Affect Wound Closure Research Articles

Development of In Vitro Dry Eye Models to Study Proliferative and Anti-Inflammatory Effects of Allogeneic Serum Eye Drops.

This study aimed to develop valid in vitro models for preclinical evaluation of proliferative and anti-inflammatory effects of human allogeneic serum eye drops for dry eye disease (DED) treatment. A DED wound healing model was developed by analyzing the influence of coating and serum concentrations on human corneal epithelial (HCE-T) wound closure. Further, intralaboratory variance, freeze-thaw cycle effects, donor variability and stability assays were conducted. Interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) were used to induce the gene expression of matrix metalloproteinase 9 (MMP9), cyclooxygenase 2 (COX2), transforming growth factor-β (TGFβ) and IL-1β. MMP9 induction was optimized using a design-of-experiments (DoE) approach and applied to examine serum under static and dynamic conditions. MMP9 protein expression was analyzed by ELISA. The DED wound healing model detected proliferative effects of serum down to 1% with a small intralaboratory variance. Serum stability was shown over six months, donor variance could be detected, and freeze-thaw cycle effects did not affect wound closure. Serum decreased MMP9 expression on the gene and protein levels. The induction method was successfully optimized using DoE modeling and transferred to a dynamic setting mimicking tear film fluidics. The DED wound healing and inflammatory DED model present useful in vitro models for the preclinical evaluation of allogeneic serum eye drops without the use of animal experiments.

Myeloid Wls expression is dispensable for skin wound healing and blood vessel regeneration.

Wnt signaling controls blood vessel growth, regression and patterning during embryonic and postnatal life. Macrophages are major producers of Wnt ligands and angiogenic growth factors. It regulates vascular development and specification during embryogenesis and wound healing. Macrophage dysregulation in wound healing impairs vessel regeneration and delay wound closure. During cutaneous wound healing, the endovascular progenitors (EVPs) proliferate and differentiate into mature endothelial (D) cells in response to signals produced by perivascular cells, including macrophages, governing blood vessels regeneration. However, the role of macrophage’s Wnt production on endothelial cells, especially the EVPs during wound healing is currently unknown. Here we used a cutaneous excisional wound model in mice with conditional deletion of Wnt secretion by myeloid cells (Wlsfl/flLysM-Cre+ ) to assess the kinetics of endothelial subpopulations (including EVP), myeloid infiltration, collagen deposition and wound closure. Deletion of Wls expression by myeloid cells did not affect wound closure and collagen deposition, indicating that myeloid Wls expression does not promote wound healing and regeneration. Myeloid-specific Wls deletion elevated the EVP population during the peak of angiogenesis, yet without affecting blood vessel density. Wounds in Wlsfl/flLysM-Cre+ animals showed unperturbed myeloid infiltration and differentiation. Overall, our data indicate that macrophage Wnt production shapes EVP kinetics without major relevance to wound healing. These findings extend the knowledge of macrophage and endothelial molecular crosstalk and position myeloid-derived Wnt production as a regulator of endovascular progenitor.

Impact of Na+ permeation on collective migration of pulmonary arterial endothelial cells.

