Fibronectin and wound healing

I have tried to briefly review the evidence (summarized in Table II) indicating that fibronectin is important in cutaneous wound healing. Fibronectin appears to be an important factor throughout this process. It promotes the spreading of platelets at the site of injury, the adhesion and migration of neutrophils, monocytes, fibroblasts, and endothelial cells into the wound region, and the migration of epidermal cells through the granulation tissue. At the level of matrix synthesis, fibronectin appears to be involved both in the organization of the granulation tissue and basement membrane. In terms of tissue remodeling, fibronectin functions as a nonimmune opsonin for phagocytosis of debris by fibroblasts, keratinocytes, and under some circumstances, macrophages. Fibronectin also enhances the phagocytosis of immune-opsonized particles by monocytes, but whether this includes phagocytosis of bacteria remains to be determined. In general, phagocytosis of bacteria has not appeared to involve fibronectin. On the contrary, the presence of fibronectin in the wound bed may promote bacterial attachment and infection. Because of the ease of experimental manipulations, wound healing experiments have been carried out on skin more frequently than other tissues. As a result, the possible role of fibronectin has not been investigated thoroughly in the repair of internal organs and tissues. Nevertheless, it seems reasonable to speculate that fibronectin plays a central role in all wound healing situations. Finally, the wound healing problems of patients with severe factor XIII deficiencies may occur because of their inability to incorporate fibronectin into blood clots.     

Modelling the interaction of keratinocytes and fibroblasts during normal and abnormal wound healing processes

The crosstalk between fibroblasts and keratinocytes is a vital component of the wound healing process, and involves the activity of a number of growth factors and cytokines. In this work, we develop a mathematical model of this crosstalk in order to elucidate the effects of these interactions on the regeneration of collagen in a wound that heals by second intention. We consider the role of four components that strongly affect this process: transforming growth factor-β, platelet-derived growth factor, interleukin-1 and keratinocyte growth factor. The impact of this network of interactions on the degradation of an initial fibrin clot, as well as its subsequent replacement by a matrix that is mainly composed of collagen, is described through an eight-component system of nonlinear partial differential equations. Numerical results, obtained in a two-dimensional domain, highlight key aspects of this multifarious process, such as re-epithelialization. The model is shown to reproduce many of the important features of normal wound healing. In addition, we use the model to simulate the treatment of two pathological cases: chronic hypoxia, which can lead to chronic wounds; and prolonged inflammation, which has been shown to lead to hypertrophic scarring. We find that our model predictions are qualitatively in agreement with previously reported observations and provide an alternative pathway for gaining insight into this complex biological process.     

Cyclophilin C-associated protein is up-regulated during wound healing

Cyclophilin C-associated protein (CyCAP) is identified from macrophages. It locates in intracellular, membrane bound and extracellular, suggesting it has an important role, however both of its regulation and function have not been elucidated. The expression of CyCAP in skin and during wound healing is also unknown. We demonstrate that CyCAP is expressed in both dermal fibroblasts and keratinocytes. In the dermis, the majority of CyCAP protein is located intracellular in a filamentous protein form while a lesser amount is in the extracellular matrix (ECM). CyCAP gene and protein expression is increased 1 day after skin wound healing in both fetal and adult rats and remains elevated level up to 1 week in adult rats. Immunohistochemistry studies demonstrate that the increased CyCAP expression locates mainly to inflammatory cells, including macrophages, monocytes and lymphocytes during wound healing. Interferon-gamma increases CyCAP gene and protein expression in cultured rat fibroblasts. We also found that wound healing is slower and less collagen is expressed in skin of CyCAP null mice. These data are the first observations of CyCAP expression in skin and during wound repair. Our data indicates that CyCAP is regulated by IFNgamma and may function on immune defense in macrophages, lymphocytes, dermal fibroblasts and keratinocytes during wound healing.     

