Initial Phase Of Wound Healing Research Articles

Preparation of Enzyme-Soluble Swim Bladder Collagen from Sea Eel (Muraenesox cinereus) and Evaluation Its Wound Healing Capacity.

In the present research, the enzyme-facilitated collagen from sea eel (Muraenesox cinereus) swim bladder was isolated, and the collagen characteristics were analyzed. Then, the collagen sponge was prepared and its potential mechanism in promoting skin wound healing in mice was further investigated. Collagen was obtained from the swim bladder of sea eels employing the pepsin extraction technique. Single-factor experiments served as the basis for the response surface method (RSM) to optimize pepsin concentration, solid-liquid ratio, and hydrolysis period. With a pepsin concentration of 2067 U/g, a solid-liquid ratio of 1:83 g/mL, and a hydrolysis period of 10 h, collagen extraction achieved a yield of 93.76%. The physicochemical analysis revealed that the extracted collagen belonged to type I collagen, and the collagen sponge displayed a fibrous structure under electron microscopy. Furthermore, in comparison to the control group, mice treated with collagen sponge dressing exhibited elevated activities of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px), and decreased levels of malondialdehyde (MDA), interleukin (IL)-1β, interleukin (IL)-6, and tumor necrosis factor (TNF)-α. The collagen sponge dressing effectively alleviated inflammation in the wound area, facilitating efficient repair and rapid healing of the skin tissue. During the initial phase of wound healing, the group treated with collagen sponge dressing exhibited an enhancement in the expressions of cluster of differentiation (CD)31, epidermal growth factor (EGF), transforming growth factor (TGF)-β1, and type I collagen, leading to an accelerated rate of wound healing. In addition, this collagen sponge dressing could also downregulate the expressions of CD31, EGF, and type I collagen to prevent scar formation in the later stage. Moreover, this collagen treatment minimized oxidative damage and inflammation during skin wound healing and facilitated blood vessel formation in the wound. Consequently, it exhibits significant potential as an ideal material for the development of a skin wound dressing.

The effect of Salvadora persica ethanolic extract on oral tissue healing in rats: An in vivo study

IntroductionInvasive surgical procedures in the oral cavity inevitably cause trauma to the soft and hard tissues. The healing process in the oral cavity tissue occurs in a complex manner involving different types of cells, maturation process, and the time of healing. Salvadora persica (miswak) has been found to exert various positive effects on the oral cavity, including antimicrobial, anti-gingivitis, anti-cariogenic, gingival healing, and teeth whitening properties. This study aims to investigate the potential of miswak as an adjunctive therapy in promoting wound healing. Materials and methods30 live Sprague-Dawley rats were used in this study. The rats’ mandibular first molar tooth was extracted, and an incision wound was made on the tongue. The extraction socket and incision wound were irrigated using normal saline and different concentrations of locally processed miswak plant extracts (0.05%, 10%, and 20%) for 7 days. The rats were sacrificed for gross examination of the tooth socket and tongue healing. Both soft tissue and alveolar bone were examined microscopically. ResultsComplete closure of the incision wound was observed on all rats’ tongues; miswak groups showed better wound healing than control and placebo groups in the oral mucosa overlying the alveolar bones. 0.05% and 20% miswak extracts showed prominent wound healing effects in the sagittal sections of the tongue, with moderate formation of connective tissue under the wound site and notable wound contraction. The 20% miswak extract group showed the highest percentage of healed oral mucosa on the alveolar bone and higher bone deposition at the alveolar base. ConclusionA concentration of 20% miswak extract enhances the initial phase of wound healing both in oral soft and hard tissues. Miswak extract at this concentration was not toxic to the tissues and had potential therapeutic effects in oral tissue healing.

