Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar.

Granulation tissue formation and contraction is an important step of second intention wound healing. Granulation tissue develops from the connective tissue surrounding the damaged or missing area and its cellular components are mainly small vessel and inflammatory cells as well as fibroblasts and myofibroblasts. As the wound closes and evolves into a scar, there is an important decrease in cellularity; in particular myofibroblasts disappear. The question arises as to which process is responsible for this cellular loss. During a previous investigation on the expression of alpha-smooth muscle actin in myofibroblasts (Darby I, Skalli O, Gabbiani G, Lab Invest, 1990, 63:21-29), we have observed that in late phases of wound healing, many myofibroblasts show changes compatible with apoptosis and suggested that this type of cell death could be responsible for the disappearance of myofibroblasts. We have now tested this hypothesis by means of morphometry at the electron microscopic level and by in situ end labeling of fragmented DNA. Our results indicate that the number of myofibroblastic and vascular cells undergoing apoptosis increases as the wound closes and support the assumption that this is the mechanism of granulation tissue evolution into a scar. The regulation of apoptotic phenomena during wound healing may be important in scar establishment and development of pathological scarring.     

瘢痕组织中α-平滑肌肌动蛋白的表达与细胞凋亡的关系

目的:观察α-平滑肌肌动蛋白在瘢痕组织中的表达,了解病理性瘢痕形成过程中凋亡所扮演的角色及其与肌成纤维细胞在真皮中变化的关系。方法:瘢痕标本来自烧伤后来我院进行整形手术的病人,同时取病人手术供皮区的正常皮肤作为对照。 8例瘢痕组织标本 (含 2例愈合较为平坦的瘢痕和 6例增生性瘢痕组织)被分成增殖期和成熟期两组。运用caspase-3mRNA及其蛋白的表达及TUNEL方法检测瘢痕及正常组织中的凋亡细胞,并以免疫组化法检测瘢痕及正常皮肤真皮内α-平滑肌肌动蛋白单克隆抗体的表达。结果:瘢痕组织中细胞凋亡的数目与正常组织明显不同。瘢痕内的TUNEL标记阳性细胞数多于正常组织;增殖期瘢痕内的细胞凋亡的数目多于成熟期。增殖期TUNEL标记阳性的细胞多于平坦瘢痕,而成熟期两者无显著差别,Caspase-3mRNA及其蛋白的表达与TUNEL标记结果具有一致性。随着瘢痕组织的成熟,α-平滑肌肌动蛋白单克隆抗体的表达逐渐降低,平坦的瘢痕组织中的表现尤为明显;增生性瘢痕中,增殖期与成熟期之间无显著差别。结论:正常伤口愈合过程中,肌成纤维细胞暂时性的表达,可引起伤口的收缩,随着真皮再塑形,含有α-平滑肌肌动蛋白的肌成纤维细胞因凋亡而消失,而病理性的愈合结局可能是它持续表达的结果。     

Scar‐free cutaneous wound healing in the leopard gecko,Eublepharis macularius

Cutaneous wounds heal with two possible outcomes: scarification or near‐perfect integumentary restoration. Whereas scar formation has been intensively investigated, less is known about the tissue‐level events characterising wounds that spontaneously heal scar‐free, particularly in non‐foetal amniotes. Here, a spatiotemporal investigation of scar‐free cutaneous wound healing following full‐thickness excisional biopsies to the tail and body of leopard geckos (Eublepharis macularius) is provided. All injuries healed without scarring. Cutaneous repair involves the development of a cell‐rich aggregate within the wound bed, similar to scarring wounds. Unlike scar formation, scar‐free healing involves a more rapid closure of the wound epithelium, and a delay in blood vessel development and collagen deposition within the wound bed. It was found that, while granulation tissue of scarring wounds is hypervascular, scar‐free wound healing conspicuously does not involve a period of exuberant blood vessel formation. In addition, during scar‐free wound healing the newly formed blood vessels are typically perivascular cell‐supported. Immunohistochemistry revealed widespread expression of both the pro‐angiogenic factor vascular endothelial growth factor A and the anti‐angiogenic factor thrombospondin‐1 within the healing wound. It was found that scar‐free wound healing is an intrinsic property of leopard gecko integument, and involves a modulation of the cutaneous scar repair program. This proportional revascularisation is an important factor in scar‐free wound healing.     

