Dinitrosyl-iron complexes with cysteine or glutathione accelerate skin wound healing in animals

The beneficial effect of NO-donors, dinitrosyl-iron complexes with cysteine or glutathione on the healing of skin wound in rats was demonstrated by hystological and hystochemical methods: dinitrosyl-iron complexes accelerated efficiently repair processes in wound tissue after a twofold injection of an aqueous solution of a dinitrosyl-iron complex into wound tissue at a total dose of 5 mmol on days 1 and 2 after skin wounding, and the granulocyte volume increased 3-4 times on the fourth day after wounding compared with the control. Higher doses of dinitrosyl-iron complex provoked an inflammation process in the wound. Similar experiments with of another NO donor S-nitrosoglutathione affected adversely the wound. S-Nitrosoglutathione was added to the wound at a total dose of 10 mmol, which ensured the administration of NO to the wound tissue in the amount equal to that introduced upon the injection of dinitrosyl-iron complex. The addition of dinitrosyl-iron complex with glutathione at a dose of 2.5 mmol was accompanied by the formation of protein-bound dinitrosyl-iron complex in wound tissue. The formation of dinitrosyl-iron complex was also observed after the injection of S-nitrosoglutathione. However, the amount of complexes was more than 25 times less than that after the administration of dinitrosyl-iron complex. The beneficial effect of dinitrosyl-iron complex on the wound was suggested to be due to the formation of a self-regulated chemical system in wound tissue, which is characterized by the mutual transformation of low-molecular dinitrosyl-iron complex and S-nitrosoglutathione. This system ensures a regulated delivery of NO to its intracellular targets without the formation of high amounts of peroxynitrite which could adversely affect the intracellular processes. It was assumed that the self-regulated system of dinitrosyl-iron complex and S-nitrosoglutathione is not formed after the addition of S-nitrosoglutathione to the wound, probably due to a low amount of intracellular iron which could provide the formation of dinitrosyl-iron complex. The rapid decomposition of S-nitrosoglutathione results in the appearance of high amounts of NO and hence peroxynitrite, which adversely affects the wound.     

Pulsed electromagnetic fields accelerate wound healing in the skin of diabetic rats

Delayed wound healing is a common complication in diabetes mellitus. From this point of view, the main purpose of the present study is to investigate the effect of extremely low frequency pulsed electromagnetic fields (ELF PEMFs) on skin wound healing in diabetic rats. In this study, diabetes was induced in male Wistar rats via a single subcutaneous injection of 65 mg/kg streptozocin (freshly dissolved in sterile saline, 0.9%). One month after the induction of diabetes, a full‐thickness dermal incision (35 mm length) was made on the right side of the paravertebral region. The wound was exposed to ELF PEMF (20 Hz, 4 ms, 8 mT) for 1 h per day. Wound healing was evaluated by measuring surface area, percentage of healing, duration of healing, and wound tensile strength. Obtained results showed that the duration of wound healing in diabetic rats in comparison with the control group was significantly increased. In contrast, the rate of healing in diabetic rats receiving PEMF was significantly greater than in the diabetic control group. The wound tensile strength also was significantly greater than the control animals. In addition, the duration of wound healing in the control group receiving PEMF was less than the sham group. Based on the above‐mentioned results we concluded that this study provides some evidence to support the use of ELF PEMFs to accelerate diabetic wound healing. Further research is needed to determine the PEMF mechanisms in acceleration of wound healing in diabetic rats. Bioelectromagnetics 31:318–323, 2010. © 2010 Wiley‐Liss, Inc.     

