Phase of repair	Approximate duration	Factors affecting duration
Inflammation
Acute phase	24-48 hours	• Tissue involved • Levels of nutrients, oxygen, cytokines, etc. • Mechanical tensions on tissues
Subacute phase	10-14 days
Proliferation
	Variable intermediate phase	As above
Remodelling
	Begins at around 2 weeks; can last for a year or more	As above

INFLAMMATORY PHASE

Inflammation is the immediate response to injury. The cardinal signs of inflammation are redness, swelling, heat and pain. The acute, or early, phase inflammatory response lasts between 24 and 48 hours and is followed by a subacute, or late, phase, which lasts between 10 and 14 days. The subacute phase can be extended if there is a continuing source of trauma or if some form of irritation, such as a foreign body or infection, is present.

Tissue injury causes both cell death and blood vessel disruption. The primary purpose of the inflammatory phase of healing is to rid the area of debris and dead tissue and to destroy any invading infection prior to the repair. This phase can be described in terms of vascular and cellular changes, which are mediated through the actions of chemical agents. Figure 4.1 shows a typical wound after 3 days.

Figure 4.1 A cutaneous wound 3 days after injury. FGF, fibroblast growth factor; IGF, insulin-like growth factor; KGF, keratinocyte growth factor; PDGF, platelet-derived growth factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor

(from Singer & Clark 1999).

VASOREGULATION AND BLOOD CLOTTING

The initial vascular reaction involves haemorrhage and fluid loss due to destruction of vessels; this is followed by vasoconstriction, vessel plugging and blood coagulation, to prevent further blood loss. These processes lead to the activation of the repair process. Blood loss into the tissues initiates platelet activity and blood coagulation directly, both of which then result in the production of chemical factors which initiate and control the healing process. In addition, the blood clot provides a provisional matrix that facilitates the migration of cells into the wound (Clark 1991b, Singer & Clark 1999).

Primary vessel constriction occurs. This is due to the release of noradrenaline (norepinephrine); the reaction lasts for only a few seconds to a few minutes. During vasoconstriction the opposing cell walls are brought into contact, and adhesion between the surfaces results. Secondary vessel vasoconstriction might follow, due to the action of serotonin, adenosine diphosphate, calcium and thrombin.

Both lymphatics and blood vessels are plugged to limit fluid loss. Initial platelet adhesion and aggregation is stimulated by the presence of thrombin (Terkeltaub & Ginsberg 1988). The platelets adhere to one another, to the vessel walls and to the interstitial extracellular matrix, leading to the build-up of relatively unstable platelet plugs. The process is continued and consolidated by the release of adhesive proteins such as fibrinogen, fibronectin, thrombospondin and von Willebrand factor by the platelets (Ginsberg et al 1988). Coagulation of extravascular blood is thought to be due to the action of platelets and intrinsic and extrinsic clotting mechanisms. Prothrombin is converted to thrombin and thus fibrinogen to fibrin, providing an early wound matrix.

Blood coagulation not only aids haemostasis through clot formation but adds to the early wound matrix and results in the generation of chemical mediators such as bradykinin (Proud & Kaplan 1988). These substances affect the local circulation, stimulate the production of further chemical mediators and act as attractants to cells such as neutrophils and monocytes.

Following this period of vasoconstriction, secondary vasodilation and increased permeability of venules occur owing to the effects of histamine, prostaglandins and hydrogen peroxide production (Issekutz 1981, Williams 1988). Subsequently, both bradykinin and the anaphyllatoxins initiate mechanisms that increase the permeability of undamaged vessels, leading to the release of plasma proteins which contribute to the generation of the extravascular clot.

CELL MIGRATION AND ACTION

Neutrophils and monocytes are the earliest cells to reach the site of injury. They migrate in response to a wide variety of chemical and mechanical stimuli, including the products of the clotting mechanism, the presence of bacteria and cell-derived factors.

The primary action of neutrophils is phagocytosis; their task is to rid the site of bacteria and of dead and dying materials. Neutrophilic margination within the vascular structures leads to the passage of neutrophils through vessel walls by amoeboid action, enabling them to reach damaged extravascular tissues. Phagocytosis is achieved by neutrophilic lysis. This results in the release of protease and collagenase, which begin the lysis of necrotic protein and collagen respectively, as shown in Figure 4.2. Infiltration of the neutrophils into the extravascular tissue ends after a couple of days, marking the end of the early phase of inflammation.

Figure 4.2 Phagocytosis. A, in phagocytosis, cells such as neutrophils and macrophages ingest large solid particles such as bacteria and dead and dying material. B, folds of the plasma membrane surround the particle to be ingested, forming a small vacuole around it, which then pinches off inside the cell. C, lysosomes fuse with the vacuole and pour their digestive enzymes (such as protease and collagenase) on to the digested/ingested material.

Macrophages are essential to the healing process and can perform the normal function of neutrophils in addition to their other tasks. Monocytes migrate from the vasculature into the tissue space and rapidly differentiate into macrophages; the factors responsible for this change have not been fully identified, but may include the presence of insoluble fibronectin (Hosein et al 1985), low oxygen tension (Hunt 1987), chemotactic agents (Ho et al 1987) and bacterial lipopolysaccharides and interferons (Riches 1996). Macrophages phagocytose pathogenic organisms, tissue debris and dying cells (including neutrophils), and release collagenase and proteoglycans, both of which are degrading enzymes that lyse necrotic material (Leibovich & Ross 1975, Tsukamoto et al 1981).

CHEMICAL FACTORS

Many growth factors that influence and control the initial inflammatory process and trigger further developments in the proliferative phase are released by cells during the stage of inflammation; some key factors are shown in Table 4.2.