The role of hydrogen peroxide and other reactive oxygen species in wound healing

Wound healing is a complex physiological process important for tissue homeostasis. An acute injury initiates massive cell migration, proliferation and differentiation, synthesis of extracellular matrix components, scar formation and remodelling. Blood flow and tissue oxygenation are parts of the complex regulation of healing. Higher organisms utilize molecular oxygen as a terminal oxidant. This way of gaining energy for vital processes such as healing leads to the production of a number of oxygen compounds that may have a defensive or informatory role. They may be harmful when present in high concentrations. Both the lack and the excess of reactive oxygen species may influence healing negatively.     

Extracellular Matrix Expression and Production in Fibroblast-Collagen Gels: Towards an <Emphasis Type="Italic">In Vitro</Emphasis> Model for Ligament Wound Healing

Ligament wound healing involves the proliferation of a dense and disorganized fibrous matrix that slowly remodels into scar tissue at the injury site. This remodeling process does not fully restore the highly aligned collagen network that exists in native tissue, and consequently repaired ligament has decreased strength and durability. In order to identify treatments that stimulate collagen alignment and strengthen ligament repair, there is a need to develop in vitro models to study fibroblast activation during ligament wound healing. The objective of this study was to measure gene expression and matrix protein accumulation in fibroblast-collagen gels that were subjected to different static stress conditions (stress-free, biaxial stress, and uniaxial stress) for three time points (1, 2 or 3 weeks). By comparing our in vitro results to prior in vivo studies, we found that stress-free gels had time-dependent changes in gene expression (col3a1, TnC) corresponding to early scar formation, and biaxial stress gels had protein levels (collagen type III, decorin) corresponding to early scar formation. This is the first study to conduct a targeted evaluation of ligament healing biomarkers in fibroblast-collagen gels, and the results suggest that biomimetic in-vitro models of early scar formation should be initially cultured under biaxial stress conditions.     

Skeletal muscle development and regeneration

In the late stages of muscle development, a unique cell population emerges that is a key player in postnatal muscle growth and muscle regeneration. The location of these cells next to the muscle fibers triggers their designation as satellite cells. During the healing of injured muscle tissue, satellite cells are capable of forming completely new muscle fibers or restoring damaged muscle fibers. A major problem in muscle healing is the formation of dysfunctional scar tissue, which leads to incomplete functional recovery. Therefore, the identification of factors that improve the process of muscle healing and reduce the formation of scar tissue is of great interest. Because satellite cells possess the capability of self-renewal, a unique feature of stem cells, they play a central role in the search for therapies to improve muscle healing. Growth factor-based and (satellite) cell-based therapies are being investigated to treat minor muscle injuries and intrinsic muscle defects. Major muscle injury that involves the loss of muscle tissue requires the use of scaffolds with or without (satellite) cells. Scaffolds are also being developed to generate muscle tissue in vitro. These approaches aim to restore the structure and function of the injured muscle without dysfunctional scarring.     

Effect of Peptide Bioregulator on Healing of Excision Wounds in Old Animals

Aging is associated with reduction of protein synthesis in cells, which leads to deceleration of proliferative processes in various tissues. Recovery of damaged skin sites was stimulated with a peptide bioregulator chondrolux. This agent is based on an extract from calf cartilage and bone tissue. Its effect on healing of excision wounds was studied in old rabbits. Morphological analysis of the wound surface during various periods of healing was carried out by electron microscopy. The results indicate that chondrolux application to the wound surface stimulated and optimized the reparative process. Active development of granulation tissue was observed as early as on day 14 after wound infliction (vs. days 21-28 in control animals). Acceleration of wound healing was paralleled by an increase in functional activities of fibroblast organelles.     

