Accelerated closure of skin wounds in mice deficient in the homeobox gene Msx2

Differences in cellular competence offer an explanation for the differences in the healing capacity of tissues of various ages and conditions. The homeobox family of genes plays key roles in governing cellular competence. Of these, we hypothesize that Msx2 is a strong candidate regulator of competence in skin wound healing because it is expressed in the skin during fetal development in the stage of scarless healing, affects postnatal digit regeneration, and is reexpressed transiently during postnatal skin wound repair. To address whether Msx2 affects cellular competence in injury repair, 3 mm full-thickness excisional wounds were created on the back of C.Cg- Msx2^tm1Rilm /Mmcd (Msx2 null) mice and the healing pattern was compared with that of the wild type mice. The results show that Msx2 null mice exhibited faster wound closure with accelerated reepithelialization plus earlier appearance of keratin markers for differentiation and an increased level of smooth muscle actin and tenascin in the granulation tissue. In vitro, keratinocytes of Msx2 null mice exhibit increased cell migration and the fibroblasts show stronger collagen gel contraction. Thus, our results suggest that Msx2 regulates the cellular competence of keratinocytes and fibroblasts in skin injury repair.     

The effects of caffeine on wound healing

The purine alkaloid caffeine is a major component of many beverages such as coffee and tea. Caffeine and its metabolites theobromine and xanthine have been shown to have antioxidant properties. Caffeine can also act as adenosine‐receptor antagonist. Although it has been shown that adenosine and antioxidants promote wound healing, the effect of caffeine on wound healing is currently unknown. To investigate the effects of caffeine on processes involved in epithelialisation, we used primary human keratinocytes, HaCaT cell line and ex vivo model of human skin. First, we tested the effects of caffeine on cell proliferation, differentiation, adhesion and migration, processes essential for normal wound epithelialisation and closure. We used 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) proliferation assay to test the effects of seven different caffeine doses ranging from 0·1 to 5 mM. We found that caffeine restricted cell proliferation of keratinocytes in a dose‐dependent manner. Furthermore, scratch wound assays performed on keratinocyte monolayers indicated dose‐dependent delays in cell migration. Interestingly, adhesion and differentiation remained unaffected in monolayer cultures treated with various doses of caffeine. Using a human ex vivo wound healing model, we tested topical application of caffeine and found that it impedes epithelialisation, confirming in vitro data. We conclude that caffeine, which is known to have antioxidant properties, impedes keratinocyte proliferation and migration, suggesting that it may have an inhibitory effect on wound healing and epithelialisation. Therefore, our findings are more in support of a role for caffeine as adenosine‐receptor antagonist that would negate the effect of adenosine in promoting wound healing.     

Comparison of human umbilical cord blood‐derived mesenchymal stem cells with healthy fibroblasts on wound‐healing activity of diabetic fibroblasts

Various types of skin substitutes composed of fibroblasts and/or keratinocytes have been used for the treatment of diabetic ulcers. However, the effects have generally not been very dramatic. Recently, human umbilical cord blood‐derived mesenchymal stromal cells (hUCB‐MSCs) have been commercialised for cartilage repair as a first cell therapy product using allogeneic stem cells. In a previous pilot study, we reported that hUCB‐MSCs have a superior wound‐healing capability compared with fibroblasts. The present study was designed to compare the treatment effect of hUCB‐MSCs with that of fibroblasts on the diabetic wound healing in vitro. Diabetic fibroblasts were cocultured with healthy fibroblasts or hUCB‐MSCs. Five groups were evaluated: group I, diabetic fibroblasts without coculture; groups II and III, diabetic fibroblasts cocultured with healthy fibroblasts or hUCB‐MSCs; and groups IV and V, no cell cocultured with healthy fibroblasts or hUCB‐MSCs. After a 3‐day incubation, cell proliferation, collagen synthesis levels and glycosaminoglycan levels, which are the major contributing factors in wound healing, were measured. As a result, a hUCB‐MSC‐treated group showed higher cell proliferation, collagen synthesis and glycosaminoglycan level than a fibroblast‐treated group. In particular, there were significant statistical differences in collagen synthesis and glycosaminoglycan levels (P = 0·029 and P = 0·019, respectively). In conclusion, these results demonstrate that hUCB‐MSCs may have a superior effect to fibroblasts in stimulating diabetic wound healing.     

