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.     

Acceleration of skin regeneration in full‐thickness burns by incorporation of bFGF‐loaded alginate microspheres into a CMCS–PVA hydrogel

Two important issues in skin tissue engineering are the vascularization and regeneration of the dermis. Basic fibroblast growth factor (bFGF) is known to promote angiogenesis and accelerate wound healing. Direct delivery of bFGF to the wound area, however, would lead to a loss of bioactivity. To this end, bFGF‐loaded alginate microspheres (Ms) were fabricated and incorporated into carboxymethyl chitosan (CMCS)–poly(vinyl alcohol) (PVA) to form a composite hydrogel. Scanning electron microscopy (SEM) results indicated that the incorporation of Ms does not significantly affect the inner structure of CMCS–PVA. In an in vitro study, the release of bFGF from Ms–CMCS–PVA in a sustained manner retained higher bioactivity over a 2‐week period. Full‐thickness burn wounds were created in the dorsal area of rats for in vivo evaluation of skin regeneration treated with CMCS–PVA hydrogel, with and without bFGF. Compared with the control, CMCS–PVA and bFGF–CMCS–PVA groups, the bFGF/Ms–CMCS–PVA group revealed significantly faster wound recovery rates, with re‐epithelialization and regeneration of the dermis. Moreover, the bFGF/Ms–CMCS–PVA group had the highest density of newly formed and mature blood vessels during the 2 mweek treatment period. The ability of the bFGF/Ms–CMCS–PVA hydrogel to accelerate wound healing in a full‐thickness burn model suggests its potential for use in dermal tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Acellular dermal matrix scaffolds coated with connective tissue growth factor accelerate diabetic wound healing by increasing fibronectin through PKC signalling pathway

The regional injection of connective tissue growth factor (CTGF) for diabetic wound healing requires multiple components and results in a substantial loss of its biological activity. Acellular dermal matrix (ADM) scaffolds are optimal candidates for delivering these factors to local ischaemic environments. In this study, we explored whether CTGF loaded on ADM scaffolds can enhance fibronectin (FN) expression to accelerate diabetic wound healing via the protein kinase C (PKC) signalling pathway. The performance of CTGF and CTGF + PKC inhibitor, which were loaded on ADM scaffolds to treat dorsal skin wounds in streptozotocin‐induced diabetic mice, was evaluated with naked ADM as a control. Wound closure showed that ADM scaffolds loaded with CTGF induced greater diabetic wound healing in the early stage of the wound in diabetic mice. Moreover, ADM scaffolds loaded with CTGF obviously increased the expression of FN both at the mRNA and protein levels, whereas the expression of FN was significantly reduced in the inhibitor group. Furthermore, the ADM + CTGF group, which produce FN, obviously promoted alpha‐smooth muscle actin and transforming growth factor‐beta expression and enhanced neovasculature and collagen synthesis at the wound sites. ADM scaffolds loaded with CTGF + PKC inhibitor delayed diabetic wound healing, indicating that FN expression was mediated by the PKC signalling pathway. Our findings offer new perspectives for the treatment of diabetic wound healing and suggest a rationale for the clinical evaluation of CTGF use in diabetic wound healing.     

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.     

In vitro comparative study of human mesenchymal stromal cells from dermis and adipose tissue for application in skin wound healing