Collective migration of endothelial cells is important for wound healing and angiogenesis. During such migration, each constituent endothelial cell coordinates its magnitude and direction of migration with its neighbors while retaining intercellular adhesion. Ensuring coordination and cohesion involves a variety of intra- and inter-cellular signaling processes. However, the role of permeation of extracellular Na+ in collective cell migration remains unclear. Here, we examined the effect of Na+ permeation in collective migration of pulmonary artery endothelial cell (PAEC) monolayers triggered by either a scratch injury or a barrier removal over 24 hours. In the scratch assay, PAEC monolayers migrated in two approximately linear phases. In the first phase, wound closure started with fast speed which then rapidly reduced within 5 hours after scratching. In the second phase, wound closure maintained at slow and stable speed from 6 to 24 hours. In the absence of extracellular Na+, the wound closure distance was reduced by >50%. Fewer cells at the leading edge protruded prominent lamellipodia. Beside transient gaps, some sustained interendothelial gaps also formed and progressively increased in size over time, and some fused with adjacent gaps. In the absence of both Na+ and scratch injury, PAEC monolayer migrated even more slowly, and interendothelial gaps obviously increased in size towards the end. Pharmacological inhibition of the epithelial Na+ channel (ENaC) using amiloride reduced wound closure distance by 30%. Inhibition of both the ENaC and the Na+/Ca2+ exchanger (NCX) using benzamil further reduced wound closure distance in the second phase and caused accumulation of floating particles in the media. Surprisingly, pharmacological inhibition of the Ca2+ release-activated Ca2+ (CRAC) channel protein 1 (Orai1) using GSK-7975A, the transient receptor potential channel protein 1 and 4 (TRPC1/4) using Pico145, or both Orai1 and TRPC1/4 using combined GSK-7975A and Pico145 treatment did not affect wound closure distance dramatically. Nevertheless, the combined treatment appeared to cause accumulation of floating particles. Note that GSK-7975A also inhibits small inward Ca2+ currents via Orai2 and Orai3 channels, whereas Pico145 also blocks TRPC4, TRPC5, and TRPC1/5 channels. By contrast, gene silence of Orai1 by shRNAs led to a 25% reduction of wound closure in the first 6 hours but had no effect afterwards. However, in the absence of extracellular Na+ or cellular injury, Orai1 did not affect PAEC collective migration. Overall, the data reveal that Na+ permeation into cells contributes to PAEC monolayer collective migration by increasing lamellipodial formation, reducing accumulation of floating particles, and improving intercellular adhesion.

Cell sheets using human amniotic fluid stem cells reduce tissue fibrosis in murine full-thickness skin wounds

The use of mesenchymal stem cell sheets is a promising strategy for skin regeneration. The injection of dissociated human amniotic fluid stem cells (hAFSCs) was recently found to accelerate cutaneous wound healing with reduced fibrotic scarring, similar to fetal wound healing. However, the use of hAFSCs in applications of cell sheet technology remains limited. The aim of this study was to determine the in vivo efficacy of in vitro-cultured hAFSC sheets in wound healing. The cell sheets were characterized by immunohistochemistry and RT-qPCR and grafted onto full-thickness wounds in BALB/c mice. The wound size was measured, and re-epithelialization, granulation tissue area, and collagen content of the regenerated wound were analyzed histologically. Although the hAFSC sheet contained abundant extracellular matrix molecules and expressed high levels of anti-fibrotic mediators, its grafting did not affect wound closure or the size of the granulation tissue area. In contrast, the organization of type I collagen bundles in the regenerated wound was markedly reduced, while the levels of type III collagen were increased after implantation of the hAFSC sheet. These results suggest that hAFSC sheets can exert anti-fibrotic properties without delaying wound closure.

Primary appendicular soft-tissue sarcoma resection: What tumour parameters affect wound closure planning?

Wound closure after wide, local excision of an appendicular soft-tissue sarcoma (STS) can be challenging. This study evaluates the value of magnetic resonance imaging (MRI)-based tumour parameters in planning wound closure. A total of 71 patients with a primary limb-based STS, excluding vascular or osseous involvement, excluding the shoulder or hand and hip or foot were included. Axial MRI images were used to measure the circumferences and cross-sectional areas of the limb, bone, and tumour. The tumour diameter, length, and depth at the level of maximal tumour dimension were measured to identify the tumour's relative contribution to the planning of optimal wound closure management through primary closure (PC) or reconstructive surgery (RS). Eighteen patients required planned wound RS. Wound complications occurred in 14% overall. Tumours, which were closed by PC, were of significantly greater depth, shorter radial diameter, and shorter tumour circumference relative to those closed by RS. On multivariate analysis, tumour depth was the greatest contributory factor in predicting type of wound closure. A quantitative analysis of MRI-based tumour parameters demonstrates tumour depth as the most predictive factor in planning for the type of wound closure and may prove beneficial in providing greater insight into planned wound management of sarcoma resection.

Mesenchymal stromal cells contract collagen more efficiently than dermal fibroblasts: Implications for cytotherapy.