Tissue Engineering and Regenerative Repair in Wound Healing

Wound healing is a highly evolved defense mechanism against infection and further injury. It is a complex process involving multiple cell types and biological pathways. Mammalian adult cutaneous wound healing is mediated by a fibroproliferative response leading to scar formation. In contrast, early to mid-gestational fetal cutaneous wound healing is more akin to regeneration and occurs without scar formation. This early observation has led to extensive research seeking to unlock the mechanism underlying fetal scarless regenerative repair. Building upon recent advances in biomaterials and stem cell applications, tissue engineering approaches are working towards a recapitulation of this phenomenon. In this review, we describe the elements that distinguish fetal scarless and adult scarring wound healing, and discuss current trends in tissue engineering aimed at achieving scarless tissue regeneration.     

Ski, a modulator of wound healing and scar formation in the rat skin and rabbit ear

We recently demonstrated that Ski is a novel wound healing-related factor that promotes fibroblast proliferation and inhibits collagen secretion. Here, we show that increasing local Ski expression by gene transfer not only significantly accelerated wound healing by relieving inflammation, accelerating re-epithelialization and increasing formation of granulation tissue, but also reduced scar formation by decreasing collagen production in rat dermal wounds. Similarly, ski gene transfer accelerated wound healing, reduced the protuberant height and volume of scars and increased collagen maturity in a hypertrophic scar model in the rabbit ear. Conversely, reducing Ski expression in the wound by RNA interference resulted in significantly slower wound healing and increased scar area in rat dermal wounds. We demonstrated that these effects of Ski are associated with transforming growth factor-β-mediated signalling pathways through both Smad2/3-dependent and Smad-independent pathways. Together, our results define a dual role for Ski in promoting wound healing and alleviating scar formation, identifying a new target for therapeutic approaches to preventing scar hyperplasia and accelerating wound healing.     

Surgical treatment of depressed scar: a simple technique

Scar formation is a process consequent to the healing of soft tissues after a trauma. However, abnormal or disturbed collagen production can cause anomalies of the cutaneous surface and textural irregularities. In the presence of a depressed scar in deep tissue, we began to use a new simple technique. In the presence of adherent scars, a small incision is performed so that an undermining scissor can enter inside. The entire cicatricial area is undermined on a subcutaneous plane which, by separating the deep scar from the superficial one, completely frees it from the present adhesions so that the existing depression is totally eliminated. In order to avoid the recreation of relapses, stitches formed in a U-shape are made in Nylon or Monocril 2-3/0 are made with a large needle and are placed close together so that a wide aversion is achieved at the margins of the scar and a deep wound closure is obtained by adhering to the undermined tissue. These stitches will then be removed about 2 weeks later.     

Immunoinflammatory responses and fibrogenesis

In response to injury, tissues adjacent to the damaged area initiate a cascade of inflammatory and matrix remodeling events that are necessary to restore tissue integrity and function. The typical features of such healing effects in adult mammals are deposition of matrix proteins, which mature to scar tissues. In general, the wound healing response demonstrates certain commonalities across organs, but there are also organ-specific mechanisms. Such organ-specific controlled healing and uncontrolled tissue scarring are partly determined by the bioactivities of resident cells and local microenvironments, which are influenced by multiple factors, including the presence of specific types of cytokines (Th1 and Th2), chemokines, growth factors, cell–cell interaction, and reorganization of matrix proteins. In this article, we briefly present the relevance of Th1 and Th2 responses and the significance of interactions between matrix-producing cells and inflammatory cells during granuloma tissue and scar tissue formation.     

