro wound bed model, cells were observed to form mostly single cell layers and in some areas multiple cell layers within 8 days, while retaining their basal, proliferative phenotype, indicating the suitability of this cell transplantation method to improve epithelial barrier restoration. This advanced cell expansion and delivery method for cutaneous cell therapy provides a flexible tool for use in clinical application. Copyright © 2017 John Wiley & Sons, Ltd.     

Fibrin biomatrix‐conjugated platelet‐derived growth factor AB accelerates wound healing in severe thermal injury

Controlled delivery of growth factors from biodegradable biomatrices could accelerate and improve impaired wound healing. The study aim was to determine whether platelet‐derived growth factor AB (PDGF.AB) with a transglutaminase (TG) crosslinking substrate site released from a fibrin biomatrix improves wound healing in severe thermal injury. The binding and release kinetics of TG‐PDGF.AB were determined in vitro. Third‐degree contact burns (dorsum of Yorkshire pigs) underwent epifascial necrosectomy 24 h post‐burn. Wound sites were covered with autologous meshed (3:1) split‐thickness skin autografts and either secured with staples or attached with sprayed fibrin sealant (FS; n = 8/group). TG‐PDGF.AB binds to the fibrin biomatrix using the TG activity of factor XIIIa, and is subsequently released through enzymatic cleavage. Three doses of TG‐PDGF.AB in FS (100 ng, 1 µg and 11 µg/ml FS) were tested. TG‐PDGF.AB was bound to the fibrin biomatrix as evidenced by western blot analysis and subsequently released by enzymatic cleavage. A significantly accelerated and improved wound healing was achieved using sprayed FS containing TG‐PDGF.AB compared to staples alone. Low concentrations (100 ng–1 µg TG‐PDGF.AB/ml final FS clot) demonstrated to be sufficient to attain a nearly complete closure of mesh interstices 14 days after grafting. TG‐PDGF.AB incorporated in FS via a specific binding technology was shown to be effective in grafted third‐degree burn wounds. The adhesive properties of the fibrin matrix in conjunction with the prolonged growth factor stimulus enabled by this binding technology could be favourable in many pathological situations associated with wound‐healing disturbances. Copyright © 2013 John Wiley & Sons, Ltd.     

Engineered skin graft with stromal vascular fraction cells encapsulated in fibrin–collagen hydrogel: A clinical study for diabetic wound healing

Despite the abundance of skin substitutes in the worldwide market, major hurdles in developing more complex tissues include the addition of skin appendages and vascular networks as the most important structure. The aim of this research was a clinical feasibility study of a novel prevascularized skin grafts containing the dermal and epidermal layer using the adipose stromal vascular fraction (SVF)‐derived endothelial cell population for vascular network regeneration. Herein, we characterized hydrogel with emphasis on biological compatibility and cell proliferation, migration, and vitality. The therapeutic potential of the prevascularized hydrogel transplanted on five human subjects as an intervention group with diabetic wounds was compared with nonvascularized skin grafts as the control on five patients. Wound planimetric and biometric analysis was performed using a Mann–Whitney nonparametric t‐test (p ≤ .05). The fibrin–collagen hydrogel was suitable for skin organotypic cell culture. There was a significant (p ≤ .05) increased in skin thickness and density in the vascular beds of the hypodermis measured with skin scanner compared with that in the control group. No significant macroscopic differences were observed between the intervention and control groups (p ≤ .05). In summary, we report for the first time the use of autologous dermal–epidermal skin grafts with intrinsic vascular plexus in a clinical feasibility study. The preliminary data showed that SVF‐based full‐thickness skin grafts are safe and accelerate the wound healing process. The next stage of the study is a full‐scale randomized clinical trial for the treatment of patients with chronic wounds.     

Rapid treatment of full‐thickness skin loss using ovine tendon collagen type I scaffold with skin cells

