In Vitro, Ex Vivo, and In Vivo Approaches for Investigation of Skin Scarring: Human and Animal Models.

Significance: The cutaneous repair process naturally results in different types of scarring that are classified as normal or pathological. Affected individuals are often affected from an esthetic, physical (functional), and psychosocial perspective. The distinct nature of scarring in humans, particularly the formation of pathological scars, makes the study of skin scarring a challenge for researchers in this area. Several established experimental models exist for studying scar formation. However, the increasing development and validation of newly emerging models have made it possible to carry out studies focused on different variables that influence this unique process. Recent Advances: Experimental models such as in vitro, ex vivo, and in vivo models have obtained different degrees of success in the reproduction of the scar formation in its native milieu and true environment. These models also differ in their ability to elucidate the molecular, cellular, and structural mechanisms involved in scarring, as well as for testing new agents and approaches for therapies. The models reviewed here, including cells derived from human skin and in vivo animal models, have contributed to the advancement of skin scarring research. Critical Issues and Future Directions: The absence of experimental models that faithfully reproduce the typical characteristics of the different types of human skin scars makes the improvement of validated models and the establishment of new ones a critical unmet need. The fields of wound healing research combined with tissue engineering have offered newer alternatives for experimental studies with the potential to provide clinically useful knowledge about scar formation.

Driving adult tissue repair via re-engagement of a pathway required for fetal healing.

Fetal cutaneous wound closure and repair differ from that in adulthood. In this work, we identify an oxidant stress sensor protein, nonselenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), that is abundantly expressed in normal fetal epidermis (and required for fetal wound closure), though not in adult epidermis, but is variably re-induced upon adult tissue wounding. NPGPx is a direct target of the miR-29 family. Following injury, abundance of miR-29 is lowered, permitting a prompt increase in NPGPx transcripts and protein expression in adult wound-edge tissue. NPGPx expression was required to mediate increased keratinocyte migration induced by miR-29 inhibition in vitro and in vivo. Increased NPGPx expression induced increased SOX2 expression and ß-catenin nuclear localization in keratinocytes. Augmenting physiologic NPGPx expression via experimentally induced miR-29 suppression, using cutaneous tissue nanotransfection or targeted lipid nanoparticle delivery of anti-sense oligonucleotides, proved to be sufficient to overcome the deleterious effects of diabetes on this specific pathway to enhance tissue repair.

Immune Cells in Cutaneous Wound Healing: A Review of Functional Data from Animal Models.

The healing of skin wounds involves the activation and recruitment of various immune cell types, many of which are believed to contribute significantly to different aspects of the repair process. Roles for immune cells have been described in practically all stages of wound healing, including hemostasis, inflammation, proliferation and scar formation/remodeling. Over the last decade, tools to deplete immune cell populations in animal models have become more advanced, leading to a surge in the number of studies examining the function of specific immune cell types in skin repair. In this review, we will summarize what is known about distinct immune cell types in cutaneous wound healing, with an emphasis on data from animal studies in which specific cell types have been targeted.

Electroceutical treatment of infected chronic wounds in a dog and a cat.

OBJECTIVES: To describe the use of an innovative printed electroceutical dressing (PED) to treat non-healing, infected chronic wounds in one dog and one cat and report outcomes. ANIMALS: A 4-year-old female spayed Mastiff and a 1-year-old female spayed domestic shorthair cat. STUDY DESIGN: Short case series. METHODS: Both cases had chronic wounds (duration: approximately 1 year for the dog and 6 3/4 months for the cat) that remained open and infected despite various wound management strategies. Both animals were treated with the PED. Observations from the records regarding wound size, antimicrobial susceptibility, and the time to healing were recorded. RESULTS: After 10 days of PED treatment in the dog and 17 days of PED treatment in the cat, the wounds had decreased in size by approximately 4.2 times in the dog and 2.5 times in the cat. Culture of punch biopsies yielded negative results. Wounds were clinically healed at 67 days in the dog and 47 days in the cat. No further treatment of the wounds was required beyond that point. CONCLUSION: Application of a PED led to closure of two chronic wounds and resolution of their persistent infection. CLINICAL SIGNIFICANCE: PEDs may provide a new treatment modality to mitigate infection and promote healing of chronic wounds.

Pre-Emptive Priming of Human Skin Improves Cutaneous Scarring and Is Superior to Immediate and Delayed Topical Anti-Scarring Treatment Post-Wounding: A Double-Blind Randomised Placebo-Controlled Clinical Trial.

