Sedlarik, K. M.

A wound is a disruption of tissue integrity that is typically associated with a loss of substance. Deeper injuries to the muscle tissue, the skeletal system, or the inner organs are defined as complicated wounds.

Every wound initiates bodily mechanisms that are designed for a single purpose: the restoration of tissue integrity through formation of new structures that more or less match the original function. Wound healing is therefore not only a locally restricted regeneration process, but is to a high degree determined by the overall condition of the afflicted organism, which again depends on diverse endogenous factors such as age, nutrition, medication, immunologic status, metabolic condition, etc.

The complex interrelationship between the wound and the patient becomes especially obvious when wound healing is impaired. As a practical consequence the individual condition of the organism should certainly be integrated more strictly into the therapy concept than is presently the case.

Every wound healing proceeds in three interrelated dynamic phases with overlapping time courses, irrespective of the wound type and the degree of tissue damage. According to morphologic changes in the course of the healing process, these phases are clinically distinguished as an inflammatory or exudative phase for the detachment of deteriorated tissue and for wound cleansing, a proliferative phase for the development of granulation tissue and a differentiation or regeneration phase for maturation, scar formation and epithelialisation (see illustration on page 6). In everyday practice, the three phases are also denoted with the abbreviated terms cleansing phase, granulation phase, and epithelialisation phase.

INFOBOX 1: THE PHASES OF WOUND HEALING

The course of wound healing is characterized by degradation or catabolic processes on one hand, and formation or anabolic processes on the other. It is a fundamental finding of modern wound healing research that anabolic activities are instigated in the connective tissue immediately after wounding and that the extent of formation processes is related to that of the degradation processes which dominate the beginning of wound repair.

The distinction between wound healing by first intention and wound healing by secondary intention refers, in comparison, to rather quantitative aspects of wound repair. Conditions for wound healing are more favourable when less tissue is damaged. The best prognosis for successful wound healing is found with smooth, closely abutting incision wounds without substantial tissue loss or presence of foreign bodies, in well-vascularised areas of the organism. In these cases, healing proceeds by first intention (per primam intentionem) as long as wound infections can be avoided.

Wound healing by secondary intention (per secundam intentionem) typically occurs in those situations when tissue defects have to be refilled or when pus formation interferes with the direct reassociation of the wound edges. Here, the wound surfaces do not lie closely adjacent to each other, but rather split, more or less away from each other. To close this wound, new tissue, the so-called granulation tissue, must be grown. The energy demand placed upon the organism on this occasion is incomparably greater than in the case of primary wound healing.

THE INFLAMMATORY / EXUDATIVE PHASE

Tissue wounding causes severance of capillaries and destroys and damages cells. Blood and plasma pass into the extravascular space.

The primary goal of the repair mechanisms is the prevention of local hemorrhage. Platelets adhere to collagenous fibres of the connective tissue and aggregate after having released various vasoactive substances. Simultaneous with platelet aggregation, the coagulation system is activated because the plug initially formed consists mostly of thrombocytes and does not allow permanent closure of the damaged vessels.

The process of coagulation can also be looked at as occuring in distinct phases. It involves numerous factors as for example, the coagulation factors of blood plasma factor I to factor XIII and is essentially characterized by successive conversion of coenzymes into enzymes. Fibrin formation at the end of the coagulation process is initiated by the catalytic activity of thrombin that cleaves fibrinogen into polypeptides. The fibrin monomers thus produced tend to aggregate spontaneously into long fibres, but are unstable as newly formed fibrin monomers. Stabilization of the fibrinous structures is mediated by the cross-linking activities of activating factor XIII. The resulting fibrin network will later serve as “scaffold” for the migrating fibroblasts.

Immediately after wounding and in an additional second phase, one or two hours later, the release of vasoactive substances, including histamine, serotonin, and cytokines causes an increase in vessel permeability with enhanced exudation of blood plasma. An oedema is formed in response to the vasoactive substances and also partially due to local acidosis in the wound area.

Today, the significance of oedema formation for wound healing is essentially seen in the following aspects: Accumulation of tissue fluid generates an aqueous milieu promoting the conversion of fibrocytes to fibroblasts or proliferative processes in general. Local acidosis with oxygen depletion and increased CO2 pressure enhances catabolism. And the local accumulation of liquid in the wound area may finally also cause dilution of toxic debris.