Novel strategies combining cell therapy, tissue engineering, and regenerative medicine have been developed to treat major skin wounds. Although mesenchymal stromal cells (MSCs) from different tissues have similar stem cell features, such as self‐renewing mesodermal differentiation potential and expression of immunophenotypic markers, they also have distinct characteristics. Therefore, we aimed to characterize the application of MSCs derived from the dermis and adipose tissue (DSCs and ASCs, respectively) in cutaneous wound healing by in vitro approaches. Human DSC and ASC were obtained and evaluated for their isolation efficiency, stemness, proliferative profile, and genetic stability over time in culture. The ability of wound closure was first assessed by direct cell scratch assay. The paracrine effects of DSC‐ and ASC‐conditioned medium in dermal fibroblasts and keratinocytes and in the induction of tubule formation were also investigated. Although the ASC isolation procedures resulted in 100 times more cells than DSC, the latter had a higher proliferation rate in culture. Both presented low frequency of nuclear alterations over time in culture and showed similar characteristics of stem cells, such as expression of immunophenotypic markers and differentiation potential. DSCs showed increased healing capacity, and their conditioned media had greater paracrine effect in closing the wound of dermal fibroblasts and keratinocytes and in inducing angiogenesis. In conclusion, the therapeutic potential of MSCs is influenced by the obtainment source. Both ASCs and DSCs are applicable for skin wound healing; however, DSCs have an improved potential and should be considered for future applications in cell therapy.     

Pharmacological priming of adipose‐derived stem cells for paracrine VEGF production with deferoxamine

Adipose‐derived stem cells (ASCs) show great potentials in applications such as therapeutic angiogenesis, regenerative medicine and tissue engineering. Pharmacological preconditioning of stem cells to boost the release of cytoprotective factors may represent an effective way to enhance their therapeutic efficacy. In this study, the aim was to determine whether deferoxamine can enhance the release of vascular endothelial growth factor (VEGF) from in vitro expanded ASCs. It is demonstrated that deferoxamine (50–300 μm ) upregulated VEGF expression in a concentration‐ and time‐dependent fashion. At the concentrations used, deferoxamine did not show any cytotoxic effects. The stimulatory effect of deferoxamine on VEGF expression was mediated by augmentation of hypoxia inducible factor‐1 in ASCs, but independent of its antioxidant properties. Moreover, deferoxamine enhanced the paracrine effects of ASCs in promoting the regenerative functions of endothelial cells (migration and in vitro wound healing activities). This study provides evidence that deferoxamine might be a useful drug with low cell toxicity for pharmacological preconditioning of ASCs to enhance their capacity of VEGF production. Copyright © 2013 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.     

Promising effects of exosomes isolated from menstrual blood‐derived mesenchymal stem cell on wound‐healing process in diabetic mouse model

Wound healing is a complicated process that contains a number of overlapping and consecutive phases, disruption in each of which can cause chronic nonhealing wounds. In the current study, we investigated the effects of exosomes as paracrine factors released from menstrual blood‐derived mesenchymal stem cells (MenSCs) on wound‐healing process in diabetic mice. The exosomes were isolated from MenSCs conditioned media using ultracentrifugation and were characterized by scanning electron microscope and western blotting assay. A full thickness excisional wound was created on the dorsal skin of each streptozotocin‐induced diabetic mouse. The mice were divided into three groups as follows: phosphate buffered saline, exosomes, and MenSC groups. We found that MenSC‐derived exosomes can resolve inflammation via induced M1–M2 macrophage polarization. It was observed that exosomes enhance neoangiogenesis through vascular endothelial growth factor A upregulation. Re‐epithelialization accelerated in the exosome‐treated mice, most likely through NF‐κB p65 subunit upregulation and activation of the NF‐κB signaling pathway. The results demonstrated that exosomes possibly cause less scar formation through decreased Col1:Col3 ratio. These notable results showed that the MenSC‐derived exosomes effectively ameliorated cutaneous nonhealing wounds. We suggest that exosomes can be employed in regenerative medicine for skin repair in difficult‐to‐heal conditions such as diabetic foot ulcer.     