The full‐thickness skin wound is a common skin complication affecting millions of people worldwide. Delayed treatment of this condition causes the loss of skin function and integrity that could lead to the development of chronic wounds or even death. This study was aimed to develop a rapid wound treatment modality using ovine tendon collagen type I (OTC‐I) bio‐scaffold with or without noncultured skin cells. Genipin (GNP) and carbodiimide (EDC) were used to cross‐link OTC‐I scaffold to improve the mechanical strength of the bio‐scaffold. The physicochemical, biomechanical, biodegradation, biocompatibility, and immunogenicity properties of OTC‐I scaffolds were investigated. The efficacy of this treatment approach was evaluated in an in vivo skin wound model. The results demonstrated that GNP cross‐linked OTC‐I scaffold (OTC‐I_GNP) had better physicochemical and mechanical properties compared with EDC cross‐linked OTC‐I scaffold (OTC‐I_EDC) and noncross‐link OTC‐I scaffold (OTC‐I_NC). OTC‐I_GNP and OTC‐I_NC demonstrated no toxic effect on cells as it promoted higher cell attachment and proliferation of both primary human epidermal keratinocytes and human dermal fibroblasts compared with OTC‐I_EDC. Both OTC‐I_GNP and OTC‐I_NC exhibited spontaneous formation of bilayer structure in vitro. Immunogenic evaluation of OTC‐I scaffolds, in vitro and in vivo, revealed no sign of immune response. Finally, implantation of OTC‐I_NC and OTC‐I_GNP scaffolds with noncultured skin cells demonstrated enhanced healing with superior skin maturity and microstructure features, resembling native skin in contrast to other treatment (without noncultured skin cells) and control group. The findings of this study, therefore, suggested that both OTC‐I scaffolds with noncultured skin cells could be promising for the rapid treatment of full‐thickness skin wound.     

Wound‐healing effect of electrospun gelatin nanofibres containing Centella asiatica extract in a rat model

Centella asiatica (CA) is a traditional herbal medicine that has been shown to exert pharmacological effects on wound healing. This study demonstrated that CA extract facilitates the wound‐repair process by promoting fibroblast proliferation and collagen synthesis and exhibits antibacterial activity. Gelatin nanofibres containing C. asiatica extract were fabricated via electrospinning and were shown to exhibit dermal wound‐healing activity in a rat model. The wound areas of rat skin treated with electrospun gelatin membranes containing C. asiatica (EGC) presented the highest recovery rate compared with those treated with gauze, neat gelatin membranes and commercial wound dressings. The results of the histopathological examination support the outcome of the wound models. Contact‐angle and water‐retention measurements confirmed that the addition of C. asiatica extract did not significantly affect the hydrophilicity of the EGC membranes. The measured weight loss revealed that the EGC membranes are biodegradable. The findings suggest that EGC membranes are a promising material for the treatment of skin wounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Evaluation of keratin biomaterial containing silver nanoparticles as a potential wound dressing in full‐thickness skin wound model in diabetic mice

Keratin is a cytoskeletal scaffolding protein essential for wound healing and tissue recovery. The aim of the study was to evaluate the potential role of insoluble fur keratin‐derived powder containing silver nanoparticles (FKDP‐AgNP) in the allogenic full‐thickness surgical skin wound model in diabetic mice. The scanning electron microscopy image evidenced that the keratin surface is covered by a single layer of silver nanoparticles. Data obtained from dynamic light scattering and micellar electrokinetic chromatography showed three fractions of silver nanoparticles with an average diameter of 130, 22.5, and 5 nm. Microbiologic results revealed that the designed insoluble FKDP‐AgNP dressing to some extent inhibit the growth of Escherichia coli and Staphylococcus aureus. In vitro assays showed that the FKDP‐AgNP dressing did not inhibit fibroblast growth or induce hemolysis. In vivo studies using a diabetic mice model confirmed biocompatible properties of the insoluble keratin dressings. FKDP‐AgNP significantly accelerated wound closure and epithelization at Days 5 and 8 (p < .05) when compared with controls. Histological examination of the inflammatory response documented that FKDP‐AgNP‐treated wounds contained predominantly macrophages, whereas their untreated variants showed mixed cell infiltrates rich in neutrophils. Wound inflammatory response based on macrophages favors tissue remodeling and healing. In conclusion, the investigated FKDP‐AgNP dressing consisting of an insoluble fraction of keratin, which is biocompatible, significantly accelerated wound healing in a diabetic mouse model.     