Characterization of a novel standardized human three‐dimensional skin wound healing model using non‐sequential fractional ultrapulsed CO2 laser treatments

Background and Objective

At present, there is no standardized in vitro human skin model for wound healing. Therefore, our aim was to establish and characterize an in vitro/ex vivo three‐dimensional (3D) wound healing model, which we employed to analyze the effects of dexpanthenol on wound healing and gene regulation.

Materials and Methods

The novel human 3D skin wound healing model using scaffold and collagen 3D organotypic skin equivalents was irradiated with a non‐sequential fractional ultrapulsed CO₂ laser. These standardized injured full‐thickness skin equivalents enable qRT–PCR, microarray, and histological studies analyzing the effect of topically or systemically applied compounds on skin wound healing.

Results

These human laser‐irradiated skin models were found to be appropriate for in vitro wound healing analysis. Topical treatment of skin wounds with a 5% dexpanthenol water‐in‐oil emulsion or two different 5% dexpanthenol oil‐in‐water emulsions clearly enhanced wound closure compared to laser‐irradiated untreated control models. To find out whether this positive effect is caused by the active substance dexpanthenol, laser‐irradiated skin models were cultured in calciumpantothenate containing medium (20 μg/ml) compared to skin equivalents cultured without calciumpantothenate. 3D models cultured in calciumpantothenate revealed considerably faster wound closure compared to the control models. Quantitative RT–PCR studies showed enhanced mRNA expression of MMP3, IL1α, keratin‐associated protein 4–12 (KRTAP4–12), and decreased expression of S100A7 in laser‐irradiated skin models cultured in medium containing calciumpantothenate.

Conclusion

This novel standardized human 3D skin wound healing model proves useful for topical pharmacological studies on wound healing and reveals new insights into molecular mechanisms of dexpanthenol‐mediated effects on wound healing. In addition, these novel 3D model systems can be used to monitor ex vivo effects of various laser systems on gene expression and morphology of human skin. Lasers Surg. Med. 47:257–265, 2015. © 2015 Wiley Periodicals, Inc.     

Reflectance confocal microscopy for the evaluation of acute epidermal wound healing

The dynamic process of wound healing is routinely evaluated by clinical or histological evaluation. Recently, a number of non‐invasive imaging techniques have been evaluated for their clinical applicability in dermatology. Among them, reflectance confocal microscopy (RCM) represents a non‐invasive imaging technique that allows the in vivo characterization of the skin at near‐histological resolution. The aim of this study was to monitor epidermal wound repair using RCM in a model of tissue damage induced by cryosurgery. For this purpose, contact cryosurgery was performed at ?32 °C for 10 seconds on the volar forearm of five healthy volunteers. Clinical and RCM evaluations were performed at nine consecutive time points. RCM allowed the visualization of edema formation and blood vessel dilatation immediately after cryosurgery, as well as morphologic features of wound repair, including the formation of finger‐like protusions of keratinocytes into the wound bed, the appearance of hairpin‐like vessels, and inflammatory cells. This pilot study illustrates that RCM represents a promising technique for quasi‐real‐time monitoring the kinetics of wound repair non‐invasively and over time, thus offering new insights into in vivo processes of cutaneous wound repair and angiogenesis, as well as potential effects of topically applied drugs on the process of tissue repair.     