With exudation of blood plasma, a number of cells are reaching the wound area that are involved in defence against infections and wound cleansing, but also secrete substances concerned in later repair aspects. These are the so-called inflammatory cells, T lymphocytes, leucocytes, especially neutrophil granulocytes, as well as monocytes or macrophages.

In particular the amoeboid neutrophil granulocytes that are part of the non-specific cellular defence system play a central role in wound cleansing and resistance to infection. Their nuclei contain proteolytic enzymes which facilitate extensive degradation of detritus and phagocytosis of bacteria. Neutrophils are attracted to the wound site by the action of several chemotactic substances.

The migration of neutrophils ceases within a few days when the wound is “clean”. However, with development of persistent infections, cell migration continues and phagocytosis is enhanced, giving rise to the clinical picture of pus formation. In this case, the first phase of wound healing is considerably delayed.

Following the neutrophils, macrophages, which play a key role in wound healing, start to migrate into the wounded area. They do not only function in wound débridement, but also secrete biologically active substances, the so-called growth factors, and thus have a lasting influence on the subsequent phases of proliferation and tissue differentiation.

Most of the macrophages are derived from hematogenic monocytes. Differentiation and activation of monocytes into macrophages takes place at the wound site. Attracted by chemotactic signals from bacterial toxins and after additional activation by neutrophil leucocytes, the cells traverse the endothelium in great numbers to reach the wound area.

Phagocytosis represents the stage of highest activity of a macrophage. But the cell is not restricted to directly attacking micro-organisms, it is also capable of mediating antigen transfer to lymphocytes. Antigens are taken up and partially degraded by the macrophage and then presented to a lymphocyte for recognition.

Far more important for wound healing are, however, the coordinating and regulating functions of macrophages that have only recently been elucidated: Macrophage mediate conversion of macromolecules into „reusable“ aminoacids and glycosides, attract further macrophages, stimulate fibroblast proliferation, initiate neovascular growth, and excrete lactate and derivatives of H₂O₂ into the wound site.

THE PROLIFERATIVE PHASE

INFOBOX 2: THE ROLE OF THE MACROPHAGES

The second phase of wound healing is dominated by cell proliferation aimed at the generation of new replacement tissue to fill and resurface the defect.

Granulation tissue was described by Letterer (1953) as a temporary primitive tissue unit that after having fulfilled its function is subjected to regression and for the most part is gradually converted into scar tissue.

Development and differentiation of granulation tissue is decisively influenced by endogenous and exogenous factors, bearing the risk of wound healing impairment, especially in deep defects.

The denotation of granulation was introduced in 1865 by Billroth and stems from the finding that pale red bodies of a vitreous transparency (Lat. granula) become apparent during development of the tissue. Each body represents a vascular tree consisting of numerous fine capillaries with loop-shaped replications at the surface. New germinal tissue adheres to these loop-shaped vessels. When granulation is not disturbed, the bodies grow in volume and number to finally form a shiny salmon-pink surface.

Formation of granulation tissue is a complex event involving leucocytes, histiocytes, plasma cells, mast cells, and in particular fibroblasts that promote tissue growth through production of collagen. Nourishment of the nascent tissue is assured by ingrowing capillaries.

The spindle-shaped fibroblasts do not arrive at the wound site via circulation, but are for the most part resting cells, already located in the injured tissue. Amino acids that are derived from the degradation of blood clots by macrophages serve as their nourishing substrates. The presence of macrophages in the wound site is thus, once again, of crucial importance.

Fibroblasts use the fibrinous net formed during coagulation as scaffold for collagen insertion. This close relationship between fibroblasts and fibrin net has in the past lead to the assumption that fibrin is converted into collagen. In actual fact, fibrin is, however, degraded during the process of collagen insertion.

Fibroblasts arrive at the site of injury after blood clots have been resolved and necrotic tissue has been re-moved. In the presence of hematomas, necrotic tissue, foreign bodies, and bacteria, migration of fibroblasts and development of capillaries is delayed. The extent of granulation tissue formation is thus directly related to the intensity of coagulation, immediate inflammation and endogenous wound débridement – repair aspects that are altogether accomplished in the first phase of wound healing.

The major function of fibroblasts is the synthesis of collagen that already begins at the second day after the injury and in primary healing reaches its peak activity between day 5 to 7. Best conditions for production are given in a slightly acidic milieu.

Collagen is one of the basic body proteins and several distinct types of collagen have been described. Scar tissue, for example, contains type III collagen, an embryonal collagen type, whereas later on in maturation type I is the dominating component.