Fibronectin involvement in granulation tissue and wound healing in rabbits

This study describes the distribution of fibronectin and its association with reticulin fibers (type III collagen) and hyaluronic acid in shallow rabbit wounds. Linear incisions were made dorsally with a surgical blade. Animals were sacrificed and 1,2,3,4,5, and 8 day wounds were examined using peroxidase-antiperoxidase to localize affinity-purified antibodies to fibronectin. Tissue samples were also stained with hematoxylin and eosin in addition to silver stains for reticulin, and Alcian blue for hyaluronic acid. After wounding, the incision filled with a fibrin clot that stained positively for fibronectin. The underlying dermis and adjacent, unwounded dermis also contained fibronectin. Epidermal cells that migrate from the wound margin between the clot and the dermis were in direct association with fibronectin in these wound components. By 72 hr, epidermal continuity was reestablished. Early granulation tissue formation was apparent just below the epidermis 5 day wounds. Fibronectin was observed in the matrix surrounding individual fibroblasts and codistributed with reticulin fibers and hyaluronic acid in both 5 and 8 day wounds. Granulation tissue of 8 day wounds stained intensely for fibronectin and extended to a greater depth in the reticular dermis. Dense fibrillar networks of fibronectin and fibroblasts were aligned parallel to the epidermis, giving the granulation tissue a highly structured and organized appearance. Fibroblasts contained fibronectin and were surrounded by less fibronectin at the wound periphery than within the granulation tissue. These findings suggest that fibronectin may be important in the reconstruction of tissues during repair by functioning as an extracellular scaffold for migrating cells.     

A secreted MMP is required for reepithelialization during wound healing

Matrix metalloproteinases (MMPs) are extracellular proteases highly expressed at wound sites. However, the precise function of MMPs during reepithelialization in vivo has been elusive in mammalian models because of the high level of redundancy among the 24 mammalian MMPs. For this reason we used Drosophila melanogaster, whose genome encodes only two MMPs-one secreted type (Mmp1) and one membrane-anchored type (Mmp2)-to study the function and regulation of the secreted class of MMPs in vivo. In the absence of redundancy, we found that the Drosophila secreted MMP, Mmp1, is required in the epidermis to facilitate reepithelialization by remodeling the basement membrane, promoting cell elongation and actin cytoskeletal reorganization, and activating extracellular signal-regulated kinase signaling. In addition, we report that the jun N-terminal kinase (JNK) pathway upregulates Mmp1 expression after wounding, but that Mmp1 is expressed independent of the JNK pathway in unwounded epidermis. When the JNK pathway is ectopically activated to overexpress Mmp1, the rate of healing is accelerated in an Mmp1-dependent manner. A primary function of Mmp1, under the control of the JNK pathway, is to promote basement membrane repair, which in turn may permit cell migration and the restoration of a continuous tissue.     

Basement membrane formation during wound healing is dependent on epidermal transplants

The purpose of the study was to compare directly the effect of healing and the formation of the basement membrane during wound healing from two autologous primary keratinocyte cultures in the liquid environment in full-thickness wounds in pigs. Wounds were either transplanted with cultured epidermal autografts (n = 26) or autologous keratinocyte suspensions (n = 24) or treated with saline alone (n = 40) and covered with a chamber. All wounds transplanted with cultured epidermal autografts and keratinocyte cell suspensions had positive "take" after transplantation. Healing times were significantly shorter for wounds treated with either cultured epidermal autografts or keratinocyte suspensions (p = 0.0001) compared with saline-treated wounds but were not different from each other (p = 0.1835). There were no differences in cytokeratin and laminin expression; however, staining with monoclonal antibody against collagen type VII showed a lower signal for cultured epidermal autografts only on days 8 and 16 compared with keratinocyte suspensions. Electron microscope evaluation showed a higher incidence of anchoring fibrils and a more mature dermal-epidermal junction in wounds treated with keratinocyte cell suspensions at day 8. These findings may be due to the single, noncontact-inhibited cells and the early formation of an in vivo neodermis to the wet wound environment. These data suggest that wounds transplanted with autologous keratinocyte suspensions in a wet environment may be an alternative method in the treatment of wounds.     

Fibronectin (FN) localizations in early wound healing of cultured frog skins

The wound healing process of frog skin fragments in epibolic cultures has provided information on FN localizations during the migration of keratinocytes. Mainly two FN localizations were studied by indirect immunodetections: Epidermal localization around keratinocytes which have acquired a fibroblastic shape. Dermal localizations of the sectioned collagen of the stratum spongiosum and stratum compactum detected at the beginning of the culture. Both localizations were observed in this epibolic wound healing process during 6 hr and 24 hr in culture and showed a differential sensitivity to cycloheximide (CHX). It was worth noting that fibronectin was permanently detected in the subcutaneous tissue of non-cultured or cultured skin fragments with or without CHX.     