The concept of pre-emptive priming of skin pre-surgery offers a novel approach in optimizing cutaneous scarring outcome. We previously showed an anti-scarring topical (epigallocatechin-3-gallate (EGCG)) is effective in improving skin scarring when applied post-surgery. The objective was to deliver an active compound at the optimal time in order to maximize its impact and improve cutaneous scarring. Therefore, pre-emptive application of anti-scarring topical pre-surgery compared with post-surgery can potentially be superior on scarring outcome. This double-blinded randomized placebo-controlled trial compares the effects of pre-emptive priming of skin with an anti-scarring topical pre-surgery versus post-surgery. Healthy volunteers (n = 40) were split into 4-groups; each undergoing different modes of application versus placebo: Group-1 = priming (7Days) pre-injury, Group-2 = priming (3D) pre-injury, Group-3 = immediate (0D) day-of-injury, Group-4 = delayed application (14D) post-injury. Excisional skin-biopsies in upper-arms were evaluated weekly with multiple quantitative devices over 8-weeks. Histological, immunohistochemical, mRNA sequencing and QRT-PCR studies were performed on tissue-biopsies. EGCG reduced mast cells at weeks-4 and 8 by gene and protein analyses (p < 0.01). Group 1 was superior to other groups (p < 0.01) in both clinical (blood flow) and laboratory parameters (elastin and immune marker expression). Additionally, there was down-regulation of angiogenic-markers by mRNA-sequencing and of CD31 and VEGF-A at weeks-4 and 8 (p < 0.01) by immunohistochemistry and at week-4 (p < 0.05) by QRT-PCR. EGCG increased antioxidant levels (HO-1) at week-4 (p < 0.01) plus elastin at week-8 (p < 0.01). In conclusion, pre-emptive priming of skin pre-injury has significant beneficial effects on surgically induced skin scarring shown by reducing mast cells, blood flow and angiogenesis plus increasing elastin content. This clinical trial was registered with ISRCTN (ISRCTN70155584).

Macrophages in Healing Wounds: Paradoxes and Paradigms.

Macrophages are prominent cells in normally healing adult skin wounds, yet their exact functions and functional significance to healing outcomes remain enigmatic. Many functional attributes are ascribed to wound macrophages, including host defense and support of the proliferation of new tissue to replace that lost by injury. Indeed, the depletion of macrophages is unmistakably detrimental to normal skin healing in adult mammals. Yet in certain systems, dermal wounds seem to heal well with limited or even no functional macrophages, creating an apparent paradox regarding the function of this cell in wounds. Recent advances in our understanding of wound macrophage phenotypes, along with new information about cellular plasticity in wounds, may provide some explanation for the apparently contradictory findings and suggest new paradigms regarding macrophage function in wounds. Continued study of this remarkable cell is needed to develop effective therapeutic options to improve healing outcomes.

A Murine Incisional Fetal Wound-Healing Model to Study Scarless and Fibrotic Skin Repair.

The ideal response to skin injury is the complete regeneration of normal tissue without scar formation. This regenerative response is known to occur at early stages of embryonic development but is lost as the skin becomes more mature. In more developed skin, the wound-healing response is suboptimal and results in the formation of scar tissue. Scar tissue can be a significant clinical concern, causing skin dysfunction as well as psychosocial issues related to poor aesthetic outcomes. Mouse models of fetal wound healing can be useful for understanding what regulatory pathways lead to skin regeneration and scarless healing in less developed skin or scarring and fibrotic healing in more developed skin. Here, a reproducible incisional wound model in developing mice is described that our lab has used repeatedly to study scarless and fibrotic fetal wound healing.

A Review of the Evidence for and against a Role for Mast Cells in Cutaneous Scarring and Fibrosis.

Scars are generated in mature skin as a result of the normal repair process, but the replacement of normal tissue with scar tissue can lead to biomechanical and functional deficiencies in the skin as well as psychological and social issues for patients that negatively affect quality of life. Abnormal scars, such as hypertrophic scars and keloids, and cutaneous fibrosis that develops in diseases such as systemic sclerosis and graft-versus-host disease can be even more challenging for patients. There is a large body of literature suggesting that inflammation promotes the deposition of scar tissue by fibroblasts. Mast cells represent one inflammatory cell type in particular that has been implicated in skin scarring and fibrosis. Most published studies in this area support a pro-fibrotic role for mast cells in the skin, as many mast cell-derived mediators stimulate fibroblast activity and studies generally indicate higher numbers of mast cells and/or mast cell activation in scars and fibrotic skin. However, some studies in mast cell-deficient mice have suggested that these cells may not play a critical role in cutaneous scarring/fibrosis. Here, we will review the data for and against mast cells as key regulators of skin fibrosis and discuss scientific gaps in the field.