Scar‐Free Wound Healing and Regeneration Following Tail Loss in the Leopard Gecko,Eublepharis macularius

Many lizards are able to undergo scar‐free wound healing and regeneration following loss of the tail. In most instances, lizard tail loss is facilitated by autotomy, an evolved mechanism that permits the tail to be self‐detached at pre‐existing fracture planes. However, it has also been reported that the tail can regenerate following surgical amputation outside the fracture plane. In this study, we used the leopard gecko, Eublepharis macularius, to investigate and compare wound healing and regeneration following autotomy at a fracture plane and amputation outside the fracture plane. Both forms of tail loss undergo a nearly identical sequence of events leading to scar‐free wound healing and regeneration. Early wound healing is characterized by transient myofibroblasts and the formation of a highly proliferative wound epithelium immunoreactive for the wound keratin marker WE6. The new tail forms from what is commonly referred to as a blastema, a mass of proliferating mesenchymal‐like cells. Blastema cells express the protease matrix metalloproteinase‐9. Apoptosis (demonstrated by activated caspase 3 immunostaining) is largely restricted to isolated cells of the original and regenerating tail tissues, although cell death also occurs within dermal structures at the original‐regenerated tissue interface and among clusters of newly formed myocytes. Furthermore, the autotomized tail is unique in demonstrating apoptosis among cells adjacent to the fracture planes. Unlike mammals, transforming growth factor‐β3 is not involved in wound healing. We demonstrate that scar‐free wound healing and regeneration are intrinsic properties of the tail, unrelated to the location or mode of tail detachment. Anat Rec, 2012. © 2012 Wiley‐Periodicals, Inc.     

Assessment of processed human amniotic membrane as a protective barrier in rat model of sciatic nerve injury

Following nerve injury, scar formation is thought to be a considerable impediment to axonal regeneration at the nerve injury site. Nerve wrapping can protect the regenerating axons, and human amniotic membrane (HAM) derived from human placenta is an effective material for that purpose. The impact of nerve wrapping with HAM on functional recovery after nerve injuries, especially after autograft repair of long gap lesions, has not been comprehensively investigated. In the current study, we investigated whether the application of HAM as a nerve wrap to a 10mm segment of transected and repaired nerve would reduce scar formation and permit better axonal regeneration and/or functional recovery in rats. The outcome was assessed with morphological and functional measures. We found that nerves wrapped with HAM had significantly fewer adhesions and less scar formation than controls. Although the final outcome, both functionally and morphologically, was not significantly improved by wrapping the nerve with HAM, the observed decrease in adhesions and scar formation might help the nerve retain its mobility and thus prevent traction injury and ischemia, which are caused by nerve tethering to the adjacent tissue during the healing process.     

自体富血小板血浆凝胶修复儿童面部Ⅱ度烧伤创面

背景：富血小板血浆可用凝血酶凝固成胶冻状,不仅可以黏合组织缺损处,还可以防止血小板流失,使血小板在局部长时间分泌生长因子,避免了目前广泛应用于临床的液态重组生长因子试剂在伤口及移植区易流失易蒸发的缺点。  目的：观察外用自体富血小板血浆在儿童面部Ⅱ度烧伤创面修复过程中的治疗作用。 方法：选择2008-01/2010-01在广西昭平县人民医院外科收治的14岁以下面部浅Ⅱ度烧伤、深Ⅱ度烧伤儿童各30例。均以左侧面部为治疗组,右侧为对照组。伤后第3天,治疗组将自体富血小板血浆凝胶直接湿敷于创面上半暴露,换药1次/d;对照组创面单用等渗盐水纱布覆盖半暴露,换药1次/d。干预7,14 d观察两组患者创面愈合率、愈合时间、疼痛与瘢痕情况及不良反应。采用目测类比评分法评估疼痛情况,改良温哥华瘢痕测量法测定深Ⅱ度创面瘢痕增生情况。 结果与结论：在浅Ⅱ度烧伤创面患者中,治疗组7 d愈合率明显高于对照组(P < 0.05),愈合时间明显短于对照组(P  < 0.05);在深Ⅱ度烧伤创面患者中,治疗组14 d愈合率明显高于对照组(P  < 0.05),愈合时间显短于对照组(P < 0.05),瘢痕指数明显低于对照组(P  < 0.05);两组患者创面疼痛评分无明显差别。说明外用自体富血小板血浆凝胶能显著加快儿童面部Ⅱ度烧伤创面愈合速度,缩短愈合时间,提高愈合质量,减少瘢痕形成,无明显不良反应。     