Green light emitting diodes accelerate wound healing: Characterization of the effect and its molecular basis in vitro and in vivo

Because light‐emitting diodes (LEDs) are low‐coherent, quasimonochromatic, and nonthermal, they are an alternative for low level laser therapy, and have photobiostimulative effects on tissue repair. However, the molecular mechanism(s) are unclear, and potential effects of blue and/or green LEDs on wound healing are still unknown. Here, we investigated the effects of red (638 nm), blue (456 nm), and green (518 nm) LEDs on wound healing. In an in vivo study, wound sizes in the skin of ob/ob mice were significantly decreased on day 7 following exposure to green LEDs, and complete reepithelialization was accelerated by red and green LEDs compared with the control mice. To better understand the molecular mechanism(s) involved, we investigated the effects of LEDs on human fibroblasts in vitro by measuring mRNA and protein levels of cytokines secreted by fibroblasts during the process of wound healing and on the migration of HaCat keratinocytes. The results suggest that some cytokines are significantly increased by exposure to LEDs, especially leptin, IL‐8, and VEGF, but only by green LEDs. The migration of HaCat keratinocytes was significantly promoted by red or green LEDs. In conclusion, we demonstrate that green LEDs promote wound healing by inducing migratory and proliferative mediators, which suggests that not only red LEDs but also green LEDs can be a new powerful therapeutic strategy for wound healing.     

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.     

Altered tissue repair in hevin-null mice: Inhibition of fibroblast migration by a matricellular SPARC homolog

Matricellular proteins such as hevin, secreted protein acidic and rich in cysteine, and thrombospondin‐2 play an important role during tissue repair through their influence on fundamental cellular activities such as adhesion, migration, proliferation, and extracellular matrix synthesis/reorganization. We have investigated the role played by hevin during excisional and incisional cutaneous wound repair in hevin‐null mice. Hevin‐null animals both close and heal their skin wounds faster than wild‐type animals, as evidenced by enhanced macrophage infiltration of wound beds at early time points, the earlier appearance of mature extracellular matrix, and the overall higher maturity score. In addition, fibrovascular invasion of polyvinyl alcohol sponges was more robust in hevin‐null mice, a result indicating that differences in cell migration might underlie the observed alterations in wound repair. Experiments in vitro showed that hevin induced the deadhesion and inhibited the migration of primary dermal fibroblasts in a Rac‐1–dependent manner. These findings indicate that the differences in wound repair between hevin‐null and wild‐type animals can be attributed in part to the deadhesive function of hevin and reduced cell migration within dermal wound beds in which this protein is expressed.     

Nucleolin enhances the proliferation and migration of heat‐denatured human dermal fibroblasts

Denatured dermis, a part of dermis in burned skin, has the ability to restore its normal morphology and functions after their surrounding microenvironment is improved. However, the cellular and molecular mechanisms by which the denatured dermis could improve wound healing are still unclear. This study aimed to investigate the role of nucleolin during the recovery of heat‐denatured human dermal fibroblasts. Nucleolin mRNA and protein expression were significantly increased time‐dependently during the recovery of heat‐denatured human dermal fibroblasts (52 °C, 30 seconds). Heat‐denaturation promoted a time‐dependent cell proliferation, migration, chemotaxis, and scratched wound healing during the recovery of human dermal fibroblasts. These effects were prevented by knockdown of nucleolin expression with small interference RNA (siRNA), whereas overexpression of nucleolin enhanced cell proliferation, migration, and chemotaxis of human dermal fibroblasts with heat‐denaturation. In addition, the expression of transforming growth factor‐beta 1(TGF‐β1) was significantly increased during the recovery of heat‐denatured dermis and human dermal fibroblasts. TGF‐β1 expression was up‐regulated by nucleolin in human dermal fibroblasts. The results suggest that nucleolin expression is up‐regulated, and play an important role in promoting cell proliferation, migration, and chemotaxis of human dermal fibroblasts during the recovery of heat‐denatured dermis with a mechanism probably related to TGF‐β1.     