Autologous platelet-rich plasma action on skin autografts in horses

ABSTRACT: This study evaluated the action of autologous platelet-rich plasma (PRP) on cutaneous wounds, containing skin autografts, in the gluteal region of horses. Seven healthy horses were used. Two 6 x 6cm cutaneous wounds were produced on each side of the gluteal region. Eight days after wound induction, grafts were performed with skin fragments harvested from the neck, as well as the application of PRP, prepared by double-centrifugation protocol. Wounds with autografts on the left side received PRP (group T), and those with autografts on the right side did not receive treatment (group C). Macroscopic and microscopic evaluations were performed, considering the integration of autografts and retraction of wound edges, as well as neovascularization, inflammatory infiltrate, young fibroblasts, collagenization, reepithelization and autografts integration. There was no difference between the groups (P > 0.05) in relation to most macroscopic and microscopic variables. However, neovascularization was significantly greater (p = 0.0191) in group T, on the 14th day after grafting. It is concluded that PRP favors the process of skin repair with autografts in horses, since it increases the neovascularization in the initial phase of wound healing. Furthermore, the PRP seems to positively influence the integration of the skin autografts and the retraction of the wound edges.

Neutrophils as a Source of Factors Increasing Duration of the Inflammatory Phase of Wound Healing in Patients with Type 2 Diabetes Mellitus

Oxidative stress and neutrophil activation leading to an increase in myeloperoxidase (MPO), elastase and neutrophil extracellular trap (NET) levels in blood are considered as the pathogenic mechanisms responsible for the development of extremity damage in people with type 2 diabetes mellitus (T2DM). The aim of this study was to analyze the relationship between factors, associated with neutrophil activation, and the duration of the initial phase of wound healing (the inflammatory phase) in T2DM patients. Patients were divided retrospectively into three groups depending on the severity of lower extent damage: group 1 (wound on toe) < group 2 (wound on foot) < group 3 (wound on lower leg). At admission to hospital, T2DM patients had significantly higher (р < 0.05) levels of blood glucose and glycated hemoglobin (groups 1−3), ESR (groups 1 and 3), blood neutrophil count (groups 2 and 3), plasma MPO concentration (groups 1−3) and blood NET concentration (group 3) and decreased levels of plasma thiols (groups 1−3) and erythrocyte glutathione peroxidase activity (groups 2 and 3) than in the control group (healthy volunteers). The length of hospital stay after surgery corresponded to the length of the inflammatory phase of the wound healing process and correlated with the number of blood neutrophils in patients before surgery (r = 0.72, p < 0.05). Leukocytic intoxication index depended on wound area (r = 0.59, р < 0.05), and it was significantly higher for groups 2 and 3 compared to the control group and group 1. The correlation found can be attributed to the increase in extracellular MPO and NETs due to the activation and degranulation of neutrophils and netosis. Thus, the duration of the inflammatory phase of wound healing depends on specific aspects of systemic inflammation increasing oxidative/halogenative stress and intoxication.

Effects of Surface Nanotopography and Calcium Chemistry of Titanium Bone Implants on Early Blood Platelet and Macrophage Cell Function

Early responses of blood platelets and immunoinflammatory cells (macrophages) to titanium (Ti) bone implants affect the subsequent biological healing of implants by modulating early tissue healing-microenvironments via the formation of temporary fibrin matrix scaffolds for stem cell migration and production of growth factors and cytokines. This study investigated the effects of nanoscale surface topography and calcium ion (Ca2+) modification of Ti surfaces on biocompatibility regulated by blood platelets and macrophages, for the future surface design of Ti bone implants with enhanced early osteogenic capacity. A nanostructured Ti surface with or without Ca2+ enrichment was prepared using the hydrothermal treatment. Immediate and early functions of platelets and macrophages modulated by modified Ti surfaces were investigated by morphological observation of platelet spreading and fibrin matrix formation, platelet growth factor release, immunostaining of macrophage phenotypes, and macrophage inflammatory cytokine production. The results showed that surface nanoscale topographical modification of Ti promotes blood platelet activation and suppresses the inflammatory response of macrophages. In addition, surface chemistry modifications with Ca2+ enhanced the platelet response-modulating function of the nanostructured Ti surface, which accelerated immediate fibrin matrix formation and platelet-derived growth factor-AB release. Thus, nanotopographical and Ca2+ modifications of implant surfaces are expected to be effective approaches that favor the initial phase of wound healing around the Ti bone implants through positive modulation of immediate blood platelet function and early macrophage immunoinflammatory response.