Curcumin reverses diabetes‐induced endothelial progenitor cell dysfunction by enhancing MnSOD expression and activity in vitro and in vivo

Diabetes mellitus (DM) causes dysfunction of endothelial progenitor cells (EPCs), resulting in impaired wound healing. EPC therapy is a potential substitute to the current treatments of chronic wounds. Because EPCs isolated from diabetic patients are dysfunctional and therefore pose an obstacle in their efficacious employment in autologous cell therapy, a strategy to rescue them prior to transplantation would be expected to improve the efficacy of autologous cell therapy multifold. Compromised reactive oxygen species scavenging ability being the main cause of EPC dysfunction (EPCD), reactive oxygen species scavengers are likely to reverse or rescue EPCD. Therefore, in this study, we evaluated the potential of curcumin in reversing DM‐induced EPCD. We found that in vitro treatment of bone marrow EPCs from diabetic mice (D‐EPC) with curcumin restored their functionality, as judged by colony formation, tubule formation, and migration assays. Most importantly, autologous transplantation of curcumin‐treated D‐EPCs onto diabetic wounds also resulted in accelerated wound healing. Furthermore, curcumin‐treated diabetic mice exhibited improved wound healing, as compared with their vehicle‐treated diabetic counterparts, underscoring the efficacy of curcumin in vivo as well. The levels and activity of manganese superoxide dismutase (MnSOD) in D‐EPCs treated in vitro with curcumin or those isolated from curcumin‐treated diabetic mice were comparable with those in non‐diabetic EPCs. Addition of methyl mercury chloride to inhibit MnSOD activity during curcumin treatment abolished the salutary effects of curcumin. Our data demonstrate that curcumin reverses DM‐induced EPCD by boosting MnSOD expression and activity and emphasizes its potential for use in autologous cell therapy for diabetic wound management.     

Unbiased and quantitative proteomics reveals highly increased angiogenesis induction by the secretome of mesenchymal stromal cells isolated from fetal rather than adult skin

Scarless wound healing and functional regeneration are typical processes of the fetus, gradually lost during postnatal life, and maximally attributed to fetal skin tissue and induced by fetal skin fibroblasts. The latter have been successfully applied to postnatal wounds, with clear advantages compared with autologous dermis grafts or adult fibroblast applications. Our goal was to functionally identify and uncover key factors and mechanisms through the analysis of secretomes, the principal players in all cell therapies based on mesenchymal stromal cells (MSCs). Cell secretomes also putatively mediate skin regenerative effects achieved in clinical applications of fetal skin fibroblasts. An innovative and unbiased approach of comparative and quantitative proteomics of cell conditioned media enabled us to gain knowledge of key molecules and processes from a translational perspective. Using banks of fetal and adult skin fibroblasts that we previously characterized as being MSCs, we discovered secretome changes by identification and comparative quantification, distinguishing secretome signatures of fetal skin MSCs putatively relevant for therapeutic microenvironment modulation. The uncovered proteins can trigger, directly and by modulation of extracellular matrix, angiogenesis, thus highlighting its key role towards scarless wound healing. The angiogenic trigger was functionally validated and corroborated in vitro, with fetal skin MSC secretomes stabilizing and inducing the formation of capillary‐like networks by endothelial cells and fetal liver MSCs, respectively. Our approach and our results may aid in the development of cell‐based and cell‐free products for skin regeneration in acute or chronic injury, and also for wound healing in the regeneration of other tissues. Copyright © 2017 John Wiley & Sons, Ltd.     

Human saliva stimulates skin and oral wound healing in vitro

Despite continuous exposure to environmental pathogens, injured mucosa within the oral cavity heals faster and almost scar free compared with skin. Saliva is thought to be one of the main contributing factors. Saliva may possibly also stimulate skin wound healing. If so, it would provide a novel therapy for treating skin wounds, for example, burns. This study aims to investigate the therapeutic wound healing potential of human saliva in vitro. Human saliva from healthy volunteers was filter sterilized before use. Two different in vitro wound models were investigated: (a) open wounds represented by 2D skin and gingiva cultures were used to assess fibroblast and keratinocyte migration and proliferation and (b) blister wounds represented by introducing freeze blisters into organotypic reconstructed human skin and gingiva. Re‐epithelialization and differentiation (keratin K10, K13, K17 expression) under the blister and inflammatory wound healing mediator secretion was assessed. Saliva‐stimulated migration of skin and oral mucosa fibroblasts and keratinocytes, but only fibroblast proliferation. Topical saliva application to the blister wound on reconstructed skin did not stimulate re‐epithelization because the blister wound contained a dense impenetrable dead epidermal layer. Saliva did promote an innate inflammatory response (increased CCL20, IL‐6, and CXCL‐8 secretion) when applied topically to the flanking viable areas of both wounded reconstructed human skin and oral mucosa without altering the skin specific keratin differentiation profile. Our results show that human saliva can stimulate oral and skin wound closure and an inflammatory response. Saliva is therefore a potential novel therapeutic for treating open skin wounds.     