High resolution ultrasound imaging for repeated measure of wound tissue morphometry, biomechanics and hemodynamics under fetal, adult and diabetic conditions.

Non-invasive, repeated interrogation of the same wound is necessary to understand the tissue repair continuum. In this work, we sought to test the significance of non-invasive high-frequency high-resolution ultrasound technology for such interrogation. High-frequency high-resolution ultrasound imaging was employed to investigate wound healing under fetal and adult conditions. Quantitative tissue cellularity and elastic strain was obtained for visualization of unresolved inflammation using Vevo strain software. Hemodynamic properties of the blood flow in the artery supplying the wound-site were studied using color Doppler flow imaging. Non-invasive monitoring of fetal and adult wound healing provided unprecedented biomechanical and functional insight. Fetal wounds showed highly accelerated closure with transient perturbation of wound tissue cellularity. Fetal hemodynamics was unique in that sharp fall in arterial pulse pressure (APP) which was rapidly restored within 48h post-wounding. In adults, APP transiently increased post-wounding before returning to the pre-wounding levels by d10 post-wounding. The pattern of change in the elasticity of wound-edge tissue of diabetics was strikingly different. Severe strain acquired during the early inflammatory phase persisted with a slower recovery of elasticity compared to that of the non-diabetic group. Wound bed of adult diabetic mice (db/db) showed persistent hypercellularity compared to littermate controls (db/+) indicative of prolonged inflammation. Normal skin strain of db/+ and db/db were asynchronous. In db/db, severe strain acquired during the early inflammatory phase persisted with a slower recovery of elasticity compared to that of non-diabetics. This study showcases a versatile clinically relevant imaging platform suitable for real-time analyses of functional wound healing.

Mast Cells in Skin Scarring: A Review of Animal and Human Research.

Mast cells (MCs) are an important immune cell type in the skin and play an active role during wound healing. MCs produce mediators that can enhance acute inflammation, stimulate re-epithelialisation as well as angiogenesis, and promote skin scarring. There is also a link between MCs and abnormal pathological cutaneous scarring, with increased numbers of MCs found in hypertrophic scars and keloid disease. However, there has been conflicting data regarding the specific role of MCs in scar formation in both animal and human studies. Whilst animal studies have proved to be valuable in studying the MC phenomenon in wound healing, the appropriate translation of these findings to cutaneous wound healing and scar formation in human subjects remains crucial to elucidate the role of these cells and target treatment effectively. Therefore, this perspective paper will focus on evaluation of the current evidence for the role of MCs in skin scarring in both animals and humans in order to identify common themes and future areas for translational research.

Inflammation as an orchestrator of cutaneous scar formation: a review of the literature.

Inflammation is a key phase in the cutaneous wound repair process. The activation of inflammatory cells is critical for preventing infection in contaminated wounds and results in the release of an array of mediators, some of which stimulate the activity of keratinocytes, endothelial cells, and fibroblasts to aid in the repair process. However, there is an abundance of data suggesting that the strength of the inflammatory response early in the healing process correlates directly with the amount of scar tissue that will eventually form. This review will summarize the literature related to inflammation and cutaneous scar formation, highlight recent discoveries, and discuss potential treatment modalities that target inflammation to minimize scarring.

Skin Biomechanics and miRNA Expression Following Chronic UVB Irradiation.

Objective: Exposure to ultraviolet (UV) light from the sun is known to accelerate the skin aging process and leads to significant alterations in skin biomechanics; however, the molecular mechanisms by which chronic UVB affects biomechanical properties of the skin have not been well described. Approach: A murine model for chronic UVB exposure was used to examine changes in epidermal barrier function, skin biomechanics, and miRNA expression as a result of UVB. Results: UVB irradiation caused skin to be weaker, less elastic, stiffer, and less pliable. Notably, these changes were not reversed after a 5-week period of recovery. Following UVB exposure, dermal collagen fibrils were significantly smaller in diameter and expression of the miR-34 family was significantly increased. Innovation: To our knowledge, this is the first study to concurrently examine alterations in skin function, miRNA expression, and tissue biomechanics in response to chronic UVB exposure. Conclusion: The data suggest that UVB alters miR-34 family expression in skin, in addition to dysregulating collagen structure with subsequent reductions in strength and elasticity. miRNAs may play a pivotal role in regulating extracellular matrix deposition and skin biomechanics following chronic UVB exposure, and thus may be a possible target for therapeutic development. However, additional studies are needed to directly probe the link between UVB exposure, miRNA production, and skin biomechanics.

