Lysophosphatidic acid stimulates epidermal growth factor‐family ectodomain shedding and paracrine signaling from human lung fibroblasts

Lysophospatidic acid (LPA) is a bioactive lipid mediator implicated in tissue repair and wound healing. It mediates diverse functional effects in fibroblasts, including proliferation, migration and contraction, but less is known about its ability to evoke paracrine signaling to other cell types involved in wound healing. We hypothesized that human pulmonary fibroblasts stimulated by LPA would exhibit ectodomain shedding of epidermal growth factor receptor (EGFR) ligands that signal to lung epithelial cells. To test this hypothesis, we used alkaline phosphatase‐tagged EGFR ligand plasmids transfected into lung fibroblasts, and enzyme‐linked immunosorbent assays to detect shedding of native ligands. LPA induced shedding of alkaline phosphatase‐tagged heparin‐binding epidermal growth factor (HB‐EGF), amphiregulin, and transforming growth factor‐a; non‐transfected fibroblasts shed amphiregulin and HBEGF under baseline conditions, and increased shedding of HB‐EGF in response to LPA. Treatment of fibroblasts with LPA resulted in elevated phosphorylation of extracellular signal‐regulated kinase 1/2, enhanced expression of mRNA for c‐fos, HB‐EGF and amphiregulin, and enhanced proliferation at 96 hours. However, none of these fibroblast responses to LPA required ectodomain shedding or EGFR activity. To test the ability of LPA to stimulate paracrine signaling from fibroblasts, we transferred conditioned medium from LPA‐stimulated cells, and found enhanced EGFR and extracellular signal‐regulated kinase 1/2 phosphorylation in reporter A549 cells in excess of what could be accounted for by transferred LPA alone. These data show that LPA mediates EGF‐family ectodomain shedding, resulting in enhanced paracrine signaling from lung fibroblasts to epithelial cells.     

Treatment with bone marrow-derived stromal cells accelerates wound healing in diabetic rats

Bone marrow stem cells participate in tissue repair processes and may have a role in wound healing. Diabetes is characterised by delayed and poor wound healing. We investigated the potential of bone marrow-derived mesenchymal stromal cells (BMSCs) to promote healing of fascial wounds in diabetic rats. After manifestation of streptozotocin (STZ)-induced diabetic state for 5 weeks in male adult Sprague-Dawley rats, healing of fascial wounds was severely compromised. Compromised wound healing in diabetic rats was characterised by excessive polymorphonuclear cell infiltration, lack of granulation tissue formation, deficit of collagen and growth factor [transforming growth factor (TGF-beta), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor PDGF-BB and keratinocyte growth factor (KGF)] expression in the wound tissue and significant decrease in biomechanical strength of wounds. Treatment with BMSC systemically or locally at the wound site improved the wound-breaking strength (WBS) of fascial wounds. The improvement in WBS was associated with an immediate and significant increase in collagen levels (types I-V) in the wound bed. In addition, treatment with BMSCs increased the expression of growth factors critical to proper repair and regeneration of the damaged tissue moderately (TGF-beta, KGF) to markedly (EGF, VEGF, PDGF-BB). These data suggest that cell therapy with BMSCs has the potential to augment healing of the diabetic wounds.     

Potential molecular mechanisms of negative pressure in promoting wound healing

Negative pressure wound therapy (NPWT) has been widely used in various lesions. This study aimed to explore the biological effects of negative pressure on the polymorphonuclear neutrophils (PMNs), macrophages, and epidermal keratinocyte cells involved in wound healing. PMNs differentiated from HL‐60, macrophages were derived from THP‐1 monocytes, and keratinocytes were cultured in vitro, and they were treated with 0, ?0.03 mp, and ?0.05 mp, respectively. Cell ultrastructure; viability; apoptosis; and protein factors such as tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), interleukin‐17 (IL‐17), and cell division cycle 42 (Cdc42) were determined by transmission electron microscopy (TEM), CCK8, flow cytometry (FCM), ELISA, and simple Western assays, respectively. After negative pressure stimulation, the cell ultrastructure of PMNs and macrophages cells was presented with a marked increase of lysosomes and a relative decrease of mitochondria. In addition, the cell viability was enhanced in PMNs and macrophages in a pressure‐dependent manner and apoptosis ratios were significantly reduced in PMNs and macrophages. In addition, under ?0.05 negative pressure, IFN‐γ and IL‐17 were significantly increased in PMNs or macrophages. Moreover, increased EGF and EGFR and Cdc42 levels in keratinocytes induced by the ?0.05 mpa were detected, indicating that the migration chemotaxis of keratinocyte cells was enhanced. Negative pressure might promote cell proliferation, accelerate inflammatory responses, and promote epithelialisation during wound healing by increasing IFN‐γ, IL‐17, Cdc42, EGF, and EGFR in PMNs, macrophages, or keratinocytes under different negative pressures.     