Neutrophils as a source of factors that increase the length of the inflammatory phase of wound healing in patients with type 2 diabetes mellitus

Oxidative stress and neutrophil activation leading to an increase in myeloperoxidase (MPO), elastase and neutrophil extracellular trap (NET) levels in blood are considered as pathogenic mechanisms responsible for the development of extremity damage in people with type 2 diabetes mellitus (T2DM). The aim of this study was to analyze the relationship between factors, associated with neutrophil activation, and the length of the initial phase of wound healing (the inflammatory phase) in T2DM patients. Patients were divided retrospectively into three groups depending on the damage extent: group 1 (wound on toe) < group 2 (wound on foot) < group 3 (wound on lower leg). Compared to the control group (healthy volunteers), T2DM patients at admission to hospital had significantly (p<0.05) increased levels of blood glucose and glycated hemoglobin (groups 1-3), ESR (groups 1 and 3), blood neutrophil count (groups 2 and 3), plasma MPO concentration (groups 1-3) and blood NET concentration (group 3) and decreased levels of plasma thiols (groups 1-3) and erythrocyte glutathione peroxidase activity (groups 2 and 3). The length of hospital stay after surgical procedures corresponded to the length of the inflammatory phase of the wound healing process and correlated with the number of blood neutrophils in patients before surgery (r=0.72, p<0.05). Leukocytic intoxication index depended on wound area (r=0.59, p<0.05), and it was significantly higher for groups 2 and 3 compared to the control group and group 1. The neutrophil count before surgery in T2DM patients with damage in the lower extremities correlated with the length of the inflammatory phase of wound healing. The correlation found can be attributed to an increase in extracellular MPO and NETs, which, in its turn, results from the activation and degranulation of neutrophils and netosis. Thus, the duration of the inflammatory phase of wound healing depends on specific aspects of systemic inflammation increasing oxidative/halogenative stress and intoxication.

The Effect of Microporous Polysaccharide Hemospheres on Wound Healing and Scarring in Wild-Type and db/db Mice.

Hemostasis, the initial phase of wound healing, sets the stage for tissue repair. Microporous polysaccharide hemosphere powder (MPH) is an FDA-approved hemostatic agent that may impact the wound-healing process. This study examined the role of MPH in murine wild-type and diabetic (db/db) wound-healing models and a foreign body response scarring model. The powder was topically applied to excisional wounds in wild-type C57BL/6 mice and db/db mice. The effect of MPH on scarring was evaluated by applying it to the expanded polytetrafluoroethylene tube implantation model. In wild-type mice, topically applied MPH increased epithelial thickness. Levels of α-smooth muscle actin (α-SMA) were decreased in MPH-treated wild-type wounds, whereas Rho-associated protein kinase 2 (ROCK2) and transforming growth factor β levels were increased. In db/db mice, topical wound MPH application decreased epithelial thickness and delayed wound closure. The db/db wounds displayed an increased collagen index. The ROCK2 was increased in a similar manner to wild-type mice, whereas α-SMA and transforming growth factor β levels were decreased. The MPH-treated expanded polytetrafluoroethylene tube mice showed increased α-SMA levels and depressed ROCK2 levels. There were no changes in histologic parameters of the foreign body response. The results suggest that MPH does not adversely impact wound healing in wild-type mice, both topically and around implants, but prolongs time to closure and diminishes thickness in db/db wounds. The MPH application alters contractile proteins in all wound models. These changes could have downstream effects on the wound healing process, and further investigation into the use of MPH in altered or impaired states of wound healing is warranted.