Nonmuscle myosin II localization is regulated by JNK during Drosophila larval wound healing

We investigated cell shape changes during wound closure in the Drosophila larval epidermis. During reepithelialization, epidermal cells permanently change shape from pentagonal or hexagonal to irregular forms. This process requires zipper, a gene encoding the Drosophila nonmuscle myosin II heavy chain. Following wounding, myosin II is localized at the wound margin and at the rear end of individual cells located within several rows from the wound hole. The c-Jun N-terminal kinase (JNK) pathway is essential for this myosin II localization. These results suggest that not only the wound leading edge but also the cells lying distal to the leading edge cells actively participate in epithelial cell sheet migration during wound hole closure.     

Chemokine changes during oral wound healing

The oral mucosa is susceptible to tissue injury from many causes, including infection, autoimmune disorders, surgical and accidental trauma, and gingival and periodontal inflammation; however, little is known about the events that influence wound healing in the mouth. Recent studies in non-oral tissues have implicated immune system-derived factors, in particular chemokines, in the wound healing process. Tissues from mice with experimental gingival wounds were studied for expression of genes for four chemokine ligands or receptors (CCL19, CCL20, CCL25, and CCR5) that are important in leukocyte trafficking or inflammation. Notably, during the peak phase of wound healing, chemokine gene expression was up-regulated for CCL19, CCL20, and CCL25, and down-regulation of CCR5, suggesting an orchestrated process of chemokine-mediated recruitment or retention of lymphocytes and macrophages into wound areas, while simultaneously suppressing a potentially adverse inflammatory response. These findings have implications for developing therapeutic strategies aimed at promoting more effective tissue healing at oral surfaces.     

Fibronectin from aged fibroblasts is defective in promoting cellular adhesion

Late passage fibroblasts show decreased cell-substrate adhesion. We provide evidence that the reduced adhesion is due to a defect in the adhesive glycoprotein fibronectin. Late passage cells become more adhesive in culture media that has been conditioned by the growth of early passage cells. Analysis of fibronectins purified from early and late passage cell conditioned media indicates that there are striking differences in their abilities to promote cell adhesion. Young cell fibronectin supports the maximal adhesion of both young and old cells. However, old cells require quantitatively more fibronectin. In contrast, old cell fibronectin is less effective in supporting the adhesion of either cell type. In addition, neither cell type achieves a normal morphology in the presence of old cell fibronectin. The results support the conclusion that the fibronectin released by late passage cells is defective and does not support normal cell-substrate interactions.     

Factors influencing myofibroblast differentiation during wound healing and fibrosis.

Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilaments bundles and the expression of α-SM actin, the actin isoform typical of contractile vascular SM cells. Myofibroblasts have been suggested to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. When granulation tissue evolves into a scar, myofibroblasts containing α-SM actin disappear, probably as a result of apoptosis. In contrast, myofibroblasts expressing α-SM actin persist in excessive scarring and in fibrotic conditions. The mechanisms leading to the development of myofibroblastic features remain to be investigated. Studies on the factors regulating the phenotype of myofibroblasts will be necessary for understanding their behavior in vivo, and possibly modifying this behavior during the different clinical settings.     

Cytophotometric evaluation of alkaline phosphatase activity in neutrophilic granulocytes and fibroblasts during wound healing in rats

The most informative indexes of alkaline phosphatase activity in neutrophil granulocytes from the peripheral blood and in neutrophil granulocytes and fibroblasts from the wound were defined in the experimental study carried out on 60 rats. These indexes were used to reveal the relationship between blood system reactions and inflammatory and regeneration processes in the wound tissue. The division of neutrophil granulocytes into three functional groups in accordance with alkaline phosphatase activity was demonstrated to be reasonable. The progress of inflammatory and regeneration processes in the wound tissues was shown to be adequately reflected in changes of both general and relative number of blood circulating neutrophil granulocytes of the third functional group characterized by high alkaline phosphatase activity. The results of the study demonstrate that the cytophotometric method is highly informative. It can be used in the clinical practice for an objective evaluation of the wound healing process as well as for an estimation of the treatment efficacy.     

The multifunctional role of fibroblasts during wound healing in Hirudo medicinalis (Annelida, Hirudinea)

Extracellular matrix components play a key role during the angiogenic process for a correct development of blood vessels: fibroblasts are the main cell type involved in the regulation of ECM protein production. In this study we characterize H. medicinalis fibroblasts and demonstrate that they take part to the regulation of angiogenesis that occurs during wound healing process. Massive proliferation and phenotypic modification are two distinctive markers of fibroblast activation. These cells, that are usually responsible for collagen production and function as an energy reservoir, are recruited during wound healing to form a collagen scaffold through a direct mechanic action and through secretion of specific proteoglycans. In addition we show that the activity of fibroblasts is modulated by EGF, a growth factor involved in wound healing in vertebrates. The formation of bundles of collagen fibrils by fibroblasts is fundamental for the development and migration of new blood vessels in lesioned areas during wound repair: administration of lovastatin in explanted leeches affects fibroblasts, damages collagen "scaffold" and indirectly causes the reduction of neo-capillary formation.     