Wound Healing Activities of Rafflesia Hasseltii Extract in Rats

The effects of topical application of Rafflesia hasseltii buds and flowers extract on the rate of wound healing and histology of healed wound were assessed. Four groups of adult male Sprague Dawley rats were experimentally wounded in the posterior neck area. A thin layer of blank placebo was applied topically to wounds of Group 1 rats. Wounds of experimental animals (Group 2 and 3) were treated with placebo containing 5% and 10% R. hasseltii buds extract, respectively. A thin layer of Intrasite gel was applied topically to wounds of Group 4 animals as reference. Macroscopically, wounds treated with placebo containing 5% and 10% R. hasseltii buds extract or Intrasite gel have been significantly accelerated the rate of wound healing compared to placebo-treated wounds. Histological analysis of healed wounds has confirmed this effect. Wounds treated with placebo containing 5%, 10% R. hasseltii buds extract or Intrasite gel showed markedly less scar width at wound enclosure and granulating tissue contained markedly more collagen and proliferating fibroblasts, but with the absence of inflammatory cells compared to wounds treated with blank placebo. In conclusion, the findings of increased rate of wound closure and contraction together with the histological findingssuggest that Rafflesia hasseltii buds extract is very effective in accelerating the wound healing process.     

Amniotic epithelial cells promote wound healing in mice through high epithelialization and engraftment

Although human amniotic epithelial cells (AMEs) are an attractive source of stem cells, their therapeutic potential in wound healing has not been fully investigated. We evaluated the therapeutic potential of AMEs for wound healing. Real‐time PCR showed that the epithelialization growth factors epidermal growth factor (EGF), platelet‐derived growth factor (PDGF)‐B and chemotactic factors interleukin‐8 (IL‐8 or CXCL8) and neutrophil‐activating protein‐2 (NAP‐2 or CXCL7) were upregulated in AMEs compared with adipose‐derived mesenchymal stem cells (ADMs). In vitro scratch wound assays revealed that AME‐derived conditioned medium substantially accelerated wound closure. Wounds in NOD/SCID mice were created by skin excision, followed by AME transplantation. AMEs implantation significantly accelerated wound healing and increased cellularity and re‐epithelialization. Transplanted AMEs exhibited high engraftment rates and expressed keratinocyte‐specific proteins and cytokeratin in the wound area, suggesting direct benefits for cutaneous closure. Taken together, these data indicate that AMEs possess therapeutic capability for wound healing through the secretion of epithelialization growth factors and enhanced engraftment properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Promotion of diabetic wound healing by collagen scaffold with collagen‐binding vascular endothelial growth factor in a diabetic rat model

Aims: Studies show that VEGF can promote tissue regeneration in diabetic wounds. The aim of this study was to evaluate the effects of a new composite biomaterial, a collagen scaffold with CBD‐VEGF, for wound healing in a diabetic rat model. Materials and methods: We produced a collagen scaffold loaded with CBD‐VEGF, which allowed VEGF to bind to the collagen scaffold. The diabetic rat model was constructed by injecting streptozocin (STZ) peritoneally and removing a 2 x 2.5 cm thick slice of skin from the back of the animal. Animals were randomly divided into 4 groups: blank control (BC Group, n = 24), collagen scaffold loaded with PBS (PBS Group, n = 24), collagen scaffold loaded with NAT‐VEGF (NAT‐VEGF Group, n = 24), and collagen scaffold loaded with CBD‐VEGF (CBD‐VEGF Group, n = 24). Wounds of the BC Group were covered with gauze and those of the PBS, NAT‐VEGF and CBD‐VEGF Groups were grafted by corresponding collagen scaffolds, respectively. Healing rates were calculated and compared among groups. Wound tissue was evaluated by histologic analysis. Results: The CBD‐VEGF group showed a higher wound healing rate, better vascularization and higher level of VEGF in the granulation tissue wound compared with NAT‐VEGF and PBS groups. Conclusions: The collagen scaffold with CBD‐VEGF promoted wound healing in a diabetic rat model, which could potentially provide better therapeutic options for the treatment of diabetic wounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Matrix‐directed differentiation of human adipose‐derived mesenchymal stem cells to dermal‐like fibroblasts that produce extracellular matrix