Different wound healing properties of dermis,adipose, and gingiva mesenchymal stromal cells

Oral wounds heal faster and with better scar quality than skin wounds. Deep skin wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic scars. Differences in wound healing and final scar quality might be related to differences in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and extrinsic signals, such as human salivary histatin, epidermal growth factor, and transforming growth factor beta1. Dermis‐, adipose‐, and gingiva‐derived MSC were compared for their regenerative potential with regards to proliferation, migration, and matrix contraction. Proliferation was assessed by cell counting and migration using a scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed in MSC populated collagen gels, and also in skin and gingival full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared to skin‐derived MSC, gingiva MSC showed greater proliferation and migration capacity, and less matrix contraction in full thickness tissue equivalents, which may partly explain the superior oral wound healing. Epidermal keratinocytes were required for enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin enhanced migration without influencing proliferation or matrix contraction in all three MSC, indicating that salivary peptides may have a beneficial effect on wound closure in general. Transforming growth factor beta1 enhanced contraction and alpha smooth muscle actin expression in all three MSC types when incorporated into collagen gels. Understanding the mechanisms responsible for the superior oral wound healing will aid us to develop advanced strategies for optimal skin regeneration, wound healing and scar formation.     

Chronic wounds – is cellular ‘reception’ at fault? Examining integrins and intracellular signalling

As with all physiologic processes, chronic wounds are associated with unique intracellular and cellular/extracellular matrix (ECM) receptor types and signalling messages. These cellular receptors mediate responses of the epidermis to provisional wound matrix and change in form and number in cases of impaired wound healing. Integrins are the major cell‐surface receptors for cell adhesion and migration and epidermal keratinocytes express several integrins that bind ECM ligands in provisional wound ECM. Integrin receptors and more particularly integrin clusters and focal adhesion points appear to influence epidermal and dermal cell matrix interactions, cell motility, cell phenotype and ultimate healing trajectory. In chronic wounds, a variety of changes in receptors have been identified: decreased integrin α5β1 receptors affect the integration of fibronectin and subsequent keratinocyte migration; integrin αvβ6 stimulate transforming growth factor (TGF)‐β and may increase the susceptibility to ulceration and fibrosis; however, TGF‐β signal receptors have been found to be dysfunctional in many chronic wounds; additionally receptor interactions result in increased senescent cells including fibroblasts, myofibroblasts and even keratinocytes – this produces a degradative ECM and wound bed and corrosive chronic wound fluid. The activation or inhibition of integrin receptors by various agents may provide an excellent means of influencing wound healing. This process offers an earlier intervention into the wound healing cascade promoting intrinsic healing and elaboration of growth factors and ECM proteins, which may be more cost effective than the traditional attempts at extrinsic addition of these agents.     

Immobilized gradients of epidermal growth factor promote accelerated and directed keratinocyte migration

Acceleration of wound closure results not only in decreased patient suffering and cost of wound treatment, but may also minimize scarring and lead to formation of a more stable closed wound. Cell migration is a critical element in wound healing, and it is believed that the ability to control the migration direction of cells will lead to accelerated closure of wounds. Thus, we have synthesized surfaces that are covalently modified with gradients of epidermal growth factor (EGF), a key molecule in the native wound-healing process, in order to create a platform that promotes directed cell migration. Standard photo-patterning techniques used herein enabled precise control over the spatial location of tethered EGF and the fabrication and quantitative characterization of gradient patterns of different types and slopes. Under serum-free conditions, human epidermal keratinocytes on immobilized EGF gradients preferentially migrated in the direction of higher EGF concentrations, and exhibited unidirectional migration speed and distance that was over five-fold greater than that observed on control surfaces. Treatment of migrating cells with an inhibitor of the EGF receptor resulted in immediate cessation of migration, thus verifying that the observed migration trends were directly attributable to keratinocyte interactions with immobilized EGF.     