Non-adenine based purines accelerate wound healing

Wound healing is a complex sequence of cellular and molecular processes that involves multiple cell types and biochemical mediators. Several growth factors have been identified that regulate tissue repair, including the neurotrophin nerve growth factor (NGF). As non-adenine based purines (NABPs) are known to promote cell proliferation and the release of growth factors, we investigated whether NABPs had an effect on wound healing. Full-thickness, excisional wound healing in healthy BALB/c mice was significantly accelerated by daily topical application of NABPs such as guanosine (50% closure by days 2.5–2.8). Co-treatment of wounds with guanosine plus anti-NGF reversed the guanosine-promoted acceleration of wound healing, indicating that this effect of guanosine is mediated, at least in part, by NGF. Selective inhibitors of the NGF-inducible serine/threonine protein kinase (protein kinase N), such as 6-methylmercaptopurine riboside abolished the acceleration of wound healing caused by guanosine, confirming that activation of this enzyme is required for this effect of guanosine. Treatment of genetically diabetic BKS.Cg-m+/+lepr db mice, which display impaired wound healing, with guanosine led to accelerated healing of skin wounds (25% closure by days 2.8–3.0). These results provide further confirmation that the NABP-mediated acceleration of cutaneous wound healing is mediated via an NGF-dependent mechanism. Thus, NABPs may offer an alternative and viable approach for the treatment of wounds in a clinical setting.     

Self-assembling peptide nanofiber scaffolds accelerate wound healing

Cutaneous wound repair regenerates skin integrity, but a chronic failure to heal results in compromised tissue function and increased morbidity. To address this, we have used an integrated approach, using nanobiotechnology to augment the rate of wound reepithelialization by combining self-assembling peptide (SAP) nanofiber scaffold and Epidermal Growth Factor (EGF). This SAP bioscaffold was tested in a bioengineered Human Skin Equivalent (HSE) tissue model that enabled wound reepithelialization to be monitored in a tissue that recapitulates molecular and cellular mechanisms of repair known to occur in human skin. We found that SAP underwent molecular self-assembly to form unique 3D structures that stably covered the surface of the wound, suggesting that this scaffold may serve as a viable wound dressing. We measured the rates of release of EGF from the SAP scaffold and determined that EGF was only released when the scaffold was in direct contact with the HSE. By measuring the length of the epithelial tongue during wound reepithelialization, we found that SAP scaffolds containing EGF accelerated the rate of wound coverage by 5 fold when compared to controls without scaffolds and by 3.5 fold when compared to the scaffold without EGF. In conclusion, our experiments demonstrated that biomaterials composed of a biofunctionalized peptidic scaffold have many properties that are well-suited for the treatment of cutaneous wounds including wound coverage, functionalization with bioactive molecules, localized growth factor release and activation of wound repair.     

beta-Adrenergic receptor antagonists accelerate skin wound healing: evidence for a catecholamine synthesis network in the epidermis

The skin is our primary defense against noxious environmental agents. Upon injury, keratinocytes migrate directionally into the wound bed to initiate re-epithelialization, essential for wound repair and restoration of barrier integrity. Keratinocytes express a high level of beta2-adrenergic receptors (beta2-ARs) that appear to play a role in cutaneous homeostasis as aberrations in either keratinocyte beta2-AR function or density are associated with various skin diseases. Here we report the novel finding that beta-AR antagonists promote wound re-epithelialization in a "chronic" human skin wound-healing model. beta-AR antagonists increase ERK phosphorylation, the rate of keratinocyte migration, electric field-directed migration, and ultimately accelerate human skin wound re-epithelialization. We demonstrate that keratinocytes express two key enzymes required for catecholamine (beta-AR agonist) synthesis, tyrosine hydroxylase and phenylethanolamine-N-methyl transferase, both localized within keratinocyte cytoplasmic vesicles. Finally, we confirm the synthesis of epinephrine by measuring the endogenously synthesized catecholamine in keratinocyte extracts. Previously, we have demonstrated that beta-AR agonists delay wound re-epithelialization. Here we report that the mechanism for the beta-AR antagonist-mediated augmentation of wound repair is due to beta2-AR blockade, preventing the binding of endogenously synthesized epinephrine. Our work describes an endogenous beta-AR mediator network in the skin that can temporally regulate skin wound repair. Further investigation of this network will improve our understanding of both the skin repair process and the multiple modes of action of one of the most frequently prescribed class of drugs, hopefully resulting in a new treatment for chronic wounds.     