Dextran derivatives modulate collagen matrix organization in dermal equivalent

Dextran derivatives can protect heparin binding growth factor implied in wound healing, such as transforming growth factor-β1 (TGF-β1) and fibroblast growth factor-2 (FGF-2). The first aim of this study was to investigate the effect of these compounds on human dermal fibroblasts in culture with or without TGF-β1. Several dextran derivatives obtained by substitution of methylcarboxylate (MC), benzylamide (B) and sulphate (Su) groups were used to determine the effects of each compound on fibroblast growth in vitro. The data indicate that sulphate groups are essential to act on the fibroblast proliferation. The dextran derivative LS21 DMCBSu has been chosen to investigate its effect on dermal wound healing process. Fibroblasts cultured in collagenous matrices named dermal equivalent were treated with the bioactive polymer alone or associated to TGF-β1 or FGF-2. Cross-sections of dermal equivalent observed by histology or immunohistochemistry, demonstrated that the bioactive polymer accelerates the collagen matrices organization and stimulates the human type-III collagen expression. This bioactive polymer induces apoptosis of myofibroblast, property which may be beneficial in treatment of hypertrophic scar. Culture media analyzed by zymography and Western blot showed that this polymer significantly increases the secretion of zymogen and active form of matrix metalloproteinase-2 (MMP-2), involved in granulation tissue formation. These data suggest that this bioactive polymer has properties which may be beneficial in the treatment of wound healing.     

Location of injury influences the mechanisms of both regeneration and repair within the MRL/MpJ mouse

The adult MRL/MpJ mouse regenerates all differentiated structures after through-and-through ear punch wounding in a scar-free process. We investigated whether this regenerative capacity was also shown by skin wounds. Dorsal skin wounds were created, harvested and archived from the same animals (MRL/MpJ and C57BL/6 mice) that received through-and-through ear punch wounds. Re-epithelialization was complete in dorsal wounds in both strains by day 5 and extensive granulation tissue was present by day 14 post-wounding. By day 21, wounds from both strains contained dense amounts of collagen that healed with a scar. The average wound area, as well as alpha-smooth muscle actin expression and macrophage influx were investigated during dorsal skin wound healing and did not significantly differ between strains. Thus, MRL/MpJ mice regenerate ear wounds in a scar-free manner, but heal dorsal skin wounds by simple repair with scar formation. A significant conclusion can be drawn from these data; mechanisms of regeneration and repair can occur within the same animal, potentially utilizing similar molecules and signalling pathways that subtly diverge dependent upon the microenvironment of the injury.     

Stromal reaction in cancer tissue: Pathophysiologic significance of the expression of matrix-degrading enzymes in relation to matrix turnover and immune/inflammatory reactions

Cancers are characterized by invasive growth and distant metastasis. Cancer cells not only destroy the pre-existing extracellular matrix, but cancer invasion per se usually induces new matrix formation by activation of stromal cells; that is, desmoplastic reaction. This process includes both matrix production and degradation; that Is, the remodeling process. The similarity between desmoplastic reactions in cancer stroma and the wound healing process has already been pointed out, and it has been well documented that matrix-degrading processes are actively involved In the wound healing process. A recent study revealed that most matrix-degrading enzymes, generally considered to be one of the main mechanisms of cancer invasion and metastasis, are originated from stromal cells. Based on these preconditions, the present review postulates that the abundant expression of matrix-degrading enzymes by fibroblasts, coupled with the abundant expression of type I procollagen, is involved in the matrix remodeling processes occurring in cancer stroma; that is, the mechanism similar to the wound healing process. Next, macrophages distributed along the invasive margin are known to express matrix-degrading enzymes/factors. Data from past studies of colon carcinoma indicate that the tissue expression of matrix metalloproteinase-9 and urokinase-type plas-mlnogen activator receptor Is inversely associated with simultaneous liver metastasis and infiltrating growth pattern. Previous clinicopathologic data have indicated that immune/Inflammatory cells are one of the factors for a favorable prognosis. This suggests that the expression of matrix-degrading enzymes/factors by these host cells may be involved in host immune/inflammatory reactions, and that the net function of these cells can be defensive towards the host. Data from past studies of colon carcinoma on the expression of the intercellular adhesion molecule-1 suggest that the interaction between macrophages, lymphocytes, and the phenotypes of venules distributed along the Invasive margin, further support the pro-inflammatory milieu there. Therefore, the matrix degradation process in cancer tissue is multifunctional: besides the Involvement in cancer invasion and metastasis, the matrix degradation process is also involved in the tissue remodeling process and in the immune/inflammatory reaction occurring in the stroma.     