The combined effect of recombinant human epidermal growth factor and erythropoietin on full‐thickness wound healing in diabetic rat model

Diabetic wound is a chronic wound in which normal process of wound healing is interrupted. Lack of blood supply, infection and lack of functional growth factors are assumed as some of the conditions that lead to non‐healing environment. Epidermal growth factor (EGF) acts primarily to stimulate epithelial cell growth across wound. Erythropoietin (EPO) is a haematopoietic factor, which stimulates the production, differentiation and maturation of erythroid precursor cells. This study hypothesised combining these two factors, non‐healing process of diabetic wound will be compensated and eventually lead to acceleration of wound healing compared with single growth factor treatment. A total of 30 diabetic Sprague–Dawley rats were divided into three treatment groups (single treatment of rh‐EPO or rh‐EGF or combined treatment on a full‐thickness skin wound). To assess the wound healing effects of the components, the wound size and the healing time were measured in each treatment groups. The skin histology was examined by light microscopy and immunohistochemical analysis of proliferating markers was performed. The combined treatment with rh‐EPO and rh‐EGF improved full‐thickness wound significantly (P < 0·05) accelerating 50% healing time with higher expression of Ki‐67 compared with single growth factor‐treated groups. The combined treatment failed to accelerate the total healing time when compared with single growth factor treatments. However, the significant improvement were found in wound size reduction in the combined treatment group on day 4 against single growth factor‐treated groups (P < 0·05). This study demonstrated that the combined treatment of rh‐EPO and rh‐EGF improved the wound healing possibly through a synergistic action of each growth factor. This application provides further insight into combined growth factor therapy on non‐healing diabetic wounds.     

Topical embryonic stem cells enhance wound healing in diabetic rats

The effects of embryonic stem cells (ESCs) on diabetic wound healing were investigated using an excisional skin wound model in 110 diabetes‐induced rats. We transplanted a clonal population of ESCs (5 × 10⁶) by topical injection into full thickness skin wounds. Four study groups were used; nondiabetic rats as a control, non‐insulin controlled diabetic rats not treated with ESCs, insulin controlled diabetic rats not treated with ESCs, and insulin controlled diabetic rats treated with ESCs. Five rats in each experimental group were sacrificed on days 1, 5, 10, 15, and 20 after wounding. Wounds images were acquired daily and wound sizes were calculated. We measured the mRNA levels of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), and fibronectin levels in extracellular matrix, and assessed wound healing by assessing histological parameters of epidermal regeneration, granulation tissue thickness, and angiogenesis. In the ESC‐treated group, wound sizes were significantly smaller than in the insulin controlled diabetic group not treated with ESCs on days 5 and 10 (p < 0.05), and EGF and VEGF levels were markedly higher on days 5 and 10, fibronectin levels on day 5 after injection. All histological scores in the ESC‐treated group were significantly higher than those of the insulin controlled diabetic group on day 5 (p < 0.05). Our results shows that topical ESCs enhance diabetic wound healing during the early stage, and suggest that ESCs transplantation offers a novel therapeutic modality for the treatment of diabetic wounds. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 1554–1562, 2011     

Epidermal growth factor‐containing wound closure enhances wound healing in non‐diabetic and diabetic rats

Background: This study was designed to elucidate the in vivo efficacy of epidermal growth factor (EGF) on wound healing in non diabetic and diabetic rats. Methods: Ninety‐six male Wistar‐Albino rats were randomly divided into six groups. Saline‐moistened gauze, pure gelatin or EGF in gelatin‐microsphere dressings were used in a dermal excision model in both normal and streptomycin‐induced diabetic rats. Wound healing was evaluated on day 7 and 14. Reduction in wound area, hydroxypyroline content and tensile strength of the wound were evaluated in each rat. Tissue samples taken from the wounds were examined histopathologically for reepithelialisation, cellular infiltration, number of fibroblasts, granulation and neovascularisation. Results: On day 7, the use of EGF‐containing dressing was observed to reduce the wound area better when compared with the other dressings tested. This effect was significant in normal rats rather than diabetic rats. The difference in reduction of wound area did not persist on day 14. No significant effect on hydroxyproline content of the wound was found with EGF‐containing dressing in either normal or diabetic rats. There was a statistically significant increase in tensile strength values of EGF‐applied non diabetic rats over the 14 day period. An increase in tensile strength was prominent in also EGF‐applied diabetic rats on day 14. Histological examination revealed higher histopathologic scores in EGF‐applied diabetic and non diabetic rats. Conclusion: These findings implicate that use of EGF in gelatin‐microsphere dressings improves wound healing both in normal and diabetic rats.     