Scarless fetal skin wound healing update

Scar formation, a physiologic process in adult wound healing, can have devastating effects for patients; a multitude of pathologic outcomes, affecting all organ systems, stems from an amplification of this process. In contrast to adult wound repair, the early‐gestation fetal skin wound heals without scar formation, a phenomenon that appears to be intrinsic to fetal skin. An intensive research effort has focused on unraveling the mechanisms that underlie scarless fetal wound healing in an attempt to improve the quality of healing in both children and adults. Unique properties of fetal cells, extracellular matrix, cytokine profile, and gene expression contribute to this scarless repair. Despite the great increase in knowledge gained over the past decades, the precise mechanisms regulating scarless fetal healing remain unknown. Herein, we describe the current proposed mechanisms underlying fetal scarless wound healing in an effort to recapitulate the fetal phenotype in the postnatal environment. Birth Defects Research (Part C) 96:237–247, 2012. © 2012 Wiley Periodicals, Inc.     

Roles of lactoferrin on skin wound healing

Skin wound healing is a complex biological process that requires the regulation of different cell types, including immune cells, keratinocytes, fibroblasts, and endothelial cells. It consists of 5 stages: hemostasis, inflammation, granulation tissue formation, re-epithelialization, and wound remodeling. While inflammation is essential for successful wound healing, prolonged or excess inflammation can result in nonhealing chronic wounds. Lactoferrin, an iron-binding glycoprotein secreted from glandular epithelial cells into body fluids, promotes skin wound healing by enhancing the initial inflammatory phase. Lactoferrin also exhibits anti-inflammatory activity that neutralizes overabundant immune response. Accumulating evidence suggests that lactoferrin directly promotes both the formation of granulation tissue and re-epithelialization. Lactoferrin stimulates the proliferation and migration of fibroblasts and keratinocytes and enhances the synthesis of extracellular matrix components, such as collagen and hyaluronan. In an in vitro model of wound contraction, lactoferrin promoted fibroblast-mediated collagen gel contraction. These observations indicate that lactoferrin supports multiple biological processes involved in wound healing.     

Serum exosomes accelerate diabetic wound healing by promoting angiogenesis and ECM formation

Nonhealing wounds in diabetes remain a global clinical and research challenge. Exosomes are primary mediators of cell paracrine action, which are shown to promote tissue repair and regeneration. In this study, we investigated the effects of serum derived exosomes (Serum-Exos) on diabetic wound healing and its possible mechanisms. Serum-Exos were isolated from blood serum of normal healthy mice and identified by transmission electron microscopy and western blot. The effects of Serum-Exos on diabetic wound healing, fibroblast growth and migration, angiogenesis and extracellular matrix (ECM) formation were investigated. Our results showed that the isolated Serum-Exos exhibited a sphere-shaped morphology with a mean diameter at 150 nm, and expressed classical markers of exosomes including HSP70, TSG101, and CD63. Treatment with Serum-Exos elevated the percentage of wound closure and shortened the time of healing in diabetic mice. Mechanistically, Serum-Exos promoted granulation tissue formation and increased the expression of CD31, fibronectin and collagen-ɑ in diabetic mice. Serum-Exos also promoted the migration of NIH/3T3 cells, which was associated with increased expression levels of PCNA, Ki67, collagen-α and fibronectin. In addition, Serum-Exos enhanced tube formation in human umbilical vein endothelial cells and induced the expression of CD31 at both protein and messenger RNA levels. Collectively, our results suggest that Serum-Exos may facilitate the wound healing in diabetic mice by promoting angiogenesis and ECM formation, and show the potential application in treating diabetic wounds.     

