Soft tissue mechanotransduction in wound healing and fibrosis

Recent evidence suggests that mechanical forces can significantly impact the biologic response to injury. Integrated mechanical and chemical signaling networks have been discovered that enable physical cues to regulate disease processes such as pathologic scar formation. Distinct molecular mechanisms control how tensional forces influence wound healing and fibrosis. Conceptual frameworks to understand cutaneous repair have expanded beyond traditional cell-cytokine models to include dynamic interactions driven by mechanical force and the extracellular matrix. Strategies to manipulate these biomechanical signaling networks have tremendous therapeutic potential to reduce scar formation and promote skin regeneration.     

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.     

Roles of wound geometry, wound size, and extracellular matrix in the healing response of bovine corneal endothelial cells in culture

It has classically been accepted that the healing of narrow wounds in epithelia occurs by the formation of a contractile actin cable, while wide wounds are resurfaced by lamellipodia-dependent migration of border cells into the denuded area. To further investigate the general validity of this idea, we performed systematic experiments of the roles of wound geometry, wound size, and extracellular matrix (ECM) in wound healing in monolayers of bovine corneal endothelial cells, a system shown here to predominantly display any of the two healing mechanisms according to the experimental conditions. We found that, in this system, it is the absence or presence of the ECM on the wound surface that determines the specific healing mode. Our observations demonstrate that, independent of their size and geometry, wounds created maintaining the ECM heal by migration of cells into the wound area, while ECM removal from the wound surface determines the predominant formation of an actin cable. While the latter mechanism is slower, the actin cable permits the maintainance of the epithelial phenotype to a larger extent during the healing process, as also confirmed by our finding of a more conserved localization of cadherin and vinculin. We also introduce a model that simulates experimental findings about the dynamics of healing mechanisms, both for the maintenance or removal of the ECM on the wound surface. The findings of this study may contribute to the understanding of physiological and pathological aspects of epithelial wound healing and to the design of therapeutic strategies.     

Isolation of wound healing/regeneration genes using restrictive fragment differential display-PCR in MRL/MPJ and C57BL/6 mice

Wound healing in mammals can take several weeks to months and the process is always accompanied by scar formation. Wound healing mechanisms that mimic regeneration are not found in most mature mammalian tissues. However, the MRL/MPJ (MRL) mouse has the unique capacity to regenerate ear hole wound completely in less than a month. To identify genes involved in wound healing without a scar, we chose to use restriction fragment differential display-PCR to isolate genes differentially expressed in the MRL (good healer) mouse and the C57BL/6 (poor healer) mouse at different stages of wound healing. We identified 36 genes that were differentially expressed in the regenerating tissue of good and poor healer strains of which several genes are also genetically linked to wound healing and thus are potential candidate genes for scarless wound healing.     

IL-10 alleviates lipopolysaccharide-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts

Hypertrophic scar (HS) is a severe fibrotic skin disease. It has always been a major problem in clinical treatment, mainly because its pathogenesis has not been well understood. The roles of bacterial contamination and prolonged wound inflammation were considered significant. IL-10 is a potent anti-inflammatory cytokine and plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL-10 alleviates lipopolysaccharide (LPS)-induced inflammatory response and skin scarring and explore the possible mechanism of scar formation. Our results showed that the expression of TLR4 and pp65 was higher in HS and HS-derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS-derived fibroblasts (NSFs). LPS could up-regulate the expression of TLR4, pp65, Col I, Col III and α-SMA in NSFs, but IL-10 could down-regulate their expression in both HSFs and LPS-induced NSFs. Blocking IL-10 receptor (IL-10R) or the phosphorylation of STAT3, their expression was up-regulated. In addition, in vitro and in vivo models results showed that IL-10 could alleviate LPS-induced fibroblast-populated collagen lattice (FPCL) contraction and scar formation. Therefore, IL-10 alleviates LPS-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of fibroblasts to myofibroblasts. Our results indicate that IL-10 can alleviate the LPS-induced harmful effect on wound healing, reduce scar contracture, scar formation and skin fibrosis. Therefore, the down-regulation of inflammation may lead to a suitable scar outcome and be a better option for improving scar quality.     