Vascular Endothelial Growth Factor and Cutaneous Scarring.

Significance: The proangiogenic mediator vascular endothelial growth factor (VEGF) plays an important role in cutaneous wound repair. Most of the work on VEGF and wound healing has focused on its role in mediating angiogenesis and how this affects wound closure rates. Less is known about how VEGF affects other phases of wound healing, including scar formation. Recent Advances: Over the last 10 years, mounting evidence suggests that VEGF plays an important role in regulating scar tissue production. Multiple studies have linked high VEGF levels with scar formation in normal, hypertrophic, and keloid scars. In addition, there is experimental evidence that VEGF inhibition can reduce scar tissue deposition. Critical Issues: While there is evidence that VEGF can promote scar formation in the skin, there are several unanswered questions that remain. First, the mechanisms by which VEGF promotes scar formation have not been completely characterized. While both indirect and direct mechanisms could be involved, clear evidence for a specific mechanism is lacking. In addition, despite the availability of anti-VEGF drugs, the potential value in targeting VEGF to attenuate scar formation clinically is not yet known. Future Directions: While there are a significant number of studies examining the effects of VEGF on angiogenesis and wound closure, much less attention has been paid to the contribution of VEGF to scar tissue production. Additional studies are required to learn more about how VEGF regulates scar formation and whether VEGF inhibition could be used clinically to manage scars.

Interleukin-33 encourages scar formation in murine fetal skin wounds.

The magnitude of the inflammatory response after skin injury is important for determining whether wounds in developing fetal skin will heal scarlessly (minimal inflammation) or with prominent scars (robust inflammation). One class of inflammatory mediators gaining attention for their role in wound inflammation is alarmins. In the current study, the alarmin interleukin-33 (IL-33) was examined in a mouse model of fetal wound healing. IL-33 expression was elevated in scar-forming embryonic day 18 wounds compared to scarless embryonic day 15 wounds. Furthermore, injection of IL-33 into embryonic day 15 wounds caused scarring when wounds were analyzed at 7 days postwounding. The introduction of IL-33 into embryonic day 15 wounds did not induce statistically significant changes in the number of neutrophils, mast cells, or macrophages in vivo. However, IL-33 treatment enhanced collagen expression in cultured fibroblasts derived from adult and fetal murine skin, suggesting that IL-33 may directly stimulate fibroblasts. In vitro studies suggested that the stimulation of collagen production by IL-33 in fibroblasts was partially dependent on NF-κB activation. Overall, the data suggest an association between IL-33 and scar formation in fetal wounds.

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

PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: 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.

New Mechanisms of ECM Production during Wound Healing: A Role for Parathyroid Hormone 2 Receptor Signaling.

Extracellular matrix deposition is required for wound healing. Studies by Sato et al. reveal a role for parathyroid hormone 2 receptor (PTH2R) in extracellular matrix production in wounds. Deficiencies in PTH2R or its ligand tuberoinfundibular peptide of 39 residues (TIP39) delayed repair, and TIP39 treatment accelerated healing. TIP39-PTH2R signaling induced decorin expression, which may explain the beneficial effects of PTH2R signaling on healing. These studies identify a novel role for PTH2R signaling in extracellular matrix production.

Inflammatory responses, matrix remodeling, and re-epithelialization after fractional CO2 laser treatment of scars.