Commercially available skin substitutes lack essential non‐immune cells for adequate tissue regeneration of non‐healing wounds. A tissue‐engineered, patient‐specific, dermal substitute could be an attractive option for regenerating chronic wounds, for which adipose‐derived mesenchymal stem cells (ADMSCs) could become an autologous source. However, ADMSCs are multipotent in nature and may differentiate into adipocytes, osteocytes and chondrocytes in vitro, and may develop into undesirable tissues upon transplantation. Therefore, ADMSCs committed to the fibroblast lineage could be a better option for in vitro or in vivo skin tissue engineering. The objective of this study was to standardize in vitro culture conditions for ADMSCs differentiation into dermal‐like fibroblasts which can synthesize extracellular matrix (ECM) proteins. Biomimetic matrix composite, deposited on tissue culture polystyrene (TCPS), and differentiation medium (DM), supplemented with fibroblast‐conditioned medium and growth factors, were used as a fibroblast‐specific niche (FSN) for cell culture. For controls, ADMSCs were cultured on bare TCPS with either DM or basal medium (BM). Culture of ADMSCs on FSN upregulated the expression of differentiation markers such as fibroblast‐specific protein‐1 (FSP‐1) and a panel of ECM molecules specific to the dermis, such as fibrillin‐1, collagen I, collagen IV and elastin. Immunostaining showed the deposition of dermal‐specific ECM, which was significantly higher in FSN compared to control. Fibroblasts derived from ADMSCs can synthesize elastin, which is an added advantage for successful skin tissue engineering as compared to fibroblasts from skin biopsy. To obtain rapid differentiation of ADMSCs to dermal‐like fibroblasts for regenerative medicine, a matrix‐directed differentiation strategy may be employed. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of advanced therapy medicinal product gingiva and skin substitutes and their in vitro wound healing potentials

Skin and oral mucosa substitutes are a therapeutic option for closing hard‐to‐heal skin and oral wounds. Our aim was to develop bi‐layered skin and gingiva substitutes, from 3 mm diameter biopsies, cultured under identical conditions, which are compliant with current European regulations for advanced therapy medicinal products. We present in vitro mode of action methods to (i) determine viability: epithelial expansion, proliferation (Ki‐67), metabolic activity (MTT assay); (ii) characterize skin and gingiva substitutes: histology and immunohistochemistry; and (iii) determine potency: soluble wound healing mediator release (enzyme‐linked immunosorbent assay). Both skin and gingiva substitutes consist of metabolically active autologous reconstructed differentiated epithelium expanding from the original biopsy sheet on a fibroblast populated connective tissue matrix (donor dermis). Gingival epithelium expanded 1.7‐fold more than skin epithelium during the 3 week culture period. The percentage of proliferating Ki‐67‐positive cells located in the basal layer of the gingiva substitute was >1.5‐fold higher than in the skin substitute. Keratins 16 and 17, which are upregulated during normal wound healing, were expressed in both the skin and gingiva substitutes. Notably, the gingiva substitute secreted higher amounts of key cytokines involved in mitogenesis, motogenesis and chemotaxis (interleukin‐6 > 23‐fold, CXCL8 > 2.5‐fold) as well as higher amounts of the anti‐fibrotic growth factor, hepatocyte growth factor (>7‐fold), compared with the skin substitute. In conclusion, while addressing the viability, characterization and potency of the tissue substitutes, important intrinsic differences between skin and gingiva were discovered that may explain in part the superior quality of wound healing observed in the oral mucosa compared with skin.     

Platelet‐derived transforming growth factor‐β1 promotes keratinocyte proliferation in cutaneous wound healing

Platelets are a recognised potent source of transforming growth factor‐β1 (TGFβ1), a cytokine known to promote wound healing and regeneration by stimulating dermal fibroblast proliferation and extracellular matrix deposition. Platelet lysate has been advocated as a novel personalised therapeutic to treat persistent wounds, although the precise platelet‐derived growth factors responsible for these beneficial effects have not been fully elucidated. The aim of this study was to investigate the specific role of platelet‐derived TGFβ1 in cutaneous wound healing. Using a transgenic mouse with a targeted deletion of TGFβ1 in megakaryocytes and platelets (TGFβ1^fl/fl.PF4‐Cre), we show for the first time that platelet‐derived TGFβ1 contributes to epidermal and dermal thickening and cellular turnover after excisional skin wounding. In vitro studies demonstrate that human dermal fibroblasts stimulated with platelet lysate containing high levels of platelet‐derived TGFβ1 did not exhibit enhanced collagen deposition or proliferation, suggesting that platelet‐derived TGFβ1 is not a key promoter of these wound healing processes. Interestingly, human keratinocytes displayed enhanced TGFβ1‐driven proliferation in response to platelet lysate, reminiscent of our in vivo findings. In summary, our novel findings define and emphasise an important role of platelet‐derived TGFβ1 in epidermal remodelling and regeneration processes during cutaneous wound healing.     

