Influence of extremely low-frequency magnetic field on the activity of antioxidant enzymes during skin wound healing in rats

The aim of this study was to evaluate the activity of the antioxidant enzymes mitochondrial and cytosolic superoxide dismutase (EC 1.15.1.1), glutathione peroxidase (POX, EC 1.11.1.9) and glutathione S-transferase (EC 3.1.2.7), as well as the concentration of malone dialdehyde (MDA), as an indicator of lipid peroxidation rate in the liver tissue homogenates and blood serum of male rats exposed to extremely low-frequency magnetic field (ELF-MF) in order to improve the healing process of an experimental cut wound on the back of each animal. The exposure to ELF-MF with frequency 40 Hz and magnetic flux density 10 mT induced an increase in POX serum activity and a decrease in MDA contents in the liver tissue, which suggests the inhibition of phospholipid peroxidation and subsequent stabilization of cellular membranes, as a result of ELF-MF action. Based on the results obtained, it seems that ELF-MF could be a useful supplement in the complex treatment of prolonged wound healing, due to the activation of endogenous enzymatic antioxidant system.     

CD44-dependent inflammation,fibrogenesis, and collagenolysis regulates extracellular matrix remodeling and tensile strength during cutaneous wound healing

Cutaneous wound healing consists of three main phases: inflammation, re-epithelialization, and tissue remodeling. During normal wound healing, these processes are tightly regulated to allow restoration of skin function and biomechanics. In many instances, healing leads to an excess accumulation of fibrillar collagen (the principal protein found in the extracellular matrix - ECM), and the formation of scar tissue, which has compromised biomechanics, tested using ramp to failure tests, compared to normal skin (Corr and Hart, 2013 [1]). Alterations in collagen accumulation and architecture have been attributed to the reduced tensile strength found in scar tissue (Brenda et al., 1999; Eleswarapu et al., 2011). Defining mechanisms that govern cellular functionality and ECM remodeling are vital to understanding normal versus pathological healing and developing approaches to prevent scarring. CD44 is a cell surface adhesion receptor expressed on nearly all cell types present in dermis. Although CD44 has been implicated in an array of inflammatory and fibrotic processes such as leukocyte recruitment, T-cell extravasation, and hyaluronic acid (the principal glycosaminoglycan found in the ECM) metabolism, the role of CD44 in cutaneous wound healing and scarring remains unknown. We demonstrate that in an excisional biopsy punch wound healing model, CD44-null mice have increased inflammatory and reduced fibrogenic responses during early phases of wound healing. At wound closure, CD44-null mice exhibit reduced collagen degradation leading to increased accumulation of fibrillar collagen, which persists after wound closure leading to reduced tensile strength resulting in a more severe scarring phenotype compared to WT mice. These data indicate that CD44 plays a previously unknown role in fibrillar collagen accumulation and wound healing during the injury response.     

An inhibitor of free radical processes decreases the rate of protein synthesis in gunshot wound tissues and decelerates the general adaptation syndrome

The dynamics of total protein and procollagen biosynthesis was studied in the muscle tissue in gunshot wound during the natural healing and after treatment with antioxidant (AO) 2,6-di-tert-butyl-4-methylphenol (BHT). AO treatment considerably decreased the rate of both total protein and procollagen synthesis in the muscle on the third day of healing. BHT notably decreased protein biosynthesis and inhibited corticosteroid production as measured from the electron spin resonance (ESR) signal of reduced adrenodoxin. AO treatment also decreased the rate of protein biosynthesis in bone marrow, thymus, and spleen. The decreased functional activity of the endocrine and immune systems suggests that AO considerably decelerates the systemic body response (general adaptation syndrome) to acute wound. Lipid peroxide radicals and the products of lipid hydroperoxide degradation are proposed as the primary mediators of the body response to stress.     

ELF MAGNETIC FIELD EFFECTS ON SOME HEMATOLOGICAL AND BIOCHEMICAL PARAMETERS OF PERIPHERAL BLOOD IN MICE

In this work we have studied some hematological and biochemical parameters of peripheral blood, as well as some histological aspects of liver and spleen during chronic exposure (1, 6, and 8 months) to extremely low-frequency magnetic fields (ELF-MF). Balb/C mice were exposed to an experimental sinusoidal magnetic wavefield of 60 Hz with a 0.11-mT intensity, generated in a system of Helmholtz coils. The results have shown no ELF-MF–cancer relationship during our experimental exposure time. However, leukopenia, hemoglobin decrease, and liver and spleen weight increase were observed. The bioeffects described could be correlated with spleen hyperfunction, which could have been produced by chronic exposure to this ELF-MF.     