Arg-Gly-Asp-containing peptides expose novel collagen receptors on fibroblasts: implications for wound healing.

Integrins are a family of cell-surface receptors intimately involved in the interactions of cells with their extracellular matrix. These receptors comprise an alpha and beta subunit in noncovalent association and many have been shown to recognize and bind an arginine-glycine-aspartate (RGD) sequence contained within their specific extracellular matrix ligand. Fibroblasts express integrin receptors belonging to two major subfamilies. Some of the members within the subfamily defined by beta 1 (VLA) are receptors for collagen but, perhaps surprisingly, the other major subfamily of integrins on fibroblasts--that defined by the alpha chain of the vitronectin receptor, alpha v--all appear to bind primarily vitronectin and/or fibronectin. In the present study we show that RGD-containing peptides expose cryptic binding sites on the alpha v-associated integrins enabling them to function as collagen receptors. The addition of RGD-containing peptides to fibroblasts cultured on type I collagen induced dramatic cell elongation and, when the cells were contained within collagen matrices, the peptides induced marked contraction of the gels. These processes were inhibited by Fab fragments of a monoclonal antibody against an alpha v integrin. Also, alpha v-associated integrins from cell lysates bound to collagen I affinity columns in the presence, but not in the absence, of RGD-containing peptides. These data suggest a novel regulatory control for integrin function. In addition, because the cryptic collagen receptors were shown to be implicated in the contraction of collagen gels, the generation of such binding forces suggests that this may be the major biological role for these integrins in processes such as wound healing.     

Tissue transglutaminase in normal and abnormal wound healing: Review article

Summary. A complex series of events involving inflammation, cell migration and proliferation, ECM stabilisation and remodelling, neovascularisation and apoptosis are crucial to the tissue response to injury. Wound healing involves the dynamic interactions of multiple cells types with components of the extracellular matrix (ECM) and growth factors. Impaired wound healing as a consequence of aging, injury or disease may lead to serious disabilities and poor quality of life. Abnormal wound healing may also lead to inflammatory and fibrotic conditions (such as renal and pulmonary fibrosis). Therefore identification of the molecular events underlying wound repair is essential to develop new effective treatments in support to patients and the wound care sector.Recent advances in the understating of the physiological functions of tissue transglutaminase a multi functional protein cross-linking enzyme which stabilises tissues have demonstrated that its biological activities interrelate with wound healing phases at multiple levels. This review describes our view of the function of tissue transglutaminase in wound repair under normal and pathological situations and highlights its potential as a strategic therapeutic target in the development of new treatments to improve wound healing and prevent scarring.     

The role of allogenic fibroblasts in an acute wound healing model

Skin is the first tissue-engineered organ to have been successfully developed in the laboratory, and it has been clinically available for use as epidermal sheets for some time. As refinements in this field of tissue engineering continue, several key issues give cause for concern. One issue is the need to form a more complete dermal analogue before grafting. To this end, fibroblasts may be used in vitro to deposit extracellular matrix components within a basic scaffold, laying down those molecules not endogenous to the material and thereby improving the quality of the skin replacement. Many studies have shown the benefits of in vitro seeding with fibroblasts, but there has been some debate regarding the longevity of such cells after allotransplantation into an immunocompetent host. In this study, the authors set out to determine the longevity of transplanted cells in an immunocompetent porcine model. A total of 24 wounds were made on four female animals, 12 of which were covered with acellular hyaluronic acid dermal matrices, and the remainder of which were covered with matrices seeded with allogenic (male) fibroblasts. After a week in vivo, the wounds were grafted with either split-thickness skin grafts or cultured epithelial autograft. Biopsy specimens were obtained from wounds at varying time intervals and assessed using genetic analysis to determine the survival of allotransplanted cells. No cells were detectable by polymerase chain reaction analysis (sensitivity, 1:100,000) after 7 days in vivo. Subsequent histologic examination demonstrated little difference in wound morphology. The authors conclude that allogenic fibroblasts do not survive transplantation in a porcine wound model.