Rice callus extracts for enhancing skin wound healing

Enhanced cell migration in the course of wound healing is required to repair damaged skin. We investigated the effects of rice callus extracts on the migration of skin keratinocytes. Rice callus extracts were obtained by using three different methods: pressurized hot water, crude ethanol, and liquid-liquid extractions. The extract obtained by using crude ethanol extraction was more effective in the migration of skin cells than that obtained by pressurized hot water extraction (PHWE). The crude ethanol extract (CEE) was further partitioned by performing liquid-liquid extraction. As phenolics are inhibitory compounds affecting cell migration, we analyzed the total phenolic content of the rice callus extracts. The level of phenolics in the n-hexane partitioned extract (n-HPE) of CEE was lower than that in all other partitioned extracts. The n-HPE was most effective in enhancing cell migration. We analyzed wound healingrelated factors including platelet derived growth factor B (PDGF-B), transforming growth factor beta 1 (TGF-β1), heparin-binding epidermal growth factor (HB-EGF), and fibroblast growth factor 2 (FGF-2) after the treatment of n-HPE. Most of the expressions of cell migration-related growth factors increased, but HB-EGF dramatically increased (6.5-fold) in keratinocytes treated with n-HPE. The results indicate that n-HPE contains more stimulating growth factors or proteins and less cell migration inhibiting factors than the other tested extracts; thus, n-HPE treatment produced the greatest enhancement of cell migration.     

Insulin-like growth factor-I in wound healing of rat skin

Endothelin-1 is involved in physiology and pathophysiology of the alimentary tract. The peptide modulates blood flow in the gastrointestinal microvasculature and regulates contractility of smooth muscles and, when present in excess, may be an important factor contributing to pathogenesis of various forms of mucosal injury and peristaltic disorders. Mechanisms that regulate endothelin concentration in the gastrointestinal tissues are unknown. Therefore, the aim of our study was to identify and characterize endothelin inactivating peptidases in the rat gastrointestinal mucosa and smooth muscle cells. We have found three high affinity and efficient endothelin-1 inactivating peptidases. The acidic (pH optimum 5.5), membrane-bound, thiorphan- (ED(50) 1.2+/-0.2 nM) and phosphoramidon (ED(50) 150+/-25 pM) sensitive, endothelin-1 inactivating peptidase (K(M) 0.12+/-0.03 microM) was present in the mucosal cells of duodenum and small intestine. The enzyme exhibited high molecular weight (>100 kDa) and characteristics similar to that of the rat and human kidney, acidic metalloendopeptidase that was recently described. Two forms of the unique, low molecular weight (100>MW>30 kDa), alkaline (pH optimum 8.5), specific (K(M) 0.5+/-0.2 microM), thiorphan- and phosphoramidon insensitive, 1,10 phenanthroline inhibitable (ED(50) 0.65+/-0.20 mM, mean+/-S.E.M.) endothelin-1 inactivating peptidase were present exclusively in the duodenal mucosal cells; soluble form in cytosol and membrane-bound form exhibiting an abundance ratio 5:1, respectively. Mucosa of the stomach and large intestine, and gastrointestinal smooth muscle cells do not contain the specific endothelin-1 inactivating peptidases. The enzymes may play a crucial role in regulation of endothelin concentration in the gastrointestinal tissues. Whether impairment of activity of the mucosal endothelin inactivating peptidases, resulting in the increase of concentration of endothelin peptides in gastrointestinal tissues, occurs in various pathological conditions is actually studied in our laboratory.     

Increased transglutaminase activity during skin wound healing in rats

Outer, middle and inner layers from wounded or unwounded rat dorsal skin were separated and extracted first with buffer and then with Triton X-100 and dithiothreitol. The extracts and residues were assayed for transglutaminase activity and tissue transglutaminase antigen. Transglutaminase activities in all skin layers are increased in the period 1-5 days after wounding. Most of the increased activity is in the buffer-soluble fraction in the inner skin layer though there is no corresponding increase in antigen in this fraction. This suggests that there is production of activated soluble tissue transglutaminase in the wounded inner layer. In the 3-5 day wounded outer layer the largest fraction of both activity and antigen is associated with the insoluble residue remaining after extraction with Triton X-100. On DEAE-cellulose chromatography Triton X-100 extracts of the inner layer of wounded skin showed a single major peak of activity, corresponding approximately with rabbit liver transglutaminase; the outer layer showed the same peak plus a different one, eluting at lower salt concentration, which is thought to be epidermal transglutaminase.     