转基因成纤维细胞向创面持续投递新型分泌型表皮细胞生长因子

目的 探索通过转基因成纤维细胞向创面持续投递表皮细胞生长因子（ＥＧＦ）促进创面愈合的新方法。方法 构建新型分泌型人ＥＧＦｃＤＮＡ并转染人成纤维细胞株ＫＭＳＴ６。将经照射的转基因细胞移植于裸鼠全厚创面。结果 稳定转染的细胞克隆可分泌有活性的ＥＧＦ。移植后可在伤口组织中检测到人ＥＧＦ,其含量缓慢下降,但至少可持续７天。结论 一次性移植少量转基因细胞可在创面愈合早期这一关键阶段持续向创面释放ＥＧＦ。     

Extracellular matrix/stromal vascular fraction gel conditioned medium accelerates wound healing in a murine model

Conditioned medium (CM) is a new treatment modality in regenerative medicine and has shown a successful outcome in wound healing. We recently introduced extracellular matrix/stromal vascular fraction gel (ECM/SVF‐gel), an adipose‐derived stem cell and adipose native extracellular matrix‐enriched product for cytotherapy. This study aimed to evaluate the effect of CM from ECM/SVF‐gel (Gel‐CM) on wound healing compared with the conventional CM from adipose tissue (Adi‐CM) and stem cell (SVF‐CM). In vitro wound healing effect of three CMs on keratinocytes and fibroblasts was evaluated in terms of proliferation property, migratory property, and extracellular matrix production. In vivo, two full‐thickness wounds were created on the back of each mice. The wounds were randomly divided to receive Gel‐CM, Adi‐CM, SVF‐CM, and PBS injection. Histologic observations and collagen content of wound skin were made. Growth factors concentration in three CMs was further quantified. In vitro, Gel‐CM promoted the proliferation and migration of keratinocytes and fibroblasts and enhanced collagen I synthesis in fibroblasts compared to Adi‐CM and SVF‐CM. In vivo, wound closure was faster, and dermal and epidermal regeneration was improved in the Gel‐CM‐treated mice compared to that in Adi‐CM and SVF‐CM‐treated mice. Moreover, The growth factors concentration (i.e., vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor‐β) in Gel‐CM were significantly higher than those in Adi‐CM and SVF‐CM. Gel‐CM generated under serum free conditions significantly enhanced wound healing effect compared to Adi‐CM and SVF‐CM by accelerating cell proliferation, migration, and production of ECM. This improved trophic effect may be attributed to the higher growth factors concentration in Gel‐CM. Gel‐CM shows potential as a novel and promising alternative to skin wound healing treatment. But limitations include the safety and immunogenicity studies of Gel‐CM still remain to be clearly clarified and more data on mechanism study are needed.     

Epidermal growth factor therapy and wound healing--past, present and future perspectives

The role ofepidermal growth factor (EGF) has been extensively investigated in normal and pathological wound healing. It is implicated in keratinocyte migration, fibroblast function and the formation of granulation tissue. Since the discovery of EGF, the first growth factor to be isolated, over 45 years ago, growth factor therapy has progressed into clinical practice in the treatment ofwounds. The investigation EGF in wound healing has progressed from the treatment of acute wounds, to its limited effect in chronic wounds. EGF is readily degraded in the chronic wound environment, but with the recent focus of research in new drug delivery systems that are able to protect and stabilise the protein, the potential healing effects of EGF are at the forefront of research. In this review, the history of EGF and wound healing research is considered, as are current and future therapeutic options.     