Immunohistochemical localization of growth factors in fetal wound healing

Fetal wound healing occurs rapidly, in a regenerative fashion, and without scar formation, by contrast with adult wound healing, where tissue repair results in scar formation which limits tissue function and growth. The extracellular matrix deposited in fetal wounds contains essentially the same structural components as that in the adult wound but there are distinct differences in the spatial and temporal distribution of these components. In particular the organization of collagen in the healed fetal wound is indistinguishable from the normal surrounding tissue. Rapidity of healing, lack of an inflammatory response, and an absence of neovascularization also distinguish fetal from adult wound healing. The mechanisms controlling these differing processes are undefined but growth factors may play a critical role. The distribution of growth factors in healing fetal wounds is unknown. We have studied, by immunohistochemistry, the localization of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), and basic fibroblast growth factor (bFGF), in fetal, neonatal, and adult mouse lip wounds. TGF beta and bFGF were present in neonatal and adult wounds, but were not detected in the fetal wounds, while PDGF was present in fetal, neonatal, and adult wounds. This pattern correlates with the known effects in vitro of these factors, the absence of an inflammatory response and neovascularization in the fetal wound, and the patterns of collagen deposition in both fetal and adult wounds. The results suggest that it may be possible to manipulate the adult wound to produce more fetal-like, scarless, wound healing.     

Effects of procoagulants on wound healing

As blood coagulation is a prelude for wound healing, a systemic haemocoagulant (Botropase) and local procoagulants (thrombin and fibrin) were evaluated on physical (wound breaking strength, wound half-closure time and period of epithelization), biochemical (granuloma-hydroxyproline and hexosamine) and histological attributes of healing wounds in albino rats. Botropase prompted all phases of tissue repair. Thrombin delayed wound contraction whereas fibrin had no discernable action. The findings that procoagulants modify healing process has bearing on their surgical use.     

Ultrastructural analysis between fetal and adult wound healing process of marsupial opossum skin

The opossum delivers a newborn baby equivalent to tremature fetus state by postpregnancy. The peculiarity is advantageous for studies of fetus, because operations to take out fetus from the uterus of a mother are not necessary. When mammalian skin is wounded by full-thickness excision, fetal and adult wound healing processes differ. Fetal-type wound healing does not leave a scar. However, studies of how the fetal wound healing process differs in detail from the adult type are not advanced. We first observed the normal skin development of the gray short-tailed opossum (Monodelphis domestica) using an electron microscope. As for normal skin, an epidermis became multi-layered, and thickened from birth through to 7 days after birth. The quantity of extracellular matrix of the dermis increased thereafter, and several types of cells were found in the dermis. To examine the wound healing, we used material from a 1 day-old newborn baby, and from another 15 days after birth, and compared the wound healing style morphologically. Differences in the constitution of cells and fine structures of the skin were observed, it was obviously suggested that change in the wound healing style from fetal-type to adult-type occurred between 1 to 15 days after birth.     

The role of focal adhesion complexes in fibroblast mechanotransduction during scar formation

Historically, great efforts have been made to elucidate the biochemical pathways that direct the complex process of wound healing; however only recently has there been recognition of the importance that mechanical signals play in the process of tissue repair and scar formation. The body's physiologic response to injury involves a dynamic interplay between mechanical forces and biochemical cues which directs a cascade of signals leading ultimately to the formation of fibrotic scar. Fibroblasts are a highly mechanosensitive cell type and are also largely responsible for the generation of the fibrotic matrix during scar formation and are thus a critical player in the process of mechanotransduction during tissue repair. Mechanotransduction is initiated at the interface between the cell membrane and the extracellular matrix where mechanical signals are first translated into a biochemical response. Focal adhesions are dynamic multi-protein complexes through which the extracellular matrix links to the intracellular cytoskeleton. These focal adhesion complexes play an integral role in the propagation of this initial mechanical cue into an extensive network of biochemical signals leading to widespread downstream effects including the influx of inflammatory cells, stimulation of angiogenesis, keratinocyte migration, fibroblast proliferation and collagen synthesis. Increasing evidence has demonstrated the importance of the biomechanical milieu in healing wounds and suggests that an integrated approach to the discovery of targets to decrease scar formation may prove more clinically efficacious than previous purely biochemical strategies.     

