the wound area. tissue swelling aids later angiogenesis by expanding and loosening the existing collagenous extracellular matrix. endothelial activation: as the wound macrophages switches from inflammatory to healing mode, it begins to secrete endothelial chemotactic and growth factors to attract adjacent endothelial cells. activated endothelial cells respond by retracting and reducing cell junctions, loosening themselves from their embedded endothelium. characteristically the activated endothelial cells show enlarged nucleoli. degradation of endothelial basement membrane: the wound macrophages, mast cells and the endothelial cells themselves secrete proteases to break down existing vascular basal lamina. vascular sprouting: with the breakdown of endothelial basement membrane, detached endothelial cells from pre-existing capillaries and post-capillary venules can divide and migrate chemotactically towards the wound, laying down new vessels in the process. vascular sprouting can be aided by ambient hypoxia and acidosis in the wound environment, as hypoxia stimulates the endothelial transcription factor, hypoxia inducible factor (hif) to transactivate angiogenic genes such as vegf and glut1. sprouted vessels can self-organise into luminal morphologies, and fusion of blind channels give rise to new capillary networks. vascular maturation: the endothelium of vessels mature by laying down new endothelial extracellular matrix, followed by basal lamina formation. lastly the vessel establishes a pericyte layer. stem cells of endothelial cells, originating from parts of uninjured blood vessels, develop pseudopodia and push through the ecm into the wound site to establish new blood vessels. endothelial cells are attracted to the wound area by fibronectin found on the fibrin scab and chemotactically by angiogenic factors released by other cells, e.g. from macrophages and platelets when in a low-oxygen environment. endothelial growth and proliferation is also directly stimulated by hypoxia, and presence of lactic acid in the wound. for example, hypoxia stimulates the endothelial transcription factor, hypoxia-inducible factor (hif) to transactivate a set of proliferative genes including vascular endothelial growth factor (vegf) and glucose transporter 1 (glut1). to migrate, endothelial cells need collagenases and plasminogen activator to degrade the clot and part of the ecm. zinc-dependent metalloproteinases digest basement membrane and ecm to allow cell migration, proliferation and angiogenesis. when macrophages and other growth factor-producing cells are no longer in a hypoxic, lactic acid-filled environment, they stop producing angiogenic factors. thus, when tissue is adequately perfused, migration and proliferation of endothelial cells is reduced. eventually blood vessels that are no longer needed die by apoptosis. fibroplasia and granulation tissue formation simultaneously with angiogenesis, fibroblasts begin accumulating in the wound site. fibroblasts begin entering the wound site two to five days after wounding as the inflammatory phase is ending, and their numbers peak at one to two weeks post-wounding. by the end of the first week, fibroblasts are the main cells in the wound. fibroplasia ends two to four weeks after wounding. as a model the mechanism of fibroplasia may be conceptualised as an analogous process to angiogenesis (see above) - only the cell type involved is fibroblasts rather than endothelial cells. initially there is a latent phase where the wound undergoes plasma exudation, inflammatory decontamination and debridement. oedema increases the wound histologic accessibility for later fibroplastic migration. second, as inflammation nears completion, macrophage and mast cells release fibroblast growth and chemotactic factors to activate fibroblasts from adjacent tissue. fibroblasts at this stage loosen themselves from surrounding cells and ecm. phagocytes further release proteases that break down the ecm of neighbouring tissue, freeing the activated fibroblasts to proliferate and migrate towards the wound. the difference between vascular sprouting and fibroblast proliferation is that the former is enhanced by hypoxia, whilst the latter is inhibited by hypoxia. the deposited fibroblastic connective tissue matures by secreting ecm into the extracellular space, forming granulation tissue (see below). lastly collagen is deposited into the ecm. in the first two or three days after injury, fibroblasts mainly migrate and proliferate, while later, they are the main cells that lay down the collagen matrix in the wound site. origins of these fibroblasts are thought to be from the adjacent uninjured cutaneous tissue (although new evidence suggests that some are derived from blood-borne, circulating adult stem cells/precursors). initially fibroblasts utilize the fibrin cross-linking fibers (well-formed by the end of the inflammatory phase) to migrate across the wound, subsequently adhering to fibronectin. fibroblasts then deposit ground substance into the wound bed, and later collagen, which they can adhere to for migration. granulation tissue functions as rudimentary tissue, and begins to appear in the wound already during the inflammatory phase, two to five days post wounding, and continues growing until the wound bed is covered. granulation tissue consists of new blood vessels, fibroblasts, inflammatory cells, endothelial cells, myofibroblasts, and the components of a new, provisional extracellular matrix (ecm). the provisional ecm is different in composition from the ecm in normal tissue and its components originate from fibroblasts. such components include fibronectin, collagen, glycosaminoglycans, elastin, glycoproteins and proteoglycans. its main components are fibronectin and hyaluronan, which create a very hydrated matrix and facilitate cell migration. later this provisional matrix is replaced with an ecm that more closely resembles that found in non-injured tissue. growth factors (pdgf, tgf-β) and fibronectin encourage proliferation, migration to the wound bed, and production of ecm molecules by fibroblasts. fibroblasts also secrete growth factors that attract epithelial cells to the wound site. hypoxia also contributes to fibroblast proliferation and excretion of growth factors, though too little oxygen will inhibit their growth and deposition of ecm components, and can lead to excessive, fibrotic scarring. collagen deposition one of fibroblasts' most important duties is the production of collagen. collagen deposition is important because it increases the strength of the wound; before it is laid down, the only thing holding the wound closed is the fibrin-fibronectin clot, which does not provide much resistance to traumatic injury. also, cells involved in inflammation, angiogenesis, and connective tissue construction attach to, grow and differentiate on the collagen