Inhibition of cyclooxygenase‐1 and ‐2 activity in keratinocytes inhibits PGE2 formation and impairs vascular endothelial growth factor release and neovascularisation in skin wounds

Inhibition of cyclooxygenase (Cox) enzymatic activity by non‐steroidal anti‐inflammatory drugs (NSAIDs) provides the molecular basis of analgesia following wounding or surgery. This study investigated the role of Cox activity in the regulation of vascular endothelial growth factor (VEGF) expression in keratinocytes and the formation of new blood vessels in acute wounds in mice. To this end, human HaCaT keratinocytes were stimulated with epidermal growth factor (EGF). EGF increased Cox‐1 mRNA in the presence of the constitutively expressed Cox‐1 protein in keratinocytes. EGF coinduced Cox‐2 and VEGF₁₆₅ mRNA and protein expression and an accumulation of prostaglandin E₂ (PGE₂) in cell culture supernatants. Inhibition of Cox isozyme activity by Cox‐1 and ‐2 siRNA or ibuprofen reduced PGE₂ and VEGF₁₆₅ release from keratinocytes. In a mouse model of excisional wound healing, Cox‐2 and VEGF₁₆₅ expression were colocalized in the granulation tissue of acute wounds. Oral treatment of mice with the Cox‐1 and ‐2 inhibitor diclofenac was associated with reduced levels of VEGF₁₆₅ protein and an impaired blood vessel formation in acute wound tissue. In summary, our data suggest that a reduction of PGE₂‐triggered VEGF₁₆₅ protein expression in wound keratinocytes is likely to contribute to the observed impairment of wound neovascularisation upon Cox inhibition.     

SCF increases in utero–labeled stem cells migration and improves wound healing

Diabetic skin wounds lack the ability to heal properly and constitute a major and significant complication of diabetes. Nontraumatic lower extremity amputations are the number one complication of diabetic skin wounds. The complexity of their pathophysiology requires an intervention at many levels to enhance healing and wound closure. Stem cells are a promising treatment for diabetic skin wounds as they have the ability to correct abnormal healing. Stem cell factor (SCF), a chemokine expressed in the skin, can induce stem cells migration, however the role of SCF in diabetic skin wound healing is still unknown. We hypothesize that SCF would correct the impairment and promote the healing of diabetic skin wounds. Our results show that SCF improved wound closure in diabetic mice and increased HIF‐1α and vascular endothelial growth factor (VEGF) expression levels in these wounds. SCF treatment also enhanced the migration of red fluorescent protein (RFP)‐labeled skin stem cells via in utero intra‐amniotic injection of lenti‐RFP at E8. Interestingly these RFP+ cells are present in the epidermis, stain negative for K15, and appear to be distinct from the already known hair follicle stem cells. These results demonstrate that SCF improves diabetic wound healing in part by increasing the recruitment of a unique stem cell population present in the skin.     

Attachment of peptide growth factors to implantable collagen

Ingrowth of fibrovascular tissue from the woundbed into collagen-based dermal substitutes and survival of cultured epithelium after transplantation may be enhanced by attachment of heparin binding growth factor 2 (HBGF2) and epidermal growth factor (EGF) to collagen. Biotinylation of collagen and the growth factors allows immobilization of HBGF2 and EGF by high affinity binding of tetravalent avidin. Biotinylated HBGF2 and EGF (B-GF) were exposed to complexes of biotinylated collagen (B-COL)-avidin (A) and detected with peroxidase-labeled avidin (AP) followed by chromagen formation on nitrocellulose paper. Binding of biotinylated HBGF2 and EGF was specific (*, P less than 0.05), proportional to the concentration of biotinylated collagen, and resistant to ionic (NaCl) displacement. Data are expressed as mean percentages of maximum binding +/- SEMs: (table; see text) Growth response of cultured human epidermal keratinocytes to HBGF2 (population doubling time, PDT = 0.70 population doublings (PD)/day) confirmed the retention of mitogenic activity after biotinylation (PDT = 0.80 PD/day). Specific binding of biotinylated HBGF2, EGF, or other biologically active molecules (antibiotics, NSAIDs) to implantable collagen may provide a mechanism for positive therapeutic modulation of wound healing, including repair of full-thickness skin wounds with cultured cell-collagen composite grafts.     