Epithelial–mesenchymal transition in the formation of hypertrophic scars and keloids

Abnormal wound healing is likely to induce the formation of hypertrophic scars and keloids, which leads to dysfunction, deformity, and mental problem in the patients. Despite the advances in prevention and management of hypertrophic scar and keloids, the mechanism underlying scar and keloid formation has not been fully elucidated. Recent insights into the role of the epithelial–mesenchymal transition (EMT) in development, wound healing, stem cell regulation, fibrosis, and tumorigenesis have increased our understanding of the pathophysiology of hypertrophic scarring and keloids and suggested new therapeutic targets. This review summarizes recent progress in the elucidation of the role of EMT in physiologic wound healing and pathologic scar formation. This knowledge will facilitate an understanding of EMT roles in scar formation and shed new light on the modulation and potential treatment of hypertrophic scars and keloids.     

Scarless fetal skin wound healing update

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

Gene profiling of the rat medial collateral ligament during early healing using microarray analysis

Ligament heals in a synchronized and complex series of events. The remodeling process may last months or years. Experimental evidence suggests the damaged ligament does not recover its normal functional properties. Specific mechanisms to prevent scar formation and to regenerate the original mechanical function remain elusive but likely involve regulation of creeping substitution. Creeping substitution creates a larger hypercellular, hypervascular, and disorganized granulation tissue mass that results in an inefficient and nonregenerative wound healing process for the ligament. Control of creeping substitution may limit the extent of this tissue compromise and reduce the time necessary for healing. The objective of this study is to better understand the mechanism behind scar formation by identifying the extracellular matrix factors and other unique genes of interest differentially expressed during rat ligament healing via microarray. For this study, rat medial collateral ligaments were either surgically transected or left intact. Ligaments were collected at day 3 or 7 postinjury and used for microarray, quantitative PCR, and/or immunohistochemistry. Results were compared with the normal intact ligament. We demonstrate that early ligament healing is characterized by the modulation of several inflammatory and extracellular matrix factors during the first week of injury. Specifically, a number of matrix metalloproteinases and collagens are differentially and significantly expressed during early ligament healing. Additionally, we demonstrate the modulation of three novel genes, periostin, collagen-triple helix repeat containing-1, and serine protease 35 in our ligament healing model. Together, control of granulation tissue creeping substitution and subsequent downstream scar formation is likely to involve these factors.     

The emerging roles of neutrophil extracellular traps in wound healing

Delayed wound healing causes problems for many patients both physically and psychologically, contributing to pain, economic burden, loss of function, and even amputation. Although many factors affect the wound healing process, abnormally prolonged or augmented inflammation in the wound site is a common cause of poor wound healing. Excessive neutrophil extracellular trap (NET) formation during this phase may amplify inflammation and hinder wound healing. However, the roles of NETs in wound healing are still unclear. Herein, we briefly introduce NET formation and discuss the possible NET-related mechanisms in wound healing. We conclude with a discussion of current studies, focusing on the roles of NETs in diabetic and normoglycemic wounds and the effectiveness of NET-targeting treatments in wound healing.Subject terms: Mechanisms of disease, Experimental models of disease     

Elastin signaling in wound repair

Skin is an important organ to the human body as it functions as an interface between the body and environment. Cutaneous injury elicits a complex wound healing process, which is an orchestration of cells, matrix components, and signaling factors that re‐establishes the barrier function of skin. In adults, an unavoidable consequence of wound healing is scar formation. However, in early fetal development, wound healing is scarless. This phenomenon is characterized by an attenuated inflammatory response, differential expression of signaling factors, and regeneration of normal skin architecture. Elastin endows a range of mechanical and cell interactive properties to skin. In adult wound healing, elastin is severely lacking and only a disorganized elastic fiber network is present after scar formation. The inherent properties of elastin make it a desirable inclusion to adult wound healing. Elastin imparts recoil and resistance and induces a range of cell activities, including cell migration and proliferation, matrix synthesis, and protease production. The effects of elastin align with the hallmarks of fetal scarless wound healing. Elastin synthesis is substantial in late stage in utero and drops to a trickle in adults. The physical and cell signaling advantages of elastin in a wound healing context creates a parallel with the innate features of fetal skin that can allow for scarless healing. Birth Defects Research (Part C) 96:248–257, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