Prostaglandin E2 differentially modulates human fetal and adult dermal fibroblast migration and contraction: implication for wound healing

Cyclooxygenase-2 is up-regulated shortly after dermal injury and it has been shown to have important activity during the repair process. Its main product in the skin, prostaglandin E2 (PGE2), modulates both inflammatory and fibrotic processes during wound healing and partially dictates the overall outcome of wound healing. PGE2 signaling has been shown to be altered during fetal wound healing. This study was designed to examine the mechanism(s) by which PGE2 regulates fibroblast migration and contraction and to determine whether these mechanisms are conserved in fetal-derived dermal fibroblasts. Fetal and adult dermal fibroblasts express all four PGE2 receptors. PGE2 inhibits fetal and adult fibroblast migration in a dose-dependent manner through the EP2/EP4-cAMP-protein kinase A pathway. However, fetal fibroblasts appear to be refractory to this effect, requiring a 10-fold higher concentration of PGE2 to achieve a similar degree of inhibition as adult fibroblasts. Inhibition of adult fibroblast migration correlated with disruption of the actin cytoskeleton. In contrast, PGE2 or a cAMP analog did not disrupt the actin cytoskeleton of fetal dermal fibroblasts. These findings were extended using a modified free-floating, fibroblast-populated collagen lattice (FPCL) contraction assay designed to measure fibroblast contraction. PGE2-inhibited FPCL contraction by adult fibroblasts, but fetal fibroblasts exhibited higher rates of FPCL contraction and a blunted response to exogenous modulation by PGE2 or a cyclase activator (forskolin). These findings indicate that fetal dermal fibroblasts are partially refractory to the effects of PGE2, a major inflammatory mediator associated with dermal wound healing. This effect may have significant and specific relevance to the scarless fetal wound-healing phenotype.     

Mesenchymal stem cell‐conditioned medium accelerates wound healing with fewer scars

Mesenchymal stem cells (MSCs) derived from umbilical cord s (UC‐MSCs) have been shown to enhance cutaneous wound healing by means of the paracrine activity. Fibroblasts are the primary cells involved in wound repair. The paracrine effects of UC‐MSCs on dermal fibroblasts have not been fully explored in vitro or in vivo. Dermal fibroblasts were treated with conditioned media from UC‐MSCs (UC‐MSC‐CM). In this model, UC‐MSC‐CM increased the proliferation and migration of dermal fibroblasts. Moreover, adult dermal fibroblasts transitioned into a phenotype with a low myofibroblast formation capacity, a decreased ratio of transforming growth factor‐β1,3 (TGF‐β1/3) and an increased ratio of matrix metalloproteinase/tissue inhibitor of metalloproteinases (MMP/TIMP). Additionally, UC‐MSC‐CM‐treated wounds showed accelerated healing with fewer scars compared with control groups. These observations suggest that UC‐MSC‐CM may be a feasible strategy to promote cutaneous repair and a potential means to realise scarless healing.     

Potential of Oncostatin M to accelerate diabetic wound healing

Oncostatin M (OSM) is a multifunctional cytokine found in a variety of pathologic conditions, which leads to excessive collagen deposition. Current studies demonstrate that OSM is also a mitogen for fibroblasts and has an anti‐inflammatory action. It was therefore hypothesised that OSM may play an important role in healing of chronic wounds that usually involve decreased fibroblast function and persist in the inflammatory stage for a long time. In a previous in vitro study, the authors showed that OSM increased wound healing activities of diabetic dermal fibroblasts. However, wound healing in vivo is a complex process involving multiple factors. Thus, the purpose of this study was to evaluate the effect of OSM on diabetic wound healing in vivo. Five diabetic mice were used in this study. Four full‐thickness round wounds were created on the back of each mouse (total 20 wounds). OSM was applied on the two left‐side wounds (n = 10) and phosphate‐buffered saline was applied on the two right‐side wounds (n = 10). After 10 days, unhealed wound areas of the OSM and control groups were compared using the stereoimage optical topometer system. Also, epithelialisation, wound contraction and reduction in wound volume in each group were compared. The OSM‐treated group showed superior results in all of the tested parameters. In particular, the unhealed wound area and the reduction in wound volume demonstrated statistically significant differences (P < 0·05). The results of this study indicate that topical application of OSM may have the potential to accelerate healing of diabetic wounds.     