Interaction of epidermal growth factor, Ca2+, and matrix metalloproteinase-9 in primary keratinocyte migration

Elevations of epidermal growth factor (EGF) and Ca²⁺ concentrations in the wound site are associated with reepithelialization during wound healing. In addition, Ca²⁺ and EGF can both induce increases in matrix metalloproteinase‐9 (MMP‐9) synthesis. However, little is known about the interplay of these events in regulating the migration properties of primary keratinocytes on collagen I, the most abundant extracellular matrix component in the skin. We found that EGF stimulated both chemokinetic and chemotactic migration of primary keratinocytes on collagen I; however, MMP‐9 was required for EGF‐stimulated chemotaxis but not EGF‐stimulated chemokinesis. Calcium at 0.5 mM stimulated chemokinetic migration of keratinocytes. Together, Ca²⁺ and EGF stimulated higher levels of chemokinesis than either stimulus alone. Furthermore, Ca²⁺ could restore the ability of keratinocytes from MMP‐9 null mice to undergo EGF‐stimulated chemotaxis. The phosphatidylinositol‐3 kinase inhibitor LY294002 inhibited both EGF‐ and Ca²⁺‐stimulated chemokinetic migration. In contrast, the MEK inhibitor PD98059 blocked Ca²⁺‐ but not EGF‐stimulated chemokinetic migration of keratinocytes. A combination of PD98059 and LY294002 was required to inhibit Ca²⁺ enhancement of EGF‐stimulated migration completely. Calcium‐stimulated chemokinesis was completely blocked by either the protein kinase C‐α inhibitor Gö6976 or the src/fyn inhibitor PP2. Using primary keratinocytes, our results showed how the combined action of Ca²⁺, EGF, and MMP‐9 regulated the contributions of extracellular‐regulated kinase and phosphatidylinositol‐3 kinase toward chemokinetic and chemotactic migration of keratinocytes.     

Acceleration of cutaneous wound healing by brassinosteroids

Brassinosteroids are plant growth hormones involved in cell growth, division, and differentiation. Their effects in animals are largely unknown, although recent studies showed that the anabolic properties of brassinosteroids are possibly mediated through the phosphoinositide 3‐kinase/protein kinase B signaling pathway. Here, we examined biological activity of homobrassinolide (HB) and its synthetic analogues in in vitro proliferation and migration assays in murine fibroblast and primary keratinocyte cell culture. HB stimulated fibroblast proliferation and migration and weakly induced keratinocyte proliferation in vitro. The effects of topical HB administration on progression of wound closure were further tested in the mouse model of cutaneous wound healing. C57BL/6J mice were given a full‐thickness dermal wound, and the rate of wound closure was assessed daily for 10 days, with adenosine receptor agonist CGS‐21680 as a positive control. Topical application of brassinosteroid significantly reduced wound size and accelerated wound healing in treated animals. mRNA levels of transforming growth factor beta and intercellular adhesion molecule 1 were significantly lower, while tumor necrosis factor alpha was nearly suppressed in the wounds from treated mice. Our data suggest that topical application of brassinosteroids accelerates wound healing by positively modulating inflammatory and reepithelialization phases of the wound repair process, in part by enhancing Akt signaling in the skin at the edges of the wound and enhancing migration of fibroblasts in the wounded area. Targeting this signaling pathway with brassinosteroids may represent a promising approach to the therapy of delayed wound healing.     

Adipose‐derived stem cells and keratinocytes in a chronic wound cell culture model: the role of hydroxyectoine

Chronic wounds represent a major socio‐economic problem in developed countries today. Wound healing is a complex biological process. It requires a well‐orchestrated interaction of mediators, resident cells and infiltrating cells. In this context, mesenchymal stem cells and keratinocytes play a crucial role in tissue regeneration. In chronic wounds these processes are disturbed and cell viability is reduced. Hydroxyectoine (HyEc) is a membrane protecting osmolyte with protein and macromolecule stabilising properties. Adipose‐derived stem cells (ASC) and keratinocytes were cultured with chronic wound fluid (CWF) and treated with HyEc. Proliferation was investigated using MTT test and migration was examined with transwell‐migration assay and scratch assay. Gene expression changes of basic fibroblast growth factor (b‐FGF), vascular endothelial growth factor (VEGF), matrix metalloproteinases‐2 (MMP‐2) and MMP‐9 were analysed by quantitative real‐time polymerase chain reaction (qRT‐PCR). CWF significantly inhibited proliferation and migration of keratinocytes. Addition of HyEc did not affect these results. Proliferation capacity of ASC was not influenced by CWF whereas migration was significantly enhanced. HyEc significantly reduced ASC migration. Expression of b‐FGF, VEGF, MMP‐2 and MMP‐9 in ASC, and b‐FGF, VEGF and MMP‐9 in keratinocytes was strongly induced by chronic wound fluid. HyEc enhanced CWF induced gene expression of VEGF in ASC and MMP‐9 in keratinocytes. CWF negatively impaired keratinocyte function, which was not influenced by HyEc. ASC migration was stimulated by CWF, whereas HyEc significantly inhibited migration of ASC. CWF induced gene expression of VEGF in ASC and MMP‐9 in keratinocytes was enhanced by HyEc, which might partly be explained by an RNA stabilising effect of HyEc.     