Development of ursolic acid-loaded polymer matrices for treatment of wounds in diabetic rats

Impaired wound healing is an important clinical problem in diabetes and results in slow wound healing which may lead to infection, sepsis, gangrene and even amputations. In the present study, polymer (collagen and arginine) based dressings were prepared to be applied as scaffolds for the control release of ursolic acid (UA), a triterpenoid that acts as an inflammatory modulator in wound healing. The activity of UA-loaded polymer matrices to treat wounds in streptozotocin diabetic induced rat was evaluated. There was a delay in wound healing in diabetic rats as compared to non-diabetic rats. The dressing exhibited potent wound healing activity as indication by the wound contraction in the excision wound model. UA-loaded polymer dressings made faster healing (21% wound area reduction) in the initial phase of wound healing in diabetic rat. The contents of hydroxyproline and hexosamine also correlated with the observed healing pattern. These findings were supported by the histopathological characteristics of healed wound sections, as greater tissue regeneration, more fibroblasts, and angiogenesis were observed in the UA-loaded polymer matrices-treated group. These results suggest that polymer-based dressings can be an effective support for UA release into diabetic wounds enhancing the healing process.

Influence of curing agent on fibrosis around silicone implants

Severe capsular contracture around silicone expander breast implants leading to pain and failure is a major clinical problem. Even though earlier studies have implicated the immunogenicity of silicone, the role of physical and chemical properties of the silicone material in excessive collagen deposition and fibrosis has been less addressed. The present study investigates whether there is any correlation between the type of curing systems i.e. addition and free radical curing and the fibrosis around silicone elastomer. The experiment carried out uses commercially available silicone ventriculo-peritoneal shunt material elastomer cured by platinum and the results are compared with results obtained in a similar study carried out by the authors using commercially available silicone tissue expander material cured by peroxide. Ultra-high molecular weight poly-ethylene (UHMWPE), the standard reference for biocompatibility evaluation, was used as the control material. The materials were implanted in rat skeletal muscle for 30 and 90 days. Inflammatory cells, myofibroblasts, cytokines, and collagen deposition at the material–tissue interface were identified by haematoxylin–eosin and Masson’s Trichrome stains and semi-quantitated based on immunohistochemical studies. Results indicate that even though the cellular response in the initial phase of wound healing was similar in both platinum and peroxide-cured materials, the collagen deposition in the proliferative phase was more around peroxide-cured material in comparison to the platinum-cured silicone elastomer. There is a need to look into the molecular mechanisms of this interaction and the possibility of using curing systems other than free radical peroxide in the manufacture of silicone elastomer expanders for breast prosthesis.

The role of stem cell factor and c-KIT in keloid pathogenesis: do tyrosine kinase inhibitors have a potential therapeutic role?

Keloids are fibroproliferative disorders characterized by increased deposition of extracellular matrix components. Stem cell factor (SCF) and its receptor c-KIT are expressed in a wide variety of cells and have also been demonstrated to be important modulators of the wound healing process. To examine the role of the SCF/c-KIT system in keloid pathogenesis. Immunohistochemical staining and Western blot analyses were used to examine localization and expression of SCF and c-KIT in keloid and normal skin tissue. This was followed by the detection of SCF and c-KIT expression in fibroblasts cultured in vitro and fibroblasts exposed to serum. To investigate the effect of epithelial-mesenchymal interactions, a two-chamber system was employed in which keratinocytes on membrane inserts were cocultured with the fibroblasts. SCF and c-KIT expression levels in all cell extracts and conditioned media were assayed by Western blotting. In another set of experiments, the effect of imatinib (Glivec(®), Gleevec(®); Novartis Pharma AG, Basel, Switzerland) on keloid fibroblasts was examined. SCF and c-KIT were upregulated in keloid scar tissue and in cultured fibroblasts stimulated with serum, highlighting their importance in the initial phase of wound healing. We further demonstrated that epithelial-mesenchymal interactions, mimicked by coculture of keratinocytes and fibroblasts in vitro, not only stimulated secretion of the soluble form of SCF in keloid cocultures but also brought about shedding of the extracellular domain of c-KIT perhaps by upregulation of tumour necrosis factor-α converting enzyme which was also upregulated in keloid scars in vivo and keloid cocultures in vitro. In addition keloid cocultures expressed increased levels of phosphorylated c-KIT highlighting an activation of the SCF/c-KIT system. Finally, we demonstrated that imatinib, a tyrosine kinase inhibitor, may be a possible therapeutic agent for keloids. These data indicate that the SCF/c-KIT system plays an important role in scar pathogenesis, and underscore the role of imatinib as a key therapeutic agent in keloid scars.

Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium

BackgroundThe mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.ResultsThe initial pattern of corneal epithelial patches in XLacZ+/- X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline) were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of XLacZ+/- eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks.ConclusionStriping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair.

Otago Glaucoma Surgery Outcome Study: Tissue Matrix Breakdown by Apoptotic Cells in Capsules Surrounding Molteno Implants

To identify cell types and extracellular matrix components in Molteno implant capsules. Histologic features including cytology, distribution of apoptotic cells, cytoantigens, collagens, basement membranes, elastic fibers, and glycoproteins were examined by light microscopy. Findings were correlated with the clinical features of 19 ocular specimens with glaucoma that had been treated with Molteno implants 11 days to 20 years previously. All but the earliest specimen capsules showed two layers: a moderately cellular outer layer of normally stained collagen and an inner avascular hypocellular layer of altered collagen. Capsules contained metabolically active fibroblasts and macrophages showing swelling, vacuolation, and apoptosis with localized loss of extracellular matrix in the inner layers of older capsules. Type I collagen was present in trace amounts. Collagens types III and VI and fibronectin were present in high concentrations in the capsules. Basement membrane material (collagen type IV and laminin) and thrombospondin were concentrated in the inner avascular layers. These results support previous findings that the normal capsule life cycle includes continual inner surface degeneration and external surface renewal. The cells and tissue matrix components of the outer capsule layer matched those involved in the initial phase of wound healing in vascular connective tissue. The tissue matrix components and widespread apoptosis found in the inner fibrodegenerative layer reflect scar tissue remodelling induced by exposure to the aqueous.

The human tri-peptide GHK and tissue remodeling

Tissue remodeling follows the initial phase of wound healing and stops inflammatory and scar-forming processes, then restores the normal tissue morphology. The human peptide Gly-(L-His)-(L-Lys) or GHK, has a copper 2+ (Cu2+) affinity similar to the copper transport site on albumin and forms GHK-Cu, a complex with Cu2+. These two molecules activate a plethora of remodeling related processes: (1) chemoattraction of repair cells such as macrophages, mast cells, capillary cells; (2) anti-inflammatory actions (suppression of free radicals, thromboxane formation, release of oxidizing iron, transforming growth factor beta-1, tumor necrosis factor alpha and protein glycation while increasing superoxide dismutase, vessel vasodilation, blocking ultraviolet damage to skin keratinocytes and improving fibroblast recovery after X-ray treatments); (3) increases protein synthesis of collagen, elastin, metalloproteinases, anti-proteases, vascular endothelial growth factor, fibroblast growth factor 2, nerve growth factor, neutrotropins 3 and 4, and erythropoietin; (4) increases the proliferation of fibroblasts and keratinocytes; nerve outgrowth, angiogenesis, and hair follicle size. GHK-Cu stimulates wound healing in numerous models and in humans. Controlled studies on aged skin demonstrated that it tightens skin, improves elasticity and firmness, reduces fine lines, wrinkles, photodamage and hyperpigmentation. GHK-Cu also improves hair transplant success, protects hepatic tissue from tetrachloromethane poisoning, blocks stomach ulcer development, and heals intestinal ulcers and bone tissue. These results are beginning to define the complex biochemical processes that regulate tissue remodeling.

Effects of Local and Whole Body Irradiation on the Appearance of Osteoblasts During Wound Healing in Tooth Extraction Sockets in Rats

Irradiation before tooth extraction delays wound healing in the alveolar socket. This study examined the influences of local and whole body irradiation before tooth extraction on appearance of osteoblasts in the alveolar bone of rat maxillary first molars because bone formation is observed at the initial phase of wound healing. Several osteoblasts were generated 3 days after tooth extraction, and the number of cells increased day by day. Morphological studies showed there were little differences between local irradiation and non-irradiated controls. In contrast, the extraction wound in the whole body irradiation group showed delayed healing, and there was poor granulation tissue and very few osteoblasts at the bottom of the socket. An ultrastructural study showed that the osteoblasts in the extraction socket of whole body irradiation rats were smaller, and had poorly developed organelles. Injection of bone marrow cells to whole body-irradiated animals immediately after tooth extraction partially restored the number of osteoblasts. New periosteal bone formations outside of sockets showed little delay in the whole body irradiation group. These findings suggest that bone formation in the wound healing of extraction socket requires bone marrow cells from hematopoietic organs such as the bone marrow as well as local sources around the alveolar socket, during the initial phase of wound healing.

