Enzymes in the ECM, notably the matrix metalloproteinases (MMPs), degrade the ECM to permit remodeling and extension of the vascular tube.
Newly formed vessels are leaky because of incomplete interendothelial junctions and because VEGF, the growth factor that drives angiogenesis, increases vascular permeability. This leakiness accounts in part for the edema that may persist in healing wounds long after the acute inflammatory response has resolved.
Activation of Fibroblasts and Deposition of Connective Tissue
The laying down of connective tissue occurs in two steps: (1) migration and proliferation of fibroblasts into the site of injury and (2) deposition of ECM proteins produced by these cells. These processes are orchestrated by locally produced cytokines and growth factors, including PDGF, FGF-2, and TGF-β. The major sources of these factors are inflammatory cells, particularly alternatively activated (M2) macrophages that infiltrate sites of injury.
In response to cytokines and growth factors, fibroblasts enter the wound from the edges and migrate toward the center. Some of these cells may differentiate into cells called myofibroblasts, which contain smooth muscle actin and have increased contractile activity, and serve to close the wound by pulling its margins toward the center. Activated fibroblasts and myofibroblasts also increase their synthetic activity and produce connective tissue proteins, mainly collagen, which is the major component of the fully developed scar.
TGF-β is the most important cytokine for the synthesis and deposition of connective tissue proteins. It is produced by most of the cells in granulation tissue, including alternatively activated macrophages. The levels of TGF-β in tissues are primarily regulated not by the transcription of the gene but by the post-transcriptional activation of latent TGF-β, the rate of secretion of the active molecule, and factors in the ECM, notably integrins, that enhance or diminish TGF-β activity. In addition, microfibrils made up of fibrillin also regulate the bioavailability of TGF-β (Chapter 6). TGF-β stimulates fibroblast migration and proliferation, increases the synthesis of collagen and fibronectin, and decreases the degradation of ECM by inhibiting metalloproteinases. TGF-β is involved not only in scar formation after injury but also in the development of fibrosis in lung, liver, and kidneys that follows chronic inflammation. TGF-β also has anti-inflammatory effects that serve to limit and terminate inflammatory responses. It does this by inhibiting lymphocyte proliferation and the activity of other leukocytes.
As healing progresses, the number of proliferating fibroblasts and new vessels decreases, but the fibroblasts progressively assume a more synthetic phenotype, and hence there is increased deposition of ECM. Collagen synthesis, in particular, is necessary for the healing wound to become strong and mechanically stable. Collagen synthesis by fibroblasts begins early in wound healing (days 3–5) and continues for several weeks, depending on the size of the wound. Net collagen accumulation depends not only on increased synthesis but also on diminished collagen degradation (discussed later). As the scar matures, there is progressive vascular regression, which eventually transforms the highly vascularized granulation tissue into a pale, largely avascular scar.
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Remodeling of Connective Tissue
After the scar is formed, it is remodeled to increase its strength and contract it. Wound strength increases because of cross-linking of collagen and increased size of collagen fibers. In addition, there is a shift of the type of collagen deposited, from type III collagen early in repair to more resilient type I collagen. In well-sutured skin wounds, strength may recover to 70% to 80% of normal skin by 3 months. Wound contraction is initially caused by myofibroblasts and later by cross-linking of collagen fibers.