All wounds undergo the same 3 steps (inflammatory, proliferative, and remodeling phases) progressing toward repair and restoration of function. Acute wounds heal in a predictable fashion, progressing through these 3 phases. Chronic wounds do not proceed past the inflammatory phase.
The inflammatory phase starts immediately after injury or incision and lasts for approximately 6 days. This phase represents the body's attempt to limit blood loss by creating a seal over the wound and is followed by removal of necrotic tissue and debris. During the inflammatory phase there is an increase in vascular permeability. Cells migrate into the tissue stimulated by chemotaxis. These cells release several cytokines and growth factors which activate migrating cells (Fig. 12-2).
The inflammatory phase of wound healing with neutrophil cellular infiltration.
Hemostasis, Inflammation, and Increased Vascular Permeability
Hemostasis is triggered by the exposure of subendothelial collagen to platelets; this is mediated by Von Willebrand factor. Platelet adhesion to the endothelium is facilitated by integrin receptor GPIIb-IIIa. This results in aggregation of platelets, vasoconstriction, and activation of the coagulation cascade. The binding of platelets results in the release of biologically active proteins from the platelets' α granules and dense bodies (eg, platelet-derived growth factor [PDGF], transforming growth factor β [TGF-β], insulin-like growth factor type I [IGF-I], fibronectin, fibrinogen, thrombospondin, Von Willebrand factor, and vasoactive amines). The increased vascular permeability observed is a result of the release of these biologically active proteins by platelets and mast cells. The most important mediators are histamine and serotonin. Prostaglandin and thromboxane A2 are released from the breakdown of cell membranes and assist in platelet aggregation and vasoconstriction.
Both the intrinsic and extrinsic clotting pathways are activated. Thrombin activates platelets and triggers fibrin formation from fibrinogen. The resulting fibrin strands trap erythrocytes and other cells in the blood to form a blood clot (Fig. 12-3). Ultimately, this results in the formation of a scaffold for inflammatory cells, fibroblasts, and endothelial cells, allowing hemostasis and the wound healing process.
Hemostasis at the wound site with deposition of a fibrin clot.
Macrophages and neutrophils are the predominant cell types to migrate into tissue in response to injury. Neutrophils (polymorphonuclear cells or PMNs) appear first, followed by macrophages. Integrin molecules, a family of cell surface receptors, are important for the migration and activation of neutrophils and macrophages.
Monocytes and endothelial cells increase the migration of PMNs into the injured tissue through the release of interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α). The PMNs release lysosomes, elastase, and other proteases, promoting further migration of PMNs and facilitating the breakdown of damaged tissue. PMNs and macrophages are phagocytes, which have the ability to remove damaged tissue and dead cells in preparation for the synthesis of new tissue.
Macrophages are very important to the wound healing process. They appear in the wound when PMNs are dwindling and induce apoptosis of the PMNs. Within 24 to 48 hours of injury, monocytes from the blood migrate into the tissue and become macrophages. Chemotactic agents from the initial response to the wound (complement c5a, thrombin, fibronectin, collagen, TGF-β) recruit monocytes into the wound. The process of transforming monocytes into macrophages is facilitated by specific integrins in the tissue, which control adhesion-mediated gene induction in monocytes. The macrophages release matrix metalloproteinases, collagenases, and other enzymes to break down the damaged tissue in preparation for regeneration. In addition, macrophages release numerous cytokines and growth factors important for the wound healing process (see Table 12-1).
Table 12-1Cytokines and Growth Factors Implicated in Tissue Repair |Favorite Table|Download (.pdf) Table 12-1 Cytokines and Growth Factors Implicated in Tissue Repair
|Factor ||Abbreviation ||Source ||Functions Regulated |
|Platelet-derived growth factor ||PDGF ||Platelets and macrophages ||Fibroblast proliferation, chemotaxis, and collagenase production |
|Transforming growth factor β ||TGF-β ||Platelets, polymorphonuclear neutrophil leukocytes, T lymphocytes, and macrophages ||Fibroblast proliferation, chemotaxis, collagen metabolism, and the action of other growth factors |
|Transforming growth factor α ||TGF-α ||Activated macrophages and many tissues ||Similar to EGF functions |
|Interleukin-1 ||IL-1 ||Macrophages ||Fibroblast proliferation |
|Tumor necrosis factor ||TNF ||Macrophages, mast cells, and T lymphocytes ||Fibroblast proliferation |
|Fibroblast growth factor ||FGF ||Brain, pituitary, macrophages, and many other tissues and cells ||Fibroblast proliferation, stimulates collagen deposition and angiogenesis |
|Epidermal growth factor ||EGF ||Saliva, urine, milk, and plasma ||Stimulates epithelial cell proliferation and granulation tissue formation |
|Insulin-like growth factor ||IGF ||Liver, plasma, and fibroblasts ||Stimulates synthesis of sulfated proteoglycans, collagen, and cell proliferation |
|Human growth factor ||HGF ||Pituitary and thus plasma ||Anabolism |
|Connective tissue growth factor ||CTGF ||Fibroblasts ||Mesenchymal cell to fibroblast differentiation |
|Hypoxia-inducible factor 1-α ||HIF-1α || ||Angiogenesis |
|Vascular endothelial growth factor ||VEGF || ||Angiogenesis |
After about 5 days, T lymphocytes appear in the wound. They stimulate other cells (primarily macrophages) and process the antigens presented by macrophages. These antigens include foreign material from bacteria, viruses, and other pathogens.