Collagen deposition in the subcutaneous tissue during wound healing in humans: a model evaluation

Wound healing encompasses coagulation, inflammation, angiogenesis, fibroplasia, contraction, epithelialisation and remodeling. A granulation tissue is produced following incision of tissue such as skin, abdominal wall or the gastrointestinal tract, and the strength of the wound is determined primarily by the collagen content early in the healing course. Few models are available to study wound healing in man. The percutaneous insertion of expanded poly-tetrafluoroethylene tubes (ePTFE) into the subcutaneous tissue has been an established model for 20 years. The procedure is performed using a local anesthesia. The model has a diameter of 2.5 mm, a length of 5-10 cm and a pore size of 90-120 microns which is substantially more than that of vascular grafts. The polymer accumulates granulation tissue, the architecture of which resembles that of a normal surgical wound. Previous studies on the use of the ePTFE model in wound healing research are summarized in detail. Histological and immunohistochemical analyses of the granulation tissue deposited in the model were undertaken. The content of amino acids following hydrolysis of the granulation tissue was determined applying spectrophotometric or HPLC assays. Collagen amounts accumulated in the model are expressed as hydroxyproline per length of ePTFE or per total protein. Following a study in rats we examined 85 healthy volunteers and 158 surgical patients in the studies. Higher contents of hydroxyproline were found 10 days after implantation as compared to 5 days with considerable inter-person variation. Regarding median values there was a 25% difference between two measurements performed on two distinct ePTFE tubes from the same person, and a 12% difference between values obtained from two different pieces of the same ePTFE. Higher accumulation levels of hydroxyproline did not result in higher variability. Deposition of proline in the model correlated closely to total protein content. The ePTFE and a modified PVA model were compared in surgical patients. No reproducible measurements of hydroxyproline deposition were obtained with the PVA model as opposed to the ePTFE model. It is concluded that the modified PVA model is inadequate for determination of collagen deposition in subcutaneous granulation tissue. We found no correlation between collagen deposition levels obtained with placement of the ePTFE model in the subcutaneous tissue of the arm and in an uncomplicated surgical wound of the groin in the same patient, respectively. Significantly higher collagen deposition levels in the model were found in the surgical wound. Conversely, there was a significant correlation between protein deposition levels obtained at the two sites. Patients undergoing minor surgery (groin hernia repair) did not differ from healthy non-traumatized volunteers as regards deposition of collagen in subcutaneous tissue of the arm, whereas patients subjected to major general surgery demonstrated a significant decline during the postoperative phase compared to a preoperative evaluation. This decline was enhanced in patients who had infectious complications. Non-smoking volunteers were found to specifically accumulate more collagen (median value 82%) than smokers matched for age and gender. Irrespective of the smoking status women accumulated significantly more collagen in the model than men. These findings were re-tested in a prospective series leading to the same conclusion. Matrix metalloproteinases (MMP-2 and MMP-9) were determined in wound fluid obtained from the subcutaneous cavities of herniotomy wounds 24 and 48 h after operation. A significant and inverse correlation was demonstrated between MMP-9 after 24 h and accumulation levels of collagen in the ePTFE tube 10 days after implantation in the wound. Finally, it was demonstrated that local application of granulocyte-macrophage colony-stimulating factor into the ePTFE model during implantation specifically and dose-dependently reduced the number of fibroblasts and deposition of collagen. The doses chosen for the experiments resulted in both a local and a systemic effect. It is concluded that the minimally invasive ePTFE model, despite a certain level of variability, presently provides one of the best possibilities of evaluation of the wound healing potential in both volunteers and patients under various conditions. We found the model convenient for the assessment of both matrix deposition during wound healing and the influence of several factors including demographic characteristics, trauma, tobacco smoking, drugs and tissue degrading components of the wound.     

A CYTOLOGICAL STUDY OF THE ALBUMIN-SECRETING CELLS OF THE HEN OVIDUCT

1. The electron and light microscope have been employed in a cytological study of the albumin-secreting cells of the hen oviduct and of fractions of this tissue obtained after homogenization and differential centrifugation. 2. These studies confirm the observation that in this tissue material corresponding to liver "microsomes" in amino acid-incorporating ability and ribonucleic acid content sediments in relatively low centrifugal fields. 3. The electron microscope studies suggest that the protein secretion of the gland is formed in intimate association with the ergastoplasm.     