Systemic translation of locally infiltrated epidermal growth factor in diabetic lower extremity wounds

Diabetic foot ulcer is one of the most frightened diabetic complications leading to amputation disability and early mortality. Diabetic wounds exhibit a complex networking of inflammatory cytokines, local proteases, and reactive oxygen and nitrogen species as a pathogenic polymicrobial biofilm, overall contributing to wound chronification and host homeostasis imbalance. Intralesional infiltration of epidermal growth factor (EGF) has emerged as a therapeutic alternative to diabetic wound healing, reaching responsive cells while avoiding the deleterious effect of proteases and the biofilm on the wound's surface. The present study shows that intralesional therapy with EGF is associated with the systemic attenuation of pro‐inflammatory markers along with redox balance recovery. A total of 11 diabetic patients with neuropathic foot ulcers were studied before and 3 weeks after starting EGF treatment. Evaluations comprised plasma levels of pro‐inflammatory, redox balance, and glycation markers. Pro‐inflammatory markers such as erythrosedimentation rate, C‐reactive protein, interleukin‐6, soluble FAS, and macrophage inflammatory protein 1‐alpha were significantly reduced by EGF therapy. Oxidative capacity, nitrite/nitrate ratio, and pentosidine were also reduced, while soluble receptor for advanced glycation end‐products significantly increased. Overall, our results indicate that the local intralesional infiltration of EGF translates in systemic anti‐inflammatory and antioxidant effects, as in attenuation of the glycation products' negative effects.     

Synergistic effect of keratinocyte transplantation and epidermal growth factor delivery on epidermal regeneration

Both keratinocyte transplantation and epidermal growth factor (EGF) delivery stimulate epidermal regeneration. In this study, we hypothesized that the combined therapy of keratinocyte transplantation and EGF delivery accelerates epidermal regeneration compared to the single therapy of either keratinocyte transplantation or EGF delivery. To test this hypothesis, we utilized fibrin matrix as a keratinocyte/EGF delivery vehicle for epidermal regeneration. Full-thickness wounds were created on the dorsum of athymic mice, and human keratinocytes and EGF in fibrin matrix were sprayed onto the wounds to regenerate epidermal layers (group 1). As controls, human keratinocytes in fibrin matrix (group 2), EGF in fibrin matrix (group 3), or fibrin matrix alone (group 4) was sprayed onto the wounds. Spraying keratinocytes suspended in fibrin matrix did not affect the keratinocyte viability, as the cell viabilities before and after spraying were not different. EGF was released from fibrin matrix for 3 days. The wounds were analyzed with histology and immunohistochemistry at 1 and 3 weeks after treatments. Compared with the control groups, initial wound closure rate was highest in group 1. Histological analyses indicated that group 1 exhibited faster and better epidermal regeneration than the other groups. Immunohistochemical analyses showed that regenerated epithelium in groups 1 and 2 stained positively for human involucrin at 3 weeks, whereas the tissue sections of the groups 3 and 4 stained negatively. Human laminin was detected at the dermal-epidermal junction of the regenerated tissues in groups 1 and 2 at 3 weeks and was not detected in groups 3 and 4. The epidermal thickness of the regenerated tissues in group 1 was significantly thicker than that of the other groups at all time points. These results suggest that the combined therapy of keratinocyte transplantation and EGF delivery is more efficacious for epidermal regeneration than each separate therapy alone.