The proliferative phase begins after the initial inflammatory response has subsided and is characterized by angiogenesis, fibroplasia, and epithelialization (Fig. 12-4).
Fibroblasts infiltrate the wound site in response to signaling from wound macrophages.
Angiogenesis is an important part of wound healing. Without new vessels to support the energy and oxygen demands of the cells taking part in healing, the wound cannot heal quickly. Angiogenesis is dependent on angiogenic growth factors. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are 2 of the most potent stimulants of angiogenesis. In addition, TGF-β, TGF-α, and hypoxia-inducible factor-1α (HIF-1α) are important for angiogenesis.
Fibroplasia is the process of collagen synthesis performed by fibroblasts for use in the extracellular matrix (ECM). Collagen synthesis starts 3 to 5 days after injury. This is the amount of time needed for undifferentiated mesenchymal cells to migrate into the tissue and differentiate into fibroblasts. It appears that the initial proliferation of fibroblasts comes from tissue-derived mesenchymal cells capable of differentiation. These cells are not as pluripotent as the mesenchymal cells found in bone marrow. The bone marrow-derived mesenchymal cells respond later.
The factors involved in the differentiation of mesenchymal cells to fibroblasts are not well characterized, but most likely involve connective tissue growth factor (CTGF). Interestingly, TGF-β and CTGF differentiate fibroblasts into the myofibroblasts responsible for wound contraction. The presence of a large number of myofibroblasts can lead to hypertrophic scars and keloids.
Epithelialization takes place very soon after an injury to the skin and replaces the clot that initially protects the wound (Fig. 12-4). Epidermal cells migrate from the basal layer of the residual epidermis or the epithelium-lined dermal appendages to form a fine layer over the wound. The migrating epidermal cells are guided by dermal integrins, allowing them to migrate between the fibrinous eschar and the underlying dermal tissue. The migrating epidermal cells use phagocytosis to remove debris as they migrate.
The ECM consists of a framework of (1) glycosaminoglycans (GAG) primarily proteoglycans (eg, hyaluronic acid); and (2) fibrous proteins such as collagen, elastin, fibronectin, and laminin. The ECM provides a lattice network for cells to migrate and differentiate. Early in wound healing a fibrin plug forms the wound matrix; this plug consists of fibrin, fibrinogen, fibronectin, and vitronectin. GAGs and proteoglycans are then synthesized, and finally collagen is synthesized by fibroblasts. GAGs form a hydrated gel that occupies a large volume and provides a matrix optimized for cell migration and collagen deposition (Table 12-2).
Table 12-2Extracellular Matrix Components |Favorite Table|Download (.pdf) Table 12-2 Extracellular Matrix Components
|Component ||Structure ||Function |
|Collagen ||Triple helical glycoprotein molecules rich in proline, hydroxyproline, and glycine ||Strength, support, and structure for all tissues and organs |
|Elastin ||Stretchable hydrophobic protein interacting with glycosylated microfibrils ||Allows tissues and structures to expand and contract |
|Fibronectin ||Specialized adhesive glycoprotein ||Mediates cell matrix adhesion |
|Laminin ||Large, complex, adhesive glycoprotein ||Binds cells to type IV collagen and heparan sulfate |
|Proteoglycans ||Heterogeneous, long glycosaminoglycan chains covalently linked to a core protein ||Moisture stores, shock absorption, sequestration of cytokines |
|Hyaluronic acid ||Very large, specialized, nonsulfated glycosaminoglycan ||Provides a fluid environment for cell movement and differentiation and binds to cytokines |
There are at least 16 types of collagen identified; each differs in its polypeptide composition. Glycine, proline, and a third amino acid, often hydroxyproline, form a tripeptide. Glycine plays an important role in the structure of collagen because of its small size, which makes it critical for the formation of the triple helix (Fig. 12-5). Integrins help direct fibroblasts and thus are important in collagen synthesis. Reconstructing the ECM is a dynamic process with degradation and synthesis happening at the same time.
The triple helical conformation of the type I procollagen molecule.
In adult scars, type III collagen is first produced, followed largely by type I. In fetal and newborn tissue, type III collagen remains the dominant type of collagen. Collagen is synthesized in fibroblasts and secreted into the ECM in a triple helix configuration called procollagen. Procollagen has propeptides attached to the carboxy and amino terminals. Specific proteases cleave the propeptides resulting in the formation of a collagen monomer. These monomers self-assemble into collagen fibrils in the ECM. Vitamin C is important for the hydroxylation of proline and the formation of stable triple helices. Vitamin C deficiency is called scurvy, a condition in which collagen becomes frail. Scurvy affects all collagen containing organs, including skin, teeth, capillaries, and bones.