Metalloproteinases and their inhibitors: regulators of wound healing

Wound healing is a dynamic process that involves a coordinated response of many cell types representing distinct tissue compartments and is fundamentally similar among tissue types. Among the many gene products that are essential for restoration of normal tissue architecture, several members of the matrix metalloproteinase (MMP) family function as positive and, at times, negative regulators of repair processes. MMPs were initially thought to only function in the resolution phase of wound healing, particularly during scar resorption; however, recent evidence suggests that they also influence other wound-healing responses, such as inflammation and re-epithelialization. In this review, we discuss what is currently known about the function of MMPs in wound healing and will provide suggestions for future research directions.     

Ascorbic acid and hydroxyproline levels in the serum and granulation tissue of rats with aseptic and infected wounds

Hydroxyproline and ascorbic acid were measured in wound tissues and ascorbic acid was determined in the blood sera of 230 mature male Wistar rats with aseptic and infected surface wounds on days 1-10 (daily), 12, and 15. The principal morphologic characteristics of the granulation tissue were assessed by semiquantitative methods. An infected wound is characterized by increased hydroxyproline levels in the granulation tissue and elevated ascorbic acid concentration in the blood serum. The granulation tissue ascorbic acid level augments during the first nine days in case of an aseptic wound and during twelve days in case of an infected wound. Morphologic and biochemical correlations are indicative of a relationship between the ascorbic acid level on the one hand and granulation tissue vascularization and fibroblast proliferation on the other.     

Computational modeling of chemo-bio-mechanical coupling: a systems-biology approach toward wound healing

Wound healing is a synchronized cascade of chemical, biological, and mechanical phenomena, which act in concert to restore the damaged tissue. An imbalance between these events can induce painful scarring. Despite intense efforts to decipher the mechanisms of wound healing, the role of mechanics remains poorly understood. Here, we establish a computational systems biology model to identify the chemical, biological, and mechanical mechanisms of scar formation. First, we introduce the generic problem of coupled chemo-bio-mechanics. Then, we introduce the model problem of wound healing in terms of a particular chemical signal, inflammation, a particular biological cell type, fibroblasts, and a particular mechanical model, isotropic hyperelasticity. We explore the cross-talk between chemical, biological, and mechanical signals and show that all three fields have a significant impact on scar formation. Our model is the first step toward rigorous multiscale, multifield modeling in wound healing. Our formulation has the potential to improve effective wound management and optimize treatment on an individualized patient-specific basis.     

Fetal wound healing: a biochemical study of scarless healing

Human fetal surgery is being successfully performed today in a small number of highly selected patients for conditions that may lead to irreversible damage to the fetus and threaten the viability of the newborn. Following surgical repair, fetal wounds heal without scarring. This study was initiated to characterize fetal wounds both histologically and biochemically. Gore-Tex tubing was implanted into the subcutaneous tissue of the back of fetal, newborn, and adult New Zealand white rabbits. Light microscopic examination of healed wounds revealed no evidence of scar formation. Electron microscopy demonstrated a striated fibrillar structure suggestive of collagen within the lumen of the Gore-Tex tubing implants. Amino acid analysis (sensitivity 40 pmol) confirmed the presence of hydroxylysine and hydroxyproline within the Gore-Tex wound chambers indicating the presence of collagen in fetal wounds. The small amount of collagen precluded the typing of the collagen using cyanogen bromide peptide analysis. The absence of scarring and the small amounts of detectable collagen suggest a high degree of reorganization of the connective tissues involved in repair. The fetal wound matrix is rich in hyaluronic acid. Topical hyaluronic acid has been associated experimentally with a reduced amount of scarring in postnatal wound healing. Hyaluronic acid extracted from human skin and scar tissue is associated with collagen and other proteins. We propose that a hyaluronic acid-collagen-protein complex may play a role in fetal wound healing.     

Effects of porcine acellular dermal matrix treatment on wound healing and scar formation: Role of Jag1 expression in epidermal stem cells

Skin wound healing involves Notch/Jagged1 signaling. However, little is known how Jag1 expression level in epidermal stem cells (ESCs) contributes to wound healing and scar formation. We applied multiple cellular and molecular techniques to examine how Jag1 expression in ESCs modulates ESCs differentiation to myofibroblasts (MFB) in vitro, interpret how Jag1 expression in ESCs is involved in wound healing and scar formation in mice, and evaluate the effects of porcine acellular dermal matrix (ADM) treatment on wound healing and scar formation. We found that Jag1, Notch1 and Hes1 expression was up-regulated in the wound tissue during the period of wound healing. Furthermore, Jag1 expression level in the ESCs was positively associated with the level of differentiation to MFB. ESC-specific knockout of Jag1 delayed wound healing and promoted scar formation in vivo. In addition, we reported that porcine ADM treatment after skin incision could accelerate wound closure and reduce scar formation in vivo. This effect was associated with decreased expression of MFB markers, including α-SMA Col-1 and Col-III in wound tissues. Finally, we confirmed that porcine ADM treatment could increase Jag1, Notch1 and Hesl expression in wound tissues. Taken together, our results suggested that ESC-specific Jag1 expression levels are critical for wound healing and scar formation, and porcine ADM treatment would be beneficial in promoting wound healing and preventing scar formation by enhancing Notch/Jagged1 signaling pathway in ESCs.     