BackgroundStem cell therapy is the next generation a well-established technique. Cell therapy with mesenchymal stem cells (MSC) has been demonstrated to enhance wound healing in diabetic mice, at least partly due to improved growth factor production. However, it is unclear whether MSC can biomechanically affect wound closure. Utilizing the well-established cell-populated collagen gel contraction model we investigated the interactions between MSC and the extracellular matrix.MethodsMurine fetal liver-derived Mesenchymal Stem Cells (MSCs) or fetal Dermal Fibroblasts (DFs) were cultured in cell–populated collagen gels (CPCGs). The effect of cell density, conditioned media, growth factors (TGF-B1, FGF, PDGF-BB), cytoskeletal disruptors (colchicine, cytochalasin-D), and relative hypoxia on gel contraction were evaluated. Finally, we also measured the expression of integrin receptors and some growth factors by MSCs within the contracting gels.ResultsOur results show that at different densities, MSCs induced a higher gel contraction compared to DFs. Higher cell density resulted in faster and more complete contraction of CPCGs. Cytoskeletal inhibitors either inhibited or prevented MSC-mediated contraction in a dose dependent fashion. Growth factors, conditioned media from both MSC and DF, and hypoxia all influenced CPCG contraction.DiscussionThe results suggest that MSCs are capable of directly contributing to wound closure through matrix contraction, and they are more effective than DF. In addition, this study demonstrates the importance of how other factors such as cell concentration, cytokines, and oxygen tension can provide potential modulation of therapies to correct wound healing impairments.

Effects of Non-thermal, Non-cavitational Ultrasound Exposure on Human Diabetic Ulcer Healing and Inflammatory Gene Expression in a Pilot Study

The purpose of this clinical study was to assess, in a limited patient population, the potential for a novel advanced wound care treatment based on low-frequency (20 kHz) low-intensity (spatial peak temporal peak intensity <100 mW/cm2; i.e., pressure amplitude of 55 kPa) ultrasound (LFLI-US), to affect wound closure rate in human diabetic foot ulcers (DFUs) and to effect changes in the relative expression of pro-inflammatory and anti-inflammatory genes. The ratio of expression of these genes, termed the M1/M2 score because it was inspired by the transition of macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) phenotypes as wound healing progresses, was previously presented as a potential healing indicator for DFUs treated with the standard of care. We previously found that non-cavitational, non-thermal LFLI-US delivered with a pulse repetition frequency of 25 Hz was effective at improving wound healing in a pilot study of 20 patients with chronic venous ulcers. In this study, we assessed the potential for weekly LFLI-US exposures to affect wound healing in patients with diabetic ulcers, and we analyzed temporal changes in the M1/M2 score in debrided diabetic wound tissue. Although this was a limited patient population of only 8 patients, wounds treated with LFLI-US exhibited a significantly faster reduction in wound size compared with sham-treated patients (p < 0.001). In addition, the value of the M1/M2 score decreased for all healing diabetic ulcers and increased for all non-healing diabetic ulcers, suggesting that the M1/M2 score could be useful as an indicator of treatment efficacy for advanced DFU treatments. Such an indicator would facilitate clinical decision making, ensuring optimal wound management and thus contributing to reduction of health care expenses. Moreover, the results presented may contribute to an understanding of the mechanisms underlying ultrasonically assisted chronic wound healing. Knowledge of these mechanisms could lead to personalized or patient-tailored treatment.

Alerting the body to tissue injury: The role of alarmins and DAMPs in cutaneous wound healing.

Tissue injury stimulates an inflammatory response that is mediated in part by alarmins. Alarmins are a group of endogenous molecules that trigger inflammation in response to damage. This class of molecules is becoming increasingly recognized for their ability to influence wound healing. This article will provide an overview of alarmins and outline the latest findings on these mediators in cutaneous wound healing. In addition to stimulating inflammatory cells, recent evidence suggests that alarmins can act on other cells in the skin to affect wound closure and the extent of scar tissue production. This review will focus on HMGB-1 and IL-33, two alarmins that have received recent attention in the wound healing field. Because a properly regulated inflammatory response is critical for optimal healing, further research must be done to fully understand the role of alarmins in the wound repair process.

An Elongated Leading Edge Facilitates Rotation Flap Closure: In Vivo Demonstration.