c-Met is essential for wound healing in the skin

Wound healing of the skin is a crucial regenerative process in adult mammals. We examined wound healing in conditional mutant mice, in which the c-Met gene that encodes the receptor of hepatocyte growth factor/scatter factor was mutated in the epidermis by cre recombinase. c-Met-deficient keratinocytes were unable to contribute to the reepithelialization of skin wounds. In conditional c-Met mutant mice, wound closure was slightly attenuated, but occurred exclusively by a few (5%) keratinocytes that had escaped recombination. This demonstrates that the wound process selected and amplified residual cells that express a functional c-Met receptor. We also cultured primary keratinocytes from the skin of conditional c-Met mutant mice and examined them in scratch wound assays. Again, closure of scratch wounds occurred by the few remaining c-Met-positive cells. Our data show that c-Met signaling not only controls cell growth and migration during embryogenesis but is also essential for the generation of the hyperproliferative epithelium in skin wounds, and thus for a fundamental regenerative process in the adult.     

The spleen and skin wound healing in Xenopus adults

In most vertebrates, the regenerative capacity to restore lost/damage tissues to original structure and functionality decreases at some time during ontogenesis. To evaluate the role of the acquired immunity in the decline of regenerative potential, we examined the cellular responses elicited in the spleen during skin repair in Xenopus adults. Modifications in the architecture were found to be induced and were remarkable 14 days postinjury when the spleen increased significantly in size. In white pulp, the periarteriolar lymphoid sheaths were associated with follicles having central light zones, morphologically similar to germinal centers. With the progress of healing, pigment‐containing cells were seen to accumulate in both white and red pulp regions. Moreover, compared to controls, the cells immunoreactive to anti‐cytokines (TNF‐α, TGF‐β1) and ‐iNOS increased from the first days after wounding. The 14th day, the positive cells formed a dense network of reticular cells in central regions of lymphoid follicles and more frequent reactive leukocytes were detected within the red pulp. A higher number of lymphoid cells immunostained with anti‐CD3ε were also observed in the perifollicular zone. The results suggest that the spleen of adult frogs is involved in skin wound healing with the expansion of lymphoid compartments. J. Morphol. 277:888–895, 2016. © 2016 Wiley Periodicals, Inc.     

Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation

Wound healing is a highly orchestrated physiological process consisting of a complex events, and scarless wound healing is highly desired for the development and application in clinical medicine. Recently, we have demonstrated that human amniotic epithelial cells (hAECs) promoted wound healing and inhibited scar formation through a paracrine mechanism. However, exosomes (Exo) are one of the most important paracrine factors. Whether exosomes derived from human amniotic epithelial cells (hAECs-Exo) have positive effects on scarless wound healing have not been reported yet. In this study, we examined the role of hAECs-Exo on wound healing in a rat model. We found that hAECs, which exhibit characteristics of both embryonic and mesenchymal stem cells, have the potential to differentiate into all three germ layers. hAECs-Exo ranged from 50 to 150 nm in diameter, and positive for exosomal markers CD9, CD63, CD81, Alix, TSG101 and HLA-G. Internalization of hAECs-Exo promoted the migration and proliferation of fibroblasts. Moreover, the deposition of extracellular matrix (ECM) were partly abolished by the treatment of high concentration of hAECs-Exo (100 μg/mL), which may be through stimulating the expression of matrix metalloproteinase-1 (MMP-1). In vivo animal experiments showed that hAECs-Exo improved the skin wound healing with well-organized collagen fibers. Taken together, These findings represent that hAECs-Exo can be used as a novel hope in cell-free therapy for scarless wound healing.