Wound healing in a fetal,adult, and scar tissue model: A comparative study

Early gestation fetal wounds heal without scar formation. Understanding the mechanism of this scarless healing may lead to new therapeutic strategies for improving adult wound healing. The aims of this study were to develop a human fetal wound model in which fetal healing can be studied and to compare this model with a human adult and scar tissue model. A burn wound (10 × 2 mm) was made in human ex vivo fetal, adult, and scar tissue under controlled and standardized conditions. Subsequently, the skin samples were cultured for 7, 14, and 21 days. Cells in the skin samples maintained their viability during the 21‐day culture period. Already after 7 days, a significantly higher median percentage of wound closure was achieved in the fetal skin model vs. the adult and scar tissue model (74% vs. 28 and 29%, respectively, p<0.05). After 21 days of culture, only fetal wounds were completely reepithelialized. Fibroblasts migrated into the wounded dermis of all three wound models during culture, but more fibroblasts were present earlier in the wound area of the fetal skin model. The fast reepithelialization and prompt presence of many fibroblasts in the fetal model suggest that rapid healing might play a role in scarless healing.     

A pre‐clinical functional assessment of an acellular scaffold intended for the treatment of hard‐to‐heal wounds

The majority of the population experience successful wound‐healing outcomes; however, 1–3% of those aged over 65 years experience delayed wound healing and wound perpetuation. These hard‐to‐heal wounds contain degraded and dysfunctional extracellular matrix (ECM); yet, the integrity of this structure is critical in the processes of normal wound healing. Here, we evaluated a novel synthetic matrix protein for its ability to act as an acellular scaffold that could replace dysfunctional ECM. In this regard, the synthetic protein was subjected to adsorption and diffusion assays using collagen and human dermal tissues; evaluated for its ability to influence keratinocyte and fibroblast attachment, migration and proliferation and assessed for its ability to influence in vivo wound healing in a porcine model. Critically, these experiments demonstrate that the matrix protein adsorbed to collagen and human dermal tissue but did not diffuse through human dermal tissue within a 24‐hour observation period, and facilitated cell attachment, migration and proliferation. In a porcine wound‐healing model, significantly smaller wound areas were observed in the test group compared with the control group following the third treatment. These data provide evidence that the synthetic matrix protein has the ability to function as an acellular scaffold for wound‐healing purposes.     

Exogenous metallothionein‐IIA promotes accelerated healing after a burn wound

Severe injury to the epidermal barrier often results in scarring and life‐long functional deficits, the outcome worsening with a number of factors including time taken to heal. We have investigated the potential of exogenous metallothionein IIA (Zn₇‐MT‐IIA), a naturally occurring small cysteine‐rich protein, to accelerate healing of burn wounds in a mouse model. Endogenous MT‐I/II expression increased in basal keratinocytes concurrent with reepithelialization after a burn injury, indicating a role for MT‐I/II in wound healing. In vitro assays of a human keratinocyte cell line indicated that, compared with saline controls, exogenous Zn₇‐MT‐IIA significantly increased cell viability by up to 30% (p<0.05), decreased apoptosis by 13% (p<0.05) and promoted keratinocyte migration by up to 14% (p<0.05), all properties that may be desirable to promote rapid wound repair. Further in vitro assays using immortalized and primary fibroblasts indicated that Zn7‐MT‐IIA did not affect fibroblast motility or contraction (p>0.05). Topical administration of exogenous Zn₇‐MT‐IIA (2 μg/mL) in vivo, immediately postburn accelerated healing, promoted faster reepithelialization (3 days: phosphate‐buffered saline (PBS), 8.9±0.3 mm diameter vs. MT‐I/II, 7.1±0.7 mm; 7 days: PBS 5.8±0.98 mm vs. MT‐I/II, 3.6±1.0 mm, p<0.05) and reduced epidermal thickness (MT‐I/II: 45±4 μm vs. PBS: 101±19 μm, p<0.05) compared with controls. Our data suggest that exogenous Zn₇‐MT‐IIA may prove a valuable therapeutic for patients with burns and other skin injuries.     