Effects of Local and Whole Body Irradiation on Appearance of Osteoclasts During Wound Healing of Tooth Extraction Sockets in Rats

We examined effects of local and whole body irradiation before tooth extraction on appearance and differentiation of osteoclasts in the alveolar bone of rat maxillary first molars. Wistar rats weighting 100 g were divided into three groups: non-irradiation group, local irradiation group, and whole body irradiation group. In the local irradiation group, a field made with lead blocks was placed over the maxillary left first molar tooth. In the whole body irradiation group, the animals were irradiated in cages. Both groups were irradiated at 8 Gy. The number of osteoclasts around the interradicular alveolar bone showed chronological changes common to non-irradiated and irradiated animals. Several osteoclasts appeared one day after tooth extraction, and the maximal peak was observed 3 days after extraction. Local irradiation had no difference from non-irradiated controls. In animals receiving whole body irradiation, tooth extraction one day after irradiation caused smaller number of osteoclasts than that 7 day after irradiation during the experimental period. Whole body-irradiated rats had small osteoclasts with only a few nuclei and narrow resorption lacunae, indicating deficiency of radioresistant osteoclast precursor cells. Injection of intact bone marrow cells to whole body-irradiated animals immediately after tooth extraction recovered to some content the number of osteoclasts. These findings suggest that bone resorption in the wound healing of alveolar socket requires radioresistant, postmitotic osteoclast precursor cells from hematopoietic organs, but not from local sources around the alveolar socket, at the initial phase of wound healing.

Expression of prothrombin, thrombin and its receptors in human scars

The coagulation system is thought to play a pivotal role during the initial phase of wound healing, but mechanisms and cells involved are only partly understood. We have therefore examined human scars for the expression of thrombin, its precursor prothrombin and the thrombin receptors, thrombomodulin (TM) and protease-activated receptor-1 (PAR-1), compared with normal skin. Biopsies of scars were obtained from primary excision sites of melanoma patients (n = 20) and were compared with normal skin distant from the scar (n = 10), using immunohistochemistry. In addition, polymerase chain reaction analyses were performed on scar versus normal tissue and on cultured keratinocytes, fibroblasts and endothelial cells before and after stimulation with selected cytokines known to be active in wound healing. Normal epidermis was stained for prothrombin, thrombin, TM and PAR-1, and dermal tissue was stained only for TM and PAR-1. In scar tissue, thrombin and TM were upregulated in the epidermis and all four molecules in the dermis, independent of the age of the scars. In tissue extracts, mRNA expression of PAR-1 and prothrombin expression were, however, unchanged and TM even slightly decreased in scars, compared with normal skin. On analysis of cultured cells, keratinocytes expressed mRNA for PAR-1, TM and prothrombin, endothelial cells for PAR-1 and TM, and fibroblasts for PAR-1. An upregulation of PAR-1 mRNA was induced in fibroblasts on exposure to tumor necrosis factor-alpha (TNF-alpha), while it remained unchanged in endothelial cells in response to TNF-alpha. A downregulation of TM was induced in endothelial cells on exposure to TNF-alpha. These findings, showing a marked modulation of thrombin, PAR-1 and TM even in older human scar tissue, suggest that the coagulation system is not only involved during clotting, but also during the inflammatory and tissue remodelling phases of wound healing.

Identification of novel AP-1 target genes in fibroblasts regulated during cutaneous wound healing.