Hypertrophic scarring and keloids: pathomechanisms and current and emerging treatment strategies

Excessive scars form as a result of aberrations of physiologic wound healing and may arise following any insult to the deep dermis. By causing pain, pruritus and contractures, excessive scarring significantly affects the patient's quality of life, both physically and psychologically. Multiple studies on hypertrophic scar and keloid formation have been conducted for decades and have led to a plethora of therapeutic strategies to prevent or attenuate excessive scar formation. However, most therapeutic approaches remain clinically unsatisfactory, most likely owing to poor understanding of the complex mechanisms underlying the processes of scarring and wound contraction. In this review we summarize the current understanding of the pathophysiology underlying keloid and hypertrophic scar formation and discuss established treatments and novel therapeutic strategies.     

Numerical modelling of the angiogenesis process in wound contraction

Angiogenesis consists of the growth of new blood vessels from the pre-existing vasculature. This phenomenon takes place in several biological processes, including wound healing. In this work, we present a mathematical model of angiogenesis applied to skin wound healing. The developed model includes biological (capillaries and fibroblasts), chemical (oxygen and angiogenic growth factor concentrations) and mechanical factors (cell traction forces and extracellular matrix deformation) that influence the evolution of the healing process. A novelty from previous works, apart from the coupling of angiogenesis and wound contraction, is the more realistic modelling of skin as a hyperelastic material. Large deformations are addressed using an updated Lagrangian approach. The coupled non-linear model is solved with the finite element method, and the process is studied over two wound geometries (circular and elliptical) of the same area. The results indicate that the elliptical wound vascularizes two days earlier than the circular wound but that they experience a similar contraction level, reducing its size by 25 %.     

Impaired wound healing in transgenic mice overexpressing the activin antagonist follistatin in the epidermis

Recently, we demonstrated a strong upregulation of activin expression after skin injury. Furthermore, overexpression of this transforming growth factor beta family member in the skin of transgenic mice caused dermal fibrosis, epidermal hyperthickening and enhanced wound repair. However, the role of endogenous activin in wound healing has not been determined. To address this question we overexpressed the soluble activin antagonist follistatin in the epidermis of transgenic mice. These animals were born with open eyes, and the adult mice had larger ears, longer tails and reduced body weight compared with non-transgenic littermates. Their skin was characterized by a mild dermal and epidermal atrophy. After injury, a severe delay in wound healing was observed. In particular, granulation tissue formation was significantly reduced, leading to a major reduction in wound breaking strength. The wounds, however, finally healed, and the resulting scar area was smaller than in control animals. These results implicate an important function of endogenous activin in the control of wound repair and scar formation.     

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.     

Regeneration and control of human fibroblast cell density by intermittently delivered pulsed electric fields

Proliferative scarring is a human disease with neither available effective treatment nor relevant animal model. One of the hypotheses for scar formation involves deregulation of fibroblast signaling and delayed apoptosis. Here, we introduce a new chemical‐free method for fibroblast density control in culture by intermittently delivered pulsed electric fields (IDPEF), which cause irreversible damage to cell membranes. Using 5–100 pulses with electric field strength of 150 V/mm, pulse duration 70 µs, and frequency of 1 Hz, we investigated the effects of PEF application on growth, death, and regeneration of normal human dermal fibroblasts in culture. We found that the fraction of fibroblasts that survive depends on the number of pulses applied and follows a Weibull distribution. We have successfully developed an IDPEF protocol that controls fibroblasts density in culture. Specifically, through application of IDPEF every 72 h for 12 days, we maintain a normal human dermal fibroblast density in the 3.1 ± 0.2 × 10⁵–1.4 ± 0.2 × 10⁵ cell/mL range. Our results suggest that IDPEFs may prove useful as a non‐chemical method for fibroblast density control in human wound healing. Biotechnol. Bioeng. 2013; 110: 1759–1768. © 2013 Wiley Periodicals, Inc.