Establishment of a long‐term hypertrophic scar model by injection of anhydrous alcohol: A rabbit model

The processes of hypertrophic scar formation are extremely complex, and current animal models have limitations in terms of the complete characterization of lesions. An ideal animal model is indispensable for exploring the complex progression of scar formation to elucidate its pathophysiology and to perform therapeutic testing. This study aimed to establish a long‐term, consistent and easily testable animal model by injecting anhydrous alcohol into the dorsal trunk dermis of rabbits. The rabbits were injected with different amounts of anhydrous alcohol. Anhydrous alcohol was infiltrated into the subcutaneous and superficial fascia. The optimal amount of anhydrous alcohol was determined by measuring the area and thickness of the scar. The typical model was established by determining the optimum dosage, and then we analysed the histological characteristics and fibrosis‐associated protein expression. The dermal scar was generated by treating with 2 ml/kg anhydrous alcohol and displayed histopathologic features that characterize human hypertrophic scarring, including a parallel collagen fibre orientation, dermal and epidermal thickening, broad collagen deposition and the loss of dermal adnexal structures. The expression of fibrotic pan‐markers was also enhanced. Moreover, the scar features and duration were compared between the anhydrous alcohol model and the rabbit ear model. Our results show that injecting anhydrous alcohol in the rabbit model thickened the dermal tissue, stimulated dermal fibroproliferation and resulted in hypertrophic scars with protein and histologic features similar to those seen in humans. Taken together, the findings from this study show that our model could be a feasible and useful tool for further research on the pathogenesis of hypertrophic scars.     

Adipogenic healing in adult mice by implantation of hollow devices in muscle

In mammals, wound healing is thought to result in the formation of scar tissue, with the exception of bony healing after fractures. Here we describe a previously unknown pattern of wound healing in which adipose rather than scar tissue is formed. Adipogenesis is normally confined to the embryo, although there are several experimental models for adipogenesis with highly specific dietary, cytokine, matrix, sex, or age requirements. The adipogenic healing described in this work provides a simple and reproducible experimental mouse model for adipogenesis without these limitations. Mice received intramuscular implants of nylon mesh material. Fibrinous material impregnated implants and within 4 weeks was replaced with highly vascular granulation tissue, typical of wound healing. Also consistent with wound healing was a reduction in vascularity of the newly formed tissue over time (P < 0.05). Lipoblasts were prevalent in granulation tissue, reaching a maximum in week 2 (P < 0.001) but falling to very low levels by week 9. These cells matured to adipocytes, with intermediate forms being seen. The identity of lipoblasts and adipocytes was confirmed by Oil Red O staining and electron microscopy. Control experiments confirmed that adipogenesis was independent of the materials used as well as of the sex and age of the animals. Rather, adipogenesis appeared to be due to replacement of fibrinous material in a space created within muscle. It is possible that adipogenic healing represents an adaptation for limiting the formation of restrictive scar tissues within muscle, and that this is the basis for the formation of traumatic lipomas in humans. Also, muscle tissue is replaced by adipose cells, seemingly derived from pluripotential satellite cells, in several degenerative muscle conditions, suggesting a role for adipogenic healing in these conditions.