Promotion of dermal regeneration using pullulan/gelatin porous skin substitute

Tissue‐engineered dermal substitutes represent a promising approach to improve wound healing and provide more sufficient regeneration, compared with current clinical standards on care of large wounds, early excision, and grafting of autografts. However, inadequate regenerative capacity, impaired regeneration/degradation profile, and high cost of current commercial tissue‐engineered dermal regeneration templates hinder their utilization, and the development of an efficient and cost‐effective tissue‐engineered dermal substitute remains a challenge. Inspired from our previously reported data on a pullulan/gelatin scaffold, here we present a new generation of a porous pullulan/gelatin scaffold (PG2) served as a dermal substitute with enhanced chemical and structural characteristics. PG2 shows excellent biocompatibility (viability, migration, and proliferation), assessed by in vitro incorporation of human dermal fibroblasts in comparison with the Integra® dermal regeneration template (Control). When applied on a mouse full‐thickness excisional wound, PG2 shows rapid scaffold degradation, more granulation tissue, more collagen deposition, and more cellularity in comparison with Control at 20 days post surgery. The faster degradation is likely due to the enhanced recruitment of inflammatory macrophages to the scaffold from the wound bed, and that leads to earlier maturation of granulation tissue with less myofibroblastic cells. Collectively, our data reveal PG2's characteristics as an applicable dermal substitute with excellent dermal regeneration, which may attenuate scar formation.     

Short‐term evaluation of electromagnetic field pretreatment of adipose‐derived stem cells to improve bone healing

An electromagnetic field is an effective stimulation tool because it promotes bone defect healing, albeit in an unknown way. Although electromagnetic fields are used for treatment after surgery, many patients prefer cell‐based tissue regeneration procedures that do not require daily treatments. This study addressed the effects of an electromagnetic field on adipose‐derived stem cells (ASCs) to investigate the feasibility of pretreatment to accelerate bone regeneration. After identifying a uniform electromagnetic field inside a solenoid coil, we observed that a 45 Hz electromagnetic field induced osteogenic marker expression via bone morphogenetic protein, transforming growth factor β, and Wnt signalling pathways based on microarray analyses. This electromagnetic field increased osteogenic gene expression, alkaline phosphate activity and nodule formation in vitro within 2 weeks, indicating that this pretreatment may provide osteogenic potential to ASCs on three‐dimensional (3D) ceramic scaffolds. This pretreatment effect of an electromagnetic field resulted in significantly better bone regeneration in a mouse calvarial defect model over 4 weeks compared to that in the untreated group. This short‐term evaluation showed that the electromagnetic field pretreatment may be a future therapeutic option for bone defect treatment. Copyright © 2012 John Wiley & Sons, Ltd.     

Bioglass enhanced wound healing ability of urine‐derived stem cells through promoting paracrine effects between stem cells and recipient cells

In cell therapy, tissue regeneration ability of stem cells relies on the paracrine effects between stem cells and recipient cells. Our recent studies demonstrated that, in tissue engineering, bioactive silicates could stimulate paracrine effects between stem cells and recipient cells, which enhanced tissue generation. Therefore, we proposed that, in cell therapy, it may be an effective method to improve tissue regeneration ability of stem cells through activating the paracrine effects between stem cells and recipient cells with bioactive silicates. As urine‐derived stem cells (USCs) have been injected for wound healing and bioglass (BG) have shown bioactivity for various types of stem cells, in this study, we activated USCs with effective BG ionic products. Then the conditioned medium of BG‐activated USCs were used to culture endothelial cells and fibroblasts as well as co‐cultures of endothelial cells and fibroblasts. Results showed that growth factor expression in BG‐activated USCs was upregulated. In addition, paracrine effects between USCs and recipient cells in wound healing were stimulated, which resulted in enhanced capillary‐like network formation of endothelial cells and matrix protein production as well as myofibroblast differentiation of fibroblasts. Finally, the BG‐activated USCs were applied on full‐thickness excisional wounds. Results confirmed that BG‐activated USCs had better wound healing ability through improving angiogenesis and collagen deposition in wound sites as compared with USCs without any treatment. Taken together, BG can be used to promote wound healing ability of USCs by enhancing paracrine effects between USCs and recipient cells.     

Adipose-derived Stromal Cells Overexpressing Vascular Endothelial Growth Factor Accelerate Mouse Excisional Wound Healing

Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12–15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10–12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired.     