Variation in the design of a rotation flap may affect wound closure tension. Lengthening the leading edge of a rotation flap has been a method of reducing the tension of closure in the primary motion. An in vitro study negating this tenant has been published. The authors set out to design an in vivo experiment to determine if lengthening the leading edge of a rotation flap has the effect of reducing closure tension in the primary motion of the repair. An animal study approved by Institutional Animal Care and Use Committee was undertaken in a pig model. A tension-measuring apparatus was designed using Teflon-coated wires and digital tensiometers. Rotation flaps of a standard design and with elongated leading edges were incised on the flanks of pigs under general anesthesia. Flap closure tensions were measured at points along the leading edge of the flap and in the secondary motion. Elongating the leading edge of a flap led to a statistically significant reduction in closure tension in the primary motion of the flap and at the flap tip. The secondary motion closure tensions were essentially unaffected. The authors confirm that elongating the leading edge of a standard rotation flap will reduce closure tension in the primary flap motion.

Angiogenesis and scar formation in healing wounds

One well described feature of wound healing is the ingrowth of new capillaries or angiogenesis. At its peak, the capillary content in healing wounds may reach three or more times that of normal uninjured tissue. This new vasculature is required to restore oxygenation and allow the growth of new tissue to fill the wound space. This review examines the assumption that a capillary content in excess of normal density is essential for adequate healing. The regulation of wound angiogenesis has been demonstrated to involve both proangiogenic and antiangiogenic stimuli, with the level of capillary growth reliant upon both sets of factors. Several studies now show that normal skin wounds heal adequately even when the angiogenic response is artificially reduced. In normal skin, a reduction of capillary growth to a level consistent with normal tissue does not affect wound closure and may even lead to highly favorable long term healing outcomes. The angiogenic response in normal wounds may exceed what is needed for optimal repair.

73. Novel mechanisms by which social isolation delays dermal wound healing in mice

It is well accepted that stress can negatively affect healing. Restraint-stressed mice exhibit delayed wound closure and have greater bacterial loads than controls, suggesting that bacterial clearance is impaired in this model. Interestingly, wound healing in isolated mice is delayed to a similar magnitude, yet these wounds have low bacterial loads. This finding challenges the notion that stress-impaired healing requires impaired bacterial clearance; here we explore its role (and necessity). Bacteria samples were collected from the skin of group-housed mice and cultured. Three housing conditions were established: group-housed, isolated 3 weeks before wounding, and isolated at wounding. Mice received two 3.5 mm dorsal punch biopsies. 25 μL of vehicle or bacteria solution was placed topically on each wound. Daily photographs monitored wound closure. Regardless of bacterial burden, group-housed mice exhibited faster wound closure than isolated mice. Artificially increasing bacterial load to that of control animals resulted in higher bacterial counts in isolates. The addition of bacteria did not affect healing rates in isolates, whereas it accelerated healing in group-housed controls. Surprisingly, similar healing delays were seen in mice that were isolated immediately or at 3 weeks prior to wounding. These data indicate that isolation-stress immediately alters tissue repair. In this model, bacterial burden did not appear to affect wound closure rates. These results indicate that social stressors negatively impact tissue repair comparably to other stressors, but through disparate mechanisms.

The effect of pH in modulating skin cell behaviour

Variations occur in the pH of cutaneous wounds which may affect wound closure, graft take and microbial infection rates. To determine how pH modulates cell behaviour in order to optimize wound care. The effects of pH on the attachment, proliferation and migration of keratinocytes and fibroblasts were investigated in vitro and in an ex vivo skin growth model. In addition, the effect of pH on keratinocyte differentiation as measured by the expression of cytokeratins 1 (K1) and K5 was studied. We demonstrated that the optimal pH for both keratinocyte and fibroblast proliferation is between pH 7.2 and 8.3. The optimal pH for growth from ex vivo skin explants was pH 8.43 which correlates with a previously reported improvement in skin graft take at higher pHs. Expression of K1 was found to be elevated in keratinocytes at a low pH. These results demonstrate that skin cells and explants proliferate and migrate at pHs higher than the physiological pH and that at lower pH keratinocytes express a differentiated keratinocyte phenotype. A better understanding of the responses of the cellular components of skin to fundamental physiological variables such as pH may help inform improved clinical wound care.