Collagen synthesis occurs in several steps. The first stage takes place in the fibroblast and involves the synthesis of polypeptide chains from glycin, proline or hydroxyproline, hydroxylysine, and an additional third of other amino acids. Three polypeptide chains are packed tightly together to form the triple helix molecule procollagen. Procollagen is then excreted into the extracellular space through cellular microtubules. Here, processing and assembly of the still soluble collagen leads to the formation of collagen fibrils and filaments of considerable tensile strength to adapt to the needs of the wound area.

In addition to collagen synthesis, fibroblasts also produce acidic hexosamine-containing mucopolysaccharides that serve as major matrix constituents, and thus contribute to the integrity of granulation tissue.

During the third week of primary healing newly formed collagen in the wound area reaches its mass maximum. Imposing a slight mechanical stress on a healing wound in the proliferative phase, however, can induce enhanced collagen formation. Transferred to clinical practice, this finding may suggest that patients should be allowed early mobilisation when the stage of wound fragility is overcome.

Vitamin C plays an important role in collagen synthesis. Without vitamin C, only inferior collagen can be produced that is then excreted into the wound area by fibroblasts in only small amounts.

In addition to vitamin C, oxygen is also indispensable for the synthesis of collagen. Oxidation of vitamin C in the presence of iron (Fe₂+) is associated with the release of great amounts of energy in the form of the superoxide anion radical (O₂-). When O₂- production exceeds the amount of available O₂ collagen synthesis is accelerated. This indicates that in practice, wound healing requires sufficient oxygen supply.

Regeneration or new growth of blood and lymph vessels begins mainly at pre-existing vessels at the edges of the wound and occurs via sprouting, cell division, and anastomosis. Circulating cells may also take part in this process. Stimulation of neovascularisation is related to macrophage and thrombocyte activity.

When capillary ingrowth reaches a sufficient level, O₂ pressure is increased to normal values by anabolic processes. Vasopermeability of newly formed capillaries is higher than that of other microvessels. This is necessary to support the increased metabolism at the site of regenerative inflammation.

In extensive secondary healing detritus removal via phagocytosis and resorption of lysed tissue debris are often not satisfactory. Development of granulation tissue therefore involves the separation (demarcation) of ischemic, non-vital parts of the tissue that are then gradually eliminated through lytic processes. During wound treatment these endogenous cleansing mechanisms may be supported via surgical, enzymatical, or physical débridement to ensure undisturbed development of granulation.

DIFFERENTIATION PHASE

INFOBOX 3: GROWTH FACTORS – THE HOPE IN WOUND HEALING

aturation of collagenous fibres is initiated approximately between 6 and 10 days after wounding. The wound contracts under the influence of specific cells, the myofibroblasts, granulation tissue becomes increasingly depleted of fluids and blood vessels, begins to strengthen, and undergoes remodelling to form scar tissue. Wound healing is then completed by epithelialisation, a process, beginning mainly at the edges of the wound, that involves the formation of new epidermal cells by mitosis and cell migration along pathways created by liquefied fibrin.

Hunt and Dunphy (1969) described wound contraction as a “remarkable physiological event that leads to spontaneous closure of open lesions”. Unlike granulation tissue formation the process of wound contraction is less depending on the general condition of the organism, thus representing an exception in the healing process.

Wound contraction, the inward movement of the intact edges of the injured tissue, decreases the dimension of the area of “incomplete repair” to its smallest possible extent. This is all the more effective, the better the skin is permitted to move against its underlying tissue. The direction of tensile strength is given by the Langer’s lines.

As opposed to the early assumption that wound contraction is caused by shrinking of the collagenous fibres, it is now well established that these shrinking processes play only an inferior role.

Wound contraction is rather mediated by fibroblasts of the granulation tissue. Under certain conditions fibroblasts can convert into a cell type that shares many structural and functional features with smooth muscle cells, including the formation of the contractile smooth muscles protein actomyosin.

Cuticularisation is the final stage in wound healing, and the processes of epithelialisation are very closely related to the developments and proceedings during wound granulation. The granulation tissue produces the chemotactic signals for migration of the epithelial cells from the edges of the wound. Effective cell migration requires an appropriate matrix: Mature granulation tissue and a slippery gliding surface are prerequisites for final epidermisation.

Re-epithelialisation is also a very complex and intricate event that could only recently be elucidated by experimental findings from research on carcinogenesis. Compensation of physiological cell loss and regenerative replacement of cells in skin defects are based on enhanced cell division.