Mesenchymal-epithelial interactions are increasingly considered to be of vital importance for epithelial homeostasis and regeneration. In skin, the transcription factor AP-1 was shown to be critically involved in the communication between keratinocytes and dermal fibroblasts. After skin injury, the release of IL-1 from keratinocytes induces the activity of the AP-1 subunits c-Jun and JunB in fibroblasts leading to a global change in gene expression. To identify AP-1 target genes in fibroblasts, which are involved in the process of cutaneous repair, we performed gene expression profiling of wild-type, c-jun- and junB-deficient fibroblasts in response to IL-1, mimicking the initial phase of wound healing. Using a 15K cDNA collection, over 1000 genes were found to be Jun-dependent and additional 300 clones showed IL-1 responsiveness. Combinatorial evaluation allowed for the dissection of the specific contribution of either AP-1 subunit to gene regulation. Besides previously identified genes that are involved in cutaneous repair, we have identified novel genes regulated during wound healing in vivo and showed their expression by fibroblasts on wound sections. The identification of novel Jun target genes should provide a basis for understanding the molecular mechanisms underlying mesenchymal-epithelial interactions and the critical contribution of AP-1 to tissue homeostasis and repair.

Growth factors regulate β-catenin-mediated TCF-dependent transcriptional activation in fibroblasts during the proliferative phase of wound healing

β-catenin is a critical regulator of cell behavior during embryogenesis and neoplastic processes. It also plays a crucial role in repair by modulating dermal fibroblast activity during the proliferative phase of cutaneous wound healing. We hypothesize that growth factors liberated during the initial phase of wound healing convey signals to induce activation of β-catenin-mediated TCF-dependent signaling during the proliferative phase. Dermal fibroblasts were isolated and cultured from mice containing a β-galactosidase reporter responsive to β-catenin–TCF transactivation (TCF-β-gal). Cells were stimulated with growth factors present at the initial phase of wound healing. EGF and TGF-β1 significantly increased β-catenin protein levels and transcriptional activity, whereas β-catenin mRNA expression was unaffected. This increase was attributed to inactivation of GSK-3β, a kinase important for β-catenin destabilization. Subcutaneous injection of EGF or TGF-β1 before wounding of TCF-β-gal mice resulted in larger scars and fibroblasts within these wounds that strongly stained for β-galactosidase, indicating significant β-catenin transcriptional activity in vivo. Thus, β-catenin-mediated signaling is activated downstream of growth factors released during the initial phase of wound repair, and may act during the proliferative phase of wound healing to integrate signals from initial phase factors into the expression of genes important during the later, remodeling phase.

This month in Gastroenterology

This month in Gastroenterology

Induction of vascular endothelial growth factor by fibrin as a dermal substrate for cultured skin substitute.

In the initial phase of wound healing, endogenous fibrin clots are known to form a provisional matrix and to promote angiogenesis. Growth factors such as vascular endothelial growth factor (VEGF) increase in wounds to stimulate angiogenesis. However, it remains unknown whether VEGF is induced when fibrin is used as a dermal substrate for cultured skin substitutes. The authors investigated the effect of fibrin gel as a dermal substrate for a cultured skin substitute, using human keratinocytes and dermal fibroblasts. A collagen-cultured skin substitute was also examined for comparison. VEGF in the culture supernatant in both types was measured by enzyme-linked immunosorbent assay, and VEGF mRNA was determined semiquantitatively by reverse-transcriptase polymerase chain reaction after 2 days of incubation. Experiments were performed using 12 cultured skin substitutes: four for histologic examination before transplantation, four for VEGF assay in vitro, and four for the transplantation to athymic mice. Three independent experiments were performed for each step. VEGF concentration in the fibrin-cultured supernatant was 84.3 +/- 11.8 pg/ml, whereas it was 27.8 +/- 4.68 pg/ml in the case of the collagen substrate. The relative levels of VEGF mRNA were 1.088 +/- 0.100 and 0.698 +/- 0.226, respectively. In in vivo transplantation, the fibrin-type cultured skin substitute showed an excellent take on the wound bed, and a normally proliferating keratinocyte layer with emergence of vascular endothelial cells in the transplanted floor was seen 3 days after transplantation. Vascular endothelial cells, which were identified using alkaline phosphatase stain, were significantly increased in the fibrin-type cultured skin substitute. The use of fibrin as a dermal substrate for cultured skin substitute increases the secretion of VEGF, improves regeneration of mature epidermal structure after in vivo transplantation, and promotes the migration of vascular endothelial cells.