Experimental models and high-throughput diagnostics for tissue regeneration

During wound healing, cells recreate functional structures to regenerate the injured tissue. Understanding the healing process is essential for the development of new concepts and the design of novel biomimetic approaches for delivery of cells, genes and growth factors to accelerate tissue regeneration. To this end, realistic experimental models and high-throughput diagnostics are necessary to understand the molecular mechanisms of healing and reveal the genetic networks that determine tissue repair versus regeneration. Following a brief overview of the biology of wound healing, this review covers the in vitro and in vivo models that are employed at present to study the healing process. Discussion then covers the application of high-throughput genomic and proteomic technologies in epithelial development, living skin substitutes and wound healing. Finally, this review provides a perspective on novel technologies that should be developed to facilitate the understanding of wound healing complications and the design of therapeutics that target the underlying deficiencies.     

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.     

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.     

Increased survival of human free fat grafts with varying densities of human adipose‐derived stem cells and platelet‐rich plasma

The high absorption rate of transplanted fat has limited the application of autogenous fat grafts in the clinical setting. Therefore, this study aimed to evaluate the effects of platelet‐rich plasma (PRP) and adipose‐derived stem cells (ASCs) on fat regeneration by investigating the impact of PRP and conditioned medium on the biological characteristics of ASCs. Fat grafts were prepared with ASCs at densities of 10⁷/ml, 10⁶/ml, 10⁵/ml, 10⁴/ml and 0/ml with and without PRP and injected subcutaneously into nude mice. Liquid overflow method, haematoxylin and eosin staining, and immunohistochemical analyses were used to examine the fat grafts. The residual fat volume of the 10⁵/ml ASC + PRP group was significantly higher than that of other treatment conditions after 90 days. Furthermore, histological examination revealed that in 10⁵/ml ASCs‐treated grafts normal adipocyte area and capillary formation were increased dramatically compared with other treatment conditions. It is concluded that fat grafts consisting of PRP and 10⁵/ml ASCs constitute an ideal transplant strategy, which may result in decreased absorption and accelerated fat regeneration. This simple and reliable method could provide a valuable and needed tool in plastic and reconstructive surgery. Copyright © 2014 John Wiley & Sons, Ltd.     

Conditioned media derived from mesenchymal stem cell cultures: The next generation for regenerative medicine

Recent studies suggest that the main driving force behind the therapeutic activity observed in mesenchymal stem cells (MSCs) are the paracrine factors secreted by these cells. These biomolecules also trigger antiapoptotic events to prevent further degeneration of the diseased organ through paracrine signalling mechanisms. In comparison with the normal physiological conditions, an increased paracrine gradient is observed within the peripheral system of diseased organs that enhances the migration of tissue‐specific MSCs towards the site of infection or injury to promote healing. Thus, upon administration of conditioned media derived from mesenchymal stem cell cultures (MSC‐CM) could contribute in maintaining the increased paracrine factor gradient between the diseased organ and the stem cell niche in order to speed up the process of recovery. Based on the principle of the paracrine signalling mechanism, MSC‐CM, also referred as the secretome of the MSCs, is a rich source of the paracrine factors and are being studied extensively for a wide range of regenerative therapies such as myocardial infarction, stroke, bone regeneration, hair growth, and wound healing. This article highlights the current technological applications and advances of MSC‐CM with the aim to appraise its future potential as a regenerative therapeutic agent.     

Progress and expectation of stem cell therapy for diabetic wound healing

Impaired wound healing presents great health risks to diabetics. Encouragingly, the current clinical successfully found out meaningful method to repair wound tissue, and stem cell therapy could be an effective method for diabetic wound healing with its ability to accelerate wound closure and avoid amputation. This minireview aims at introducing stem cell therapy for facilitating tissue repair in diabetic wounds, discussing the possible therapeutic mechanism and clinical application status and problems.