Epidermal cells that are metabolically active and capable of promoting wound healing reactions obviously contain an unlimited potential for mitosis. Their proliferative activity is usually controlled by tissue specific inhibitors, called epidermal chalones, but is fully effective in the case of a lesion.

A dermal injury causes a local decrease in chalone level due to the loss of several chalone producing cells in the wound site. This promotes a correspondingly high mitotic activity in the basal cell layer of the epidermis and initiates the necessary cell proliferation to resurface the denuded area.

Only the cells in the monostratal basal cell layer are able to synthesise DNA and thus to proliferate. Division of these stem cells is always asymmetric and results in the generation of two cell types of different prospective potentials; one set of cells remains in the basal cell layer, its descendants shift away from their place of birth while they undergo successive transformation, they “migrate”.

During physiological maturation of the epidermis migration is directed towards the surface of the skin. Regenerative cell replacement, however, proceeds via linear cell movement in direction of the adjacent wound edges. In both cases, the driving force for cell movement is obviously the continuous proliferative activity of the stem cells.

Only abrasions heal according to the pattern of physiological regeneration; the result is a correspondingly intact and normally structured skin. In any other skin injury, in particular in full thickness wounds, replacement of tissue defects is accomplished by cell migration starting from the edges of the wound and from remaining integumentary appendages. This re-epithelialisation does not produce a fully restored skin substitute, but leads to a thin, poorly vascularised replacement tissue that lacks essential epidermal components as glandular or pigment cells and important characteristics, such as sufficient neurotisation.

Epithelial migration starting at the edges of the wound is initiated as soon as the integrity of the upper epidermal layers is disrupted. Its most striking feature is the odd ability of the epidermal cells, that have been torn apart, to migrate across the wound surface in an active amoeboid movement resembling that of single cell organisms, and thus to close the wound defect. This is, however, in this form only accomplished in fissured surface wounds; they may already be covered and closed within 24 to 48 hours.

In all other skin injuries, migration of the epithelial cells at the edges of the wound depends on previous refilling of the defect with germinative wound tissue, the granulation tissue. Epithelial cells from the edges of the wound are not prone to descend into wound recesses and craters; they require a smooth, moist gliding surface.

Today, the mechanical causes and functions of cell migration that begin about 36 hours after wounding can, for the most part, be explained. Interaction between the coagulum, that is initially formed at the wound site and contains the specific ferment plasminogen, and a cytokine, that is derived from the destroyed epidermal cells, leads to the formation of plasmin. This enzyme degrades wound protein, liquefies the thrombus and the epidermal layers below the top horny layer, and thus generates a matrix that allows the amoeboid gliding movement of the migrating cells.

INFOBOX 4: QUANTITATIVE CLASSIFICATION OF WOUND HEALING

As the amoeboid abilities of the epithelial cells from the edge of the wound necessitate an appropriately moist matrix with sufficient blood supply, cell migration is severely delayed if not impaired by desiccation of the surface layers of the granulation tissue (Montagna 1962; Turner 1991).

It is also not unusual that epithelial islands appear far away from the wound edges in the middle of the granulation tissue, e.g. in full thickness skin wounds, which evidently cannot have developed from remaining dermal appendages. This is rather due to spontaneous epithelial dispersion, as it has already been described by Melchior in 1955. The epithelial islands serve again as source for further cell migration and thus contribute to enhanced resurfacing of the granulation tissue.

Migration of the epithelial cells from the edges of the wound does not occur in a steady, incessant manner, but is an intermittent process, probably depending on the respective conditions of wound granulation and the reception of chemotactic signals.

A first episode of epithelial growth at the edges of the wound is followed by a phase of thickening and stocking of the initially monolayered epithelial cover, the necessary augmentation of epithelial cells being ensured by cell division. The soon multilayered epithelial layers become at the same time more resistant and dense.

With advancing epithelialisation of the mature granulation tissue the modelling, plastic influence of the epithelial cover becomes apparent. Due to the early onset of wound contraction the injury does no longer possess its initial extension, but has by now shrunk to about half of its original dimension. The granulation tissue that is covered with a lawn of young epithelial cells is in an impressive manner levelled to the skin surface (Jorns 1962). The closure of the skin defect is thus accomplished on the outside. Maturation of the scar tissue, however, may yet take several more months.

PD Dr. med. habil. Karel M. Sedlarik
Leiter der Abteilung Medizin
PAUL HARTMANN AG
Postfach 1420
89504 Heidenheim