Influence of Fertilization Practices on Live Oak Wound Closure

A 2-year study was conducted in southwest Florida to evaluate the effects of quick vs. slow-release nitrogen sources commonly used in landscapes on wound closure in live oak [Quercus virginiana (Mill.)]. Fertilized trees displayed nonsignificant shoot growth and leaf color when compared to nonfertilized ones. Neither fertilizer type, nor rate of application appeared to affect wound closure rates over the two year measurement period. This study showed increased replication will be needed when studying landscape trees, since trends in known primary growth response to fertilization were present but were not statistically significant. Fertilization effects on secondary growth including diameter increase and wound closure rate, were correspondingly not significant. Secondary growth responses may be difficult to demonstrate in live oak when using landscape trees especially at lower nitrogen rates currently being recommended in the industry.

Cellular senescence and matrix metalloproteinase activity in chronic wounds. Relevance to debridement and new technologies.

A prolonged inflammatory response may adversely affect wound closure. Delayed wound closure and extended exposure to chronic wound fluid may also affect cellular activity in a wound bed and result in cellular senescence. Prolonged inflammation and cellular senescence may adversely affect the efficacy of topically-applied biological agents, including growth factors. Appropriate wound bed preparation and debridement are necessary to improve clinical outcomes of new technologies.

Prostaglandin E(2) regulates wound closure in airway epithelium.

Repair of the airway epithelium after injury is critical for the maintenance of barrier function and the limitation of airway hyperreactivity. Airway epithelial cells (AECs) metabolize arachidonic acid to biologically active eicosanoids via the enzyme cyclooxygenase (COX). We investigated whether stimulating or inhibiting COX metabolites would affect wound closure in monolayers of cultured AECs. Inhibiting COX with indomethacin resulted in a dose-dependent inhibition of wound closure in human and feline AECs. Specific inhibitors for both COX-1 and COX-2 isoforms impaired wound healing. Inhibitors of 5-lipoxygenase did not affect wound closure in these cells. The addition of prostaglandin E(2) (PGE(2)) eliminated the inhibition due to indomethacin treatment, and the exogenous application of PGE(2) stimulated wound closure in a dose-dependent manner. Inhibition of COX with indomethacin only at initial time points resulted in a sustained inhibition of wound closure, indicating that prostanoids are involved in early wound repair processes such as spreading and migration. These differences in wound closure may be important if arachidonic acid metabolism and eicosanoid concentrations are altered in disease states such as asthma.

The mechanism of excisional fetal wound repair in vitro is responsive to growth factors.

We have investigated the ability of fetal rat skin to heal an excisional wound in vitro. Skin from the backs of E17-E19 rats was wounded using a 1-mm diameter cutting needle and suspended in culture on a 6-pin cradle for 72 h. Neither contraction nor epithelial closure was observed within wounds created in skin from E19 embryos. In contrast, wounds in E17 skin contracted to 35-50% of their original area over 72 h, although, in the absence of serum, complete wound closure was not observed. Addition of FBS at the time of culture resulted in the movement of the epithelium over the dermal margins of the wound to effect complete closure. Histological sections through these healed wounds revealed an epithelial bridge spanning the dermal margins of the wound. A similar mechanism of repair was observed in the presence of day 14 adult wound fluid. The response of wounds in E17 skin to a range of growth factors was then assessed in an attempt to reproduce the serum response under defined conditions. Insulin-like growth factor I or epidermal growth factor did not significantly affect wound closure. Basic fibroblast growth factor, transforming growth factor-beta, or platelet-derived growth factor did promote wound closure although, in contrast to the serum-induced response, wound histology revealed repair had been achieved by dermal fibroblasts that occupied the space between the epithelial margins of the healed wound. We have therefore shown that the epithelial component of fetal wound repair proceeds in organ-cultured fetal skin in the absence of an adhesive substrate over which to migrate and is dependent on the source of trophic factors. The inability of skin taken from the E19 embryo to heal in vitro suggests a developmental switch in the mechanism of wound epithelialization.