matrix laid down by fibroblasts. type iii collagen and fibronectin generally begin to be produced in appreciable amounts at somewhere between approximately 10 hours and 3 days, depending mainly on wound size. their deposition peaks at one to three weeks. they are the predominating tensile substances until the later phase of maturation, in which they are replaced by the stronger type i collagen. even as fibroblasts are producing new collagen, collagenases and other factors degrade it. shortly after wounding, synthesis exceeds degradation so collagen levels in the wound rise, but later production and degradation become equal so there is no net collagen gain. this homeostasis signals the onset of the later maturation phase. granulation gradually ceases and fibroblasts decrease in number in the wound once their work is done. at the end of the granulation phase, fibroblasts begin to commit apoptosis, converting granulation tissue from an environment rich in cells to one that consists mainly of collagen. epithelialization the formation of granulation tissue into an open wound allows the reepithelialization phase to take place, as epithelial cells migrate across the new tissue to form a barrier between the wound and the environment. basal keratinocytes from the wound edges and dermal appendages such as hair follicles, sweat glands and sebacious (oil) glands are the main cells responsible for the epithelialization phase of wound healing. they advance in a sheet across the wound site and proliferate at its edges, ceasing movement when they meet in the middle. in healing that results in a scar, sweat glands, hair follicles and nerves do not form. with the lack of hair follicles, nerves and sweat glands, the wound, and the resulting healing scar, provide a challenge to the body with regards to temperature control. keratinocytes migrate without first proliferating. migration can begin as early as a few hours after wounding. however, epithelial cells require viable tissue to migrate across, so if the wound is deep it must first be filled with granulation tissue. thus the time of onset of migration is variable and may occur about one day after wounding. cells on the wound margins proliferate on the second and third day post-wounding in order to provide more cells for migration. if the basement membrane is not breached, epithelial cells are replaced within three days by division and upward migration of cells in the stratum basale in the same fashion that occurs in uninjured skin. however, if the basement membrane is ruined at the wound site, reepithelization must occur from the wound margins and from skin appendages such as hair follicles and sweat and oil glands that enter the dermis that are lined with viable keratinocytes. if the wound is very deep, skin appendages may also be ruined and migration can only occur from wound edges. migration of keratinocytes over the wound site is stimulated by lack of contact inhibition and by chemicals such as nitric oxide. before they begin to migrate, cells must dissolve their desmosomes and hemidesmosomes, which normally anchor the cells by intermediate filaments in their cytoskeleton to other cells and to the ecm. transmembrane receptor proteins called integrins, which are made of glycoproteins and normally anchor the cell to the basement membrane by its cytoskeleton, are released from the cell's intermediate filaments and relocate to actin filaments to serve as attachments to the ecm for pseudopodia during migration. thus keratinocytes detach from the basement membrane and are able to enter the wound bed. before they begin migrating, keratinocytes change shape, becoming longer and flatter and extending cellular processes like lamellipodia and wide processes that look like ruffles. actin filaments and pseudopodia form. during migration, integrins on the pseudopod attach to the ecm, and the actin filaments in the projection pull the cell along. the interaction with molecules in the ecm through integrins further promotes the formation of actin filaments, lamellipodia, and filopodia. epithelial cells climb over one another in order to migrate. this growing sheet of epithelial cells is often called the epithelial tongue. the first cells to attach to the basement membrane form the stratum basale. these basal cells continue to migrate across the wound bed, and epithelial cells above them slide along as well. the more quickly this migration occurs, the less of a scar there will be. fibrin, collagen, and fibronectin in the ecm may further signal cells to divide and migrate. like fibroblasts, migrating keratinocytes use the fibronectin cross-linked with fibrin that was deposited in inflammation as an attachment site to crawl across. a scab covering a healing wound as keratinocytes migrate, they move over granulation tissue but stay underneath the scab, thereby separating the scab from the underlying tissue. epithelial cells have the ability to phagocytize debris such as dead tissue and bacterial matter that would otherwise obstruct their path. because they must dissolve any scab that forms, keratinocyte migration is best enhanced by a moist environment, since a dry one leads to formation of a bigger, tougher scab. to make their way along the tissue, keratinocytes must dissolve the clot, debris, and parts of the ecm in order to get through. they secrete plasminogen activator, which activates plasminogen, turning it into plasmin to dissolve the scab. cells can only migrate over living tissue, so they must excrete collagenases and proteases like matrix metalloproteinases (mmps) to dissolve damaged parts of the ecm in their way, particularly at the front of the migrating sheet. keratinocytes also dissolve the basement membrane, using instead the new ecm laid down by fibroblasts to crawl across. as keratinocytes continue migrating, new epithelial cells must be formed at the wound edges to replace them and to provide more cells for the advancing sheet. proliferation behind migrating keratinocytes normally begins a few days after wounding and occurs at a rate that is 17 times higher in this stage of epithelialization than in normal tissues. until the entire wound area is resurfaced, the only epithelial cells to proliferate are at the wound edges. growth factors, stimulated by integrins and mmps, cause cells to proliferate at the wound edges. keratinocytes themselves also produce and secrete factors, including growth factors and basement membrane proteins, which aid both in epithelialization and in other phases of healing. growth factors are also important for the innate immune defense of skin wounds by stimulation of the production of antimicrobial peptides and neutrophil chemotactic cytokines in keratinocytes. keratinocytes continue migrating across the wound bed until cells from either side meet in the middle, at which point contact inhibition causes them to