ORIGINAL RESEARCH – BASIC SCIENCE: Primary dermal fibroblasts derived from sdc‐1 deficient mice migrate faster and have altered αv integrin function

The goal of this study is to determine whether dermal fibroblasts lacking syndecan‐1 (sdc1) show differences in integrin expression and function that could contribute to the delayed skin and corneal wound healing phenotypes seen in sdc‐1 null mice. Using primary dermal fibroblasts, we show that after 3 days in culture no differences in α‐smooth muscle actin were detected but sdc‐1 null cells expressed significantly more αv and β1 integrin than wildtype (wt) cells. Transforming growth factor β1 (TGFβ1) treatment at day 3 increased αv‐ and β1‐integrin expression in sdc‐1 null cells at day 5 whereas wt cells showed increased expression only of αv‐integrin. Using time‐lapse studies, we showed that the sdc‐1 null fibroblasts migrate faster than wt fibroblasts, treatment with TGFβ1 increased these migration differences, and treatment with a TGFβ1 antagonist caused sdc‐1 null fibroblasts to slow down and migrate at the same rate as untreated wt cells. Cell spreading studies on replated fibroblasts showed altered cell spreading and focal adhesion formation on vitronectin and fibronectin‐coated surfaces. Additional time lapse studies with β1‐ and αv‐integrin antibody antagonists, showed that wt fibroblasts expressing sdc‐1 had activated integrins on their surface that impeded their migration whereas the null cells expressed αv‐containing integrins which were less adhesive and enhanced cell migration. Surface expression studies showed increased surface expression of α2β1 and α3β1 on the sdc‐1 null fibroblasts compared with wt fibroblasts but no significant differences in surface expression of α5β1, αvβ3, or αvβ5. Taken together, our data indicates that sdc‐1 functions in the activation of αv‐containing integrins and support the hypothesis that impaired wound healing phenotypes seen in sdc‐1 null mice could be due to integrin‐mediated defects in fibroblast migration after injury.     

Exogenous calreticulin improves diabetic wound healing

A serious consequence of diabetes mellitus is impaired wound healing, which largely resists treatment. We previously reported that topical application of calreticulin (CRT), an endoplasmic reticulum chaperone protein, markedly enhanced the rate and quality of wound healing in an experimental porcine model of cutaneous repair. Consistent with these in vivo effects, in vitro CRT induced the migration and proliferation of normal human cells critical to the wound healing process. These functions are particularly deficient in poor healing diabetic wounds. Using a genetically engineered diabetic mouse (db/db) in a full‐thickness excisional wound healing model, we now show that topical application of CRT induces a statistically significant decrease in the time to complete wound closure compared with untreated wounds by 5.6 days (17.6 vs. 23.2). Quantitative analysis of the wounds shows that CRT increases the rate of reepithelialization at days 7 and 10 and increases the amount of granulation tissue at day 7 persisting to day 14. Furthermore, CRT treatment induces the regrowth of pigmented hair follicles observed on day 28. In vitro, fibroblasts isolated from diabetic compared with wild‐type mouse skin and human fibroblasts cultured under hyperglycemic compared with normal glucose conditions proliferate and strongly migrate in response to CRT compared with untreated controls. The in vitro effects of CRT on these functions are consistent with CRT's potent effects on wound healing in the diabetic mouse. These studies implicate CRT as a potential powerful topical therapeutic agent for the treatment of diabetic and other chronic wounds.