Role of neuropeptides in skin inflammation and its involvement in diabetic wound healing

Importance of the field: In 2010, the world prevalence of diabetes is 6.4%, affecting 285 million adults. Diabetic patients are at risk of developing neuropathy and delayed wound healing that can culminate in incurable diabetic foot ulcerations (DFUs) or even foot amputation.

Areas covered in this review: The contrast between cellular and molecular events of wound healing and diabetic wound healing processes is characterized. Neuropeptides released from the autonomous nervous system and skin cells reveal a major role in the immunity of wound healing. Therefore, the signaling pathways that induce pro/anti-inflammatory cytokines expression and its involvement in diabetic wound healing are discussed. The involvement of neuropeptides in the activation, growth, migration and maturation of skin cells, like keratinocytes, Langerhans cells, macrophages and mast cells, are described.

What the reader will gain: This review attempts to address the role of neuropeptides in skin inflammation, focusing on signal transduction, inflammatory mediators and pro/anti-inflammatory function, occurring in each cell type, as well as, its connection with diabetic wound healing.

Take home message: Understanding the role of neuropeptides in the skin, their application on skin wounds could be a potential therapy for skin pathologies, like the problematic and prevalent DFUs.     

An in vitro priming step increases the expression of numerous epidermal growth and migration mediators in a tissue‐engineering construct

An FDA‐approved, prototypic, living, bilayered skin construct (BSC) has been used for non‐healing wounds. Using this particular construct as proof of principle, we hypothesized that an in vitro ‘priming’ step may enhance its repertoire of expression of key mediators and genes. The priming step used here was incubation in Dulbecco's modified Eagle's medium (DMEM) for 24 h at 37°C and 5% CO₂, with or without construct meshing. Microarray and ingenuity pathway analysis (IPA) showed that >1000 genes were overexpressed by the priming step, including interleukin 6 (IL‐6), which plays important roles in wound healing. Genes highly overexpressed by priming were those involved in epidermal proliferation and migration. Quantitative real‐time PCR (qRT–PCR), immunostaining and western blots verified the results. An epiboly assay (epidermal migration over dermis) showed that BSC epiboly was inhibited by IL‐6 neutralizing antibody. Back wounds of nude mice were treated with primed or control BSCs for 3 days prior to harvesting; primed BSCs showed a significantly (p = 0.006) greater level of epidermal migration vs unprimed. Our study demonstrates that an in vitro priming step induces wound healing‐related genes in the BSC, leading to a construct that could prove more effective in stimulating wound healing. Copyright © 2014 John Wiley & Sons, Ltd.     

Functionalized PVA–silk blended nanofibrous mats promote diabetic wound healing via regulation of extracellular matrix and tissue remodelling

Chronic cutaneous ulcers, a complex pathophysiological diabetic condition, represent a critical clinical challenge in the current diabetes mellitus pandemic. Consequently, there is a compelling need for bioactive dressings that can trigger healing processes for complete wound repair. Silk fibroin (SF), a natural protein polymer from mulberry and non‐mulberry silkworms, has properties that support accelerated wound healing rate. SF from non‐mulberry variety possesses additional cell‐binding motifs (arginine, glycine, and aspartate), offering cell–material interactions. This study is aimed to investigate wound healing efficacy of dressings made up of various SF varieties blended with poly(vinyl alcohol) biopolymer in alloxan‐induced diabetic rabbit model. The nanofibrous mats have been developed using electrospinning and functionalized with growth factors and LL‐37 antimicrobial peptide for sustained delivery. Following post 14‐day treatment, non‐mulberry SF (NMSF)‐based dressings healed the wounds faster, in comparison with their mulberry Bombyx mori SF, poly(vinyl alcohol), and control counterparts (p < .01). NMSF‐based dressings also supported faster granulation tissue development, angiogenesis, and reepithelialization of wounds. Gene expression study of matrix metalloproteinases and collagen proteins affirmed higher extent of tissue remodelling during the repair process. Furthermore, there was organized extracellular matrix deposition (collagen type I, collagen type III, elastin, and reticulin) and higher wound breaking strength in NMSF compared with other groups after 4 weeks. These results validated the potential of NMSF‐based bioactive dressings to regulate extracellular matrix deposition leading to faster and complete repair of chronic diabetic cutaneous wounds.