Wound Healing: A Review I. The Biology of Wound Healing SHELDON V. POLLACK, M.D.
The self-repairing capability o f the skin may be studied experimentally in both humans and small animals. A great deal o f work has been done with animal models and the findings therefrom are important to dermatologic surgeons. In this paper some aspects o f the biology o f wound healing are reviewed and in subsequent papers several other aspects o f the phenomenon o f wound heal ing will be reviewed in the same way.
F o l l o w i n g d i s r u p t i v e i n j u r y to the skin by acci dent or surgical design, a predictable, orderly se quence of biological events occurs. At the moment of the injury, blood enters the wound, bringing with it not only cellular elements but also various proteins, of which fibrinogen is very important. A fibrin network is formed from these precursor molecules and serves not only in hemostasis but, in the case of incisions, also to unite weakly wound edges. Subsequently, that net work of fibrin is destined to play a role in both epithelization and in fibroblast migration, as will be seen later. Within a few hours, the wound surface becomes dry due to clotting of blood and evaporation of moisture. A scab forms on the surface. Winter1 found that in the domestic pig, drying of the exposed dermal tissue be gins immediately after removal of epidermis. The sur face of the dermis continues to dry out for about 18 hours and a “ water table” is established approxi mately 0.3 mm below the surface. As a result, the most superficial portion of the wound that is able to support cellular life lies just beneath the desiccated region. Consequently, epidermal migration often takes place not across the surface of the wound, but through the fibrous protein of the superficial corium either from adnexal epithelium if left or from the nests of epithelial cells from the edges. It is likely that migrating epider mal cells secrete a collagenolytic enzyme that helps
Dr. Pollack is Clinical Instructor in Dermatology, New York Uni versity School of Medicine, New York, New York. Address reprint requests to Dr. Sheldon Pollack, Chemosurgery Section, Skin and Cancer Unit, New York University Medical Center, 566 First Avenue, New York, New York 10016.
them in this difficult transit.2 Collagenases have been found in skins of amphibians,3 in human corneal epithelium,4 and in mammalian skin5. If external fac tors are such that much tissue is excessively dehy drated, epidermal cells may have to reach deeper into the dermis in order to obtain a sustaining milieu. This results in slower wound healing because of the longer time required for complete epithelization. Proper dress ing of wounds, a subject to be discussed in a later article, is important in this regard. Following blood-clot formation, and likely as a re sult of the release of vaso-active substances from in jured tissue, nearby intact blood vessels become “ leaky.” This results in increased blood flow to the wound, leading to the availability of substances and nutrients that will later be required in the healing pro cess. In addition, the exudate that oozes into a wounded area contains albumin, globulin, and im munoglobulins. Such a fluid also provides an environ ment that enables immigrating leukocytes to debride wounds and ingest bacteria. Polymorphonuclear leukocytes appear first, within six hours, and engulf any bacteria that may be present. Most of them move towards the surface and become trapped in the upper portion of the wound where dehydration is greatest and brings death to them. They are readily identifiable there in biopsy specimens even after many days, shrunken in cytoplasm but in the main not autolysed. It is likely too that they serve as a barrier against bacte rial contamination during the early phase of wound healing. Polymorphonuclear leukocytes have a short life span, but within five days a second kind of leuko
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cyte, the macrophage, appears and becomes predomi nant. Tissue macrophages derive from monocytes that enter the wound via the bloodstream. Unlike neu trophils, they have a long life span and are demonstra ble in healing wounds into late phases of the woundhealing process. Macrophages have the important function of de bridement by phagocytosis of necrotic tissue, foreign material, and dead cells. They also appear to have some other duties in wound healing6 and if they are absent, wound repair suffers. Macrophages play a metabolic role by digesting ingested material and by excreting products of that digestion into the surround ing environment. This “ recycling” of useful substrate materials is an efficient way of supplying necessary building blocks such as amino acids and simple sugars required for wound repair. Macrophages also release chemotactic factors that attract more of them to the area and stimulatory substances that cause multiplica tion of fibroblasts and neoangiogenesis. It has been shown that if macrophages are eliminated from newly made incisions by specific anti-serum, wound repair tends to be inhibited and development of wound strength to be poor.6 After a wound has been debrided and a supportive environment created, the stage is set for elimination of the tissue defect. This requires progressive coopera tion of epidermal cells to resurface the heeding wound in step with fibroblasts as they reconstitute collagen. EPITHELIZATION
Within 12 hours of wounding, the normal orderly ar rangement of adjacent epidermis is disrupted by changes in cellular morphology and function. Epider mal cells adjacent to the wound, both basal and suprabasalar, become somewhat flattened, lose many of their junctional complexes, and develop “ ruffled bor ders” in the form of pseudopod-like extensions of their cytoplasms. Other cellular changes are appearances of inclusion bodies that most likely are phago lysosomes,7 prominent rough endoplasmic reticulum, and well-developed Golgi complexes. A “ cortical band” of filaments, 40-80 angstroms in diameter, also develops8 at the periphery of migrating epithelial cells and selectively fixes anti-actin antibodies. This con tractile protein is not demonstrable in normal epithelium adjacent to the wound and disappears from the epithelium after healing.9 This phenomenon sug gests that epidermal-cell migration during wound heal ing is accomplished by means of a newly formed con tractile microfilamentous apparatus. Having undergone these physical changes, epider mal cells no longer carry on their previous function of keratin formation and enter into different functions of migration, and multiplication by division. The mecha 390
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nism by which migration of epithelial cells is activated is not known precisely, but many investigators conjec ture that it is a result of loss of whatever makes for “ contact inhibition.” In his “ co-aptation theory,” Weiss10 suggested that cells have specific stereochemi cal bonds (templates) which become saturated when contact with homologous cells occurs. When these templates are saturated an equilibrium develops that is disturbed when a wound is created and then cells re sume their inherent propensity to move. Epidermal cells migrate in sheets. Desmosomes be tween cells are present within these sheets, but they are smaller and fewer in number than those seen in resting epithelial cells. Their migration seems to com bine ameboid motion of individual cells and mass movement of the sheets of them.11 In wounds that ex tend wide and deep in the corium, epithelization must develop from the periphery alone, but in more superfi cial injury it proceeds from the epidermal appendages (hair follicles and sweat glands). As a result, in superfi cial abrasions, second-degree burns, donor sites of split-thickness skin grafts, and dermabraded areas, migration of cells is as short as are the distances be tween appendages, and epithelization is therefore rapid. In deep, broad defects, such as occur in thirddegree burns, extensive Mohs’ surgery, and decubitus ulcers, epithelization developing from the edges of such defects takes a much longer time. Epidermal cells are guided in their migration by the network of fibrin strands that function as a scaffolding over which the cells creep onto a wound.12 This is termed “ contact guidance.” As mentioned earlier, if the surface of the wound is too desiccated, epidermal cells have to