Calpain activity is essential in skin wound healing and contributes to scar formation

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.     

Mathematical Modeling in Wound Healing,Bone Regeneration and Tissue Engineering

The processes of wound healing and bone regeneration and problems in tissue engineering have been an active area for mathematical modeling in the last decade. Here we review a selection of recent models which aim at deriving strategies for improved healing. In wound healing, the models have particularly focused on the inflammatory response in order to improve the healing of chronic wound. For bone regeneration, the mathematical models have been applied to design optimal and new treatment strategies for normal and specific cases of impaired fracture healing. For the field of tissue engineering, we focus on mathematical models that analyze the interplay between cells and their biochemical cues within the scaffold to ensure optimal nutrient transport and maximal tissue production. Finally, we briefly comment on numerical issues arising from simulations of these mathematical models.     

A comparative analysis of healing of surgical cleft lip corrected in utero and neonates.

We studied the healing process in surgically created cleft lips in fetal mice and compared it with that in newborn mice with cleft lips. Our purpose was to determine the time for optimal healing, defined as minimal scarring, for a repaired cleft lip. Full-thickness paramedian lip incisions were made in NMRI mice in utero, in 2- and 4-day-old neonates, and in adults (n = 10 in each experimental and control group). The healing process was studied by biochemical analysis of hyaluronic acid and hydroxyproline content in the repaired cleft tissue. We found that the production of hyaluronic acid remained stable during the healing period and was similar in all experimental groups. However, there was an unexplained but consistent depression in the hyaluronic acid content of fetal tissue 2 days after repair. Hydroxyproline was present in the fetal healing tissue, but in a low concentration, starting 4 days after surgical incision of the lip. The production of hydroxyproline in 2-day-old neonates was similar to that in the fetuses throughout the healing period (p less than 0.0005). However, the production of hydroxyproline increased in 4-day-old neonatal and adult tissues. In conclusion, we found an optimal healing period for mice with minimal collagen production in the late fetal stage, and this lasted 2 days after birth.     

Intrinsic fibroblast-mediated remodeling of damaged collagenous matrices in vivo

Numerous studies have examined wound healing and tissue repair after a complete tissue rupture and reported provisional matrix and scar tissue formation in the injury gap. The initial phases of the repair are largely mediated by the coagulation response and a principally extrinsic inflammatory response followed by type III collagen deposition to form scar tissue that may be later remodeled. In this study, we examine subfailure (Grade II sprain) damage to collagenous matrices in which no gross tissue gap is present and a localized concentration of provisional matrix or scar tissue does not form. This results in extracellular matrix remodeling that relies heavily upon type I collagen, and associated proteoglycans, and less heavily on type III scar tissue collagen. For instance, following subfailure tissue damage, collagen I and III expression was suppressed after 1 day, but by day 7 expression of both genes was significantly increased over controls, with collagen I expression significantly larger than type III expression. Concurrent with increased collagen expression were significantly increased expression of the collagen fibrillogenesis supporting proteoglycans fibromodulin, lumican, decorin, the large aggregating proteoglycan versican, and proteases cathepsin K and L. Interestingly, this remodeling process appears intrinsic with little or no inflammation response as damaged tissues show no changes in macrophage or neutrophils levels following injury and expression of the inflammatory markers, tumor necrosis factor-alpha and tartrate-resistant acid phosphatase were unchanged. Hence, since inflammation plays a large role in wound healing by inducing cell migration and proliferation, and controlling extracellular matrix scar formation, its absence leaves fibroblasts to principally direct tissue remodeling. Therefore, following a Grade II subfailure injury to the collagen matrix, we conclude that tissue remodeling is fibroblast-mediated and occurs without scar tissue formation, but instead with type I collagen fibrillogenesis to repair the tissue. As such, this system provides unique insight into acute tissue damage and offers a potentially powerful model to examine fibroblast behavior.