Osteogenesis imperfecta (OI) is a disorder of collagen type I. In type I OI, 1 out of 2 genes producing procollagen is defective, resulting in only half the quantity of collagen type I pro-α1 chains. Other types of OI involve gene mutations that alter the amino acid sequence and prohibit the formation of mature triple helices. This leads to brittle bones and is important to consider in a child with multiple fractures consistent with child abuse.
Ehlers–Danlos syndrome is another condition that leads to defective collagen production. It can affect collagen type I, III, V, and other parts of collagen synthesis. Ehlers–Danlos results in unusually elastic skin and joints, and can also lead to fragile blood vessels and otosclerosis (Table 12-3).
Table 12-3Molecular Structure of Collagen Types I Through VI |Favorite Table|Download (.pdf) Table 12-3 Molecular Structure of Collagen Types I Through VI
|Type ||Molecular Configurationa ||Distribution |
|I ||[α1(I)]2α2(1) ||All connective tissues except cartilage and basement membranes |
|II ||[α1(II)]3 ||Cartilages and vitreous humor |
|III ||[α1(III)]3 ||Distensible connective tissues (eg, fetal skin, blood vessels, and uterus) |
|IV ||[α1(IV)]2α2 (IV) ||Basement membranes |
|V ||[α1(V)]2α2 (V) ||Essentially all tissues |
|VI ||[α1(VI), α2 (VI), α3 (VI)] ||Essentially all tissues |
GAG are also known as mucopolysaccharides. They are produced by fibroblasts or cells in the fibroblast family (eg, chondroblasts, osteoblasts). The addition of a protein moiety to a GAG is called a proteoglycan (Fig. 12-6). Proteoglycans have negatively charged side chains because of their sulfate or carboxyl groups. Na+ is bound to the side chains and is followed by water. The large amount of water incorporated into the ECM provides tissue turgor and the ability to withstand pressure. Proteoglycans play an important role in controlling migration of cells, nutrients, hormones, and metabolites. Because of their many different side chains and core proteins, they have the ability to alter the ECM environment.
The structure of a representative proteoglycan (KS, keratan sulfate; CS, chondroitin sulfate) demonstrating the “bottle-brush” architecture.
As previously mentioned, the GAG, hyaluronic acid, is predominant in fetal tissue and appears to facilitate a perfect deposition of collagen. Hyaluronic acid is composed of nonsulfated disaccharide units, and does not have an attached protein moiety. It provides an ideal environment for cell migration into the wound. In adult tissue where sulfated GAGs predominate, a denser scar is formed (Table 12-4).
Table 12-4Glycosaminoglycans and Their Tissue Distributions |Favorite Table|Download (.pdf) Table 12-4 Glycosaminoglycans and Their Tissue Distributions
|Repeating Disaccharide (A-B) |
|Glycosaminoglycan ||Molecular Weight (Da) ||Monosaccharide A ||Monosaccharide B ||Linked to Protein ||Tissue Distribution |
|Hyaluronic acid ||4000-8 × 108 ||D-Glucuronic acid ||N-Acetyl-D-glucosamine ||– ||Most connective tissues, skin, cartilage, and synovial fluid |
|Chondroitin sulfate ||5000-50,000 ||D-Glucuronic acid ||N-Acetyl-D-galactosamine ||+ ||Cartilage, bone, and skin |
|Dermatan nitrate ||15,000-40,000 ||D-Glucuronic acid or L-iduronic acid ||N-Acetyl-D-galactosamine ||+ ||Skin and blood vessels |
|Heparan sulfate ||5000-12,000 ||D-Glucuronic acid or L-iduronic acid ||N-Acetyl-D-glucosamine ||+ ||Lungs, arteries, and cell surfaces |
|Heparin ||6000-25,000 ||D-Glucuronic acid or L-iduronic acid ||N-Acetyl-D-glucosamine ||+ ||Skin, lungs, liver, and mast cells |
|Keratan sulfate ||4000-19,000 ||D-Galactose ||N-Acetyl-D-glucosamine ||+ ||Cartilage and intervertebral disc |
Collagen is synthesized by fibroblasts as part of the healing process. There is a dynamic balance between breakdown and synthesis. The balance is weighted heavily toward synthesis in the beginning stages of healing. As part of the maturation of the wound, excessive collagen is broken down by collagenases and the scar becomes less prominent.
The wound also contracts and becomes slightly smaller and less visible. Myofibroblasts are specialized fibroblasts responsible for wound contraction. They are present between 1 and 4 weeks after the injury but peak around 2 weeks. In acute wounds, they undergo apoptosis and then disappear after they have completed the wound contraction.
Research has shown that TGF-β and CTGF help to differentiate the fibroblast into a myofibroblast. When hypertrophic scars form, myofibroblasts are present beyond the usual time period or are present in excessive amounts. This may be related to ongoing autocrine TGF-β stimulation.
The wound strength increases rapidly between 1 and 6 weeks after injury. At 3 weeks, the breaking strength increases as a result of cross-linking of collagen. The wound reaches close to its final strength after 2 months. At a year, the wound strength plateaus and is approximately 70% that of normal skin.