Table 4.2 Key cytokines affecting tissue repair (adapted from Singer ft Clark 1999)

Cytokine groups	Arising from	Targets tissues and effects
Epidermal growth factors
EGF	Platelets	Epidermal and mesenchymal structures:
TGF-α	Macrophages, epidermal cells	cell motilityand proliferation
Heparin binding EGT	Macrophages
Fibroblast growth factors	Macrophages, endothelial cells, fibroblasts	Wound vascularization, fibroblast proliferation, epidermal cell proliferation and motility
Transforming growth factor-β family	Platelets, macrophages	Fibrosis and increased tensile strength
Platelet-derived growth factor	Platelets, macrophages	Fibroblast proliferation and chemoattraction; macrophage chemoattraction and activation
lnterleukin-1	Neutrophils	Production of growth factors
Insulin-like growth factor-1	Fibroblasts, epidermal cells	Granulation tissue formation; re-epithelialisation
Tumour necrosis factor	Neutrophils	Production of growth factors
lnterferon-α/β	Lymphocytes, fibroblasts	Macrophage activation, inhibit fibroblast proliferation
Thromboxane A₂	Destroyed cells	Potent vasoconstrictor

EGF, epidermal growth factors; EGT, epidermal growth factor; TGF, transforming growth factor.

Macrophages release factors that attract fibroblasts to the area (Tsukamoto et al 1981) and enhance collagen deposition (Clark 1985, Weeks 1972). Platelets release growth factors that contribute to the control of fibrin deposition, fibroplasia and angiogenesis through their action on a variety of cells. Platelets also release fibronectin, fibrinogen, thrombospondin and von Willebrand factor (Ginsberg et al 1988); these are necessary for the aggregation of platelets and for their binding to tissue structure. In addition, serotonin, adenosine diphosphate, calcium and thromboxin are released; these are necessary for blood vessel constriction to prevent haemorrhage.

Dead and dying cells release substances that influence the development of the neomatrix; these include a variety of tissue factors, lactic acid, lactate dehydrogenase, calcium, lysosomal enzymes and fibroblast growth factor (Clark 1991a). In addition, prostaglandins (PG) are produced by almost all cells of the body following damage, due to alterations in the phospholipid content of the cell walls (Janssen et al 1991); some types of PG are proinflammatory, increasing vascular permeability, sensitising pain receptors and attracting leucocytes to the area. Other classes of PG are anti-inflammatory. Both can be involved in early stages of repair.

PROLIFERATIVE PHASE

Granulation tissue is formed during the proliferative phase. This is a temporary structure that evolves after a period of a couple of days and comprises neomatrix, neovasculature, macrophages and fibroblasts. Granulation tissue precedes the development of mature scar tissue. ‘Fibroplasia’ is a term that encompasses the processes of fibroblast proliferation and migration, and the development of the collagenous and non-collagenous matrices.

Fibroplasia

Fibroblasts produce and organize the major extracellular components of the granulation tissue. They migrate into the wound in response to both chemical and physical attractants (Clark 1990, McCarthy et al 1996, Repesh et al 1982). The fibroblast is primarily responsible for the deposition of the new matrix. Once present within the wound, fibroblasts synthesize hyaluronic acid, fibronectin and types I and III collagen; these form the early extracellular matrix. Changes take place as the matrix matures: the amount of hyaluronic acid and fibrinogen is gradually reduced, type I collagen becomes the predominant component, and proteoglycans are deposited.

Hyaluronic acid, present only in early wound healing, appears to facilitate cell motility and might be important in fibroblast proliferation (Lark et al 1985, Toole 1981). Fibronectin has many functions within a wound, including acting as a chemoattractant to cells such as fibroblasts and endothelial cells, augmenting the attachment of fibroblasts to fibrin, facilitating the migration of fibroblasts, and possibly providing a template for collagen deposition (Clark 1996). Proteoglycans contribute to tissue resilience and help to regulate cell motility and growth, and the deposition of collagen.

Collagen is a generic term covering a number of different types of glycoprotein found in the extracellular matrix. Collagen provides a rigid network, which facilitates further healing. The types of collagen within a wound, and their quantities, are gradually modified with time. Type III (embryonic collagen) is gradually absorbed and replaced by type I collagen, which is mature fibrillar collagen. Type IV collagen might be produced as a part of the basement membrane when skin damage occurs, and type V collagen is deposited around cells, forming a structural support.

Two primary factors affect collagen metabolism and, therefore, production. The first is the effect of the cytokines; Table 4.3 lists some of the cytokines believed to affect collagen metabolism. There appears to be a balance between the stimulatory and inhibitory effects of these substances, leading to optimal healing with neither over- nor underproduction of collagen.

Table 4.3 Cytokines controlling collagen production

Cytokine	Action	Reference
TGF-β	Induces collagen synthesis	Ignotz & Massague 1986
IL-1	Induces collagen synthesis	Prostlethwaite et al 1988
TNF	Induces collagen synthesis	Duncan & Berman 1989
IFN	Decreases collagen synthesis	Czaja et al 1987
TNF-α	Decreases collagen synthesis	Scharffetter et al 1989
PGE2	Decreases collagen synthesis	Nicholas et al 1991

IFN, interferons; IL-1, interleukin-1; PGE₂, prostaglandin E₂; TGF, transforming growth factor; TNF, tumour necrosis factor.

The second factor influencing collagen metabolism is the nature of the extracellular matrix (Kulozik et al 1991, Mauch & Krieg 1990). The extracellular matrix provides both a structural scaffold for the tissue and signalling for the cells. Reduced collagen synthesis results from cell contact with mature, type I collagen, upon which the production of collagenase is activated.

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