BACKGROUND AND OBJECTIVE: Fractional CO2 laser therapy has been used to improve scar pliability and appearance; however, a variety of treatment protocols have been utilized with varied outcomes. Understanding the relationship between laser power and extent of initial tissue ablation and time frame for remodeling could help determine an optimum power and frequency for laser treatment. The characteristics of initial injury caused by fractional CO2 laser treatment, the rates of dermal remodeling and re-epithelialization, and the extent of inflammation as a function of laser stacking were assessed in this study in a porcine scar model. MATERIALS AND METHODS: Full-thickness burn wounds were created on female Red Duroc pigs followed by immediate excision of the eschar and split-thickness autografting. Three months after injury, the resultant scars were treated with a fractional CO2 laser with 70 mJ of energy delivered as either a single pulse or stacked for three consecutive pulses. Immediately prior to laser treatment and at 1, 24, 96, and 168 hours post-laser treatment, transepidermal water loss (TEWL), erythema, and microscopic characteristics of laser injury were measured. In addition, markers for inflammatory cytokines, extracellular matrix proteins, and re-epithelialization were quantified at all time points using qRT-PCR. RESULTS: Both treatments produced erythema in the scar that peaked 24 hours after treatment then decreased to basal levels by 168 hours. TEWL increased after laser treatment and returned to normal levels between 24 and 96 hours later. Stacking of the pulses did not significantly increase the depth of ablated wells or extend the presence of erythema. Interleukin 6 and monocyte chemoattractant protein-1 were found to increase significantly 1 hour after treatment but returned to baseline by 24 hours post laser. In contrast, expression of transforming growth factor ß1 and transforming growth factor ß3 increased slowly after treatment with a more modest increase than interleukin 6 and monocyte chemoattractant protein-1. CONCLUSIONS: In the current study, the properties of the ablative zones were not directly proportional to the total amount of energy applied to the porcine scars with the use of triple stacking, resulting in only minor increases to microthermal zone (MTZ) depth and width versus a single pulse. Re-epithelialization and re-establishment of epidermal barrier function were observed in laser treated scars by 48 hours post therapy. Finally, many of the inflammatory genes up-regulated by the laser ablation returned to baseline within 1 week. As a whole, these results suggest that microthermal zones created by FXCO2 treatment re-epithelialize rapidly with the inflammatory response to the laser induced injury largely resolved within 1 week post treatment. Further study is needed to understand the relationship between laser stacking and MTZ properties in human scars in order to evaluate the clinical applicability of the stacking technique. Lasers Surg. Med. 49:675-685, 2017. © 2017 Wiley Periodicals, Inc.

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. FROM THE CLINICAL EDITOR: In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs.

Vascular Endothelial Growth Factor and Angiogenesis in the Regulation of Cutaneous Wound Repair.

Significance: Angiogenesis, the growth of new blood vessels from existing vessels, is an important aspect of the repair process. Restoration of blood flow to damaged tissues provides oxygen and nutrients required to support the growth and function of reparative cells. Vascular endothelial growth factor (VEGF) is one of the most potent proangiogenic growth factors in the skin, and the amount of VEGF present in a wound can significantly impact healing. Recent Advances: The activity of VEGF was once considered to be specific for endothelial cells lining the inside of blood vessels, partly because VEGF receptor (VEGFR) expression was believed to be restricted to endothelial cells. It is now known, however, that VEGFRs can be expressed by a variety of other cell types involved in wound repair. For example, keratinocytes and macrophages, which both carry out important functions during wound healing, express VEGFRs and are capable of responding directly to VEGF. Critical Issues: The mechanisms by which VEGF promotes angiogenesis are well established. Recent studies, however, indicate that VEGF can directly affect the activity of several nonendothelial cell types present in the skin. The implications of these extra-angiogenic effects of VEGF on wound repair are not yet known, but they suggest that this growth factor may play a more complex role during wound healing than previously believed. Future Directions: Despite the large number of studies focusing on VEGF and wound healing, it is clear that the current knowledge of how VEGF contributes to the repair of skin wounds is incomplete. Further research is needed to obtain a more comprehensive understanding of VEGF activities during the wound healing process.

The Importance of Mast Cells in Dermal Scarring.

Significance: Mast cells are resident inflammatory cells present in high numbers in the skin. They are one of the first cell types to respond to damage and they do so by quickly releasing a variety of preformed mediators that are stored within mast cell granules. Mast cells are not only active early on, where they help induce inflammation, but they also stimulate the proliferation of several important cell types and influence the production and remodeling of collagen. Recent Advances: Recent studies have highlighted the importance of mast cells in determining the amount of scar tissue that forms as a result of the repair process. Mast cells are found in low numbers and in a less activated state in scarless wounds, whereas high numbers of activated mast cells are associated with scarring and fibrosis. Furthermore, animals that lack mast cells or have been treated with degranulation inhibitors or drugs that block the activity of mast cell proteases have been shown to heal with reduced scar tissue. Critical Issues: Despite evidence suggesting that mast cells regulate scar tissue development, the entire range of mast cell activities during wound repair and scar formation has not been completely characterized. In addition, the potential therapeutic benefits of targeting mast cells clinically have yet to be fully explored. Future Directions: More studies are needed to determine whether inhibiting mast cell activation and blocking the function of mast cell mediators are viable options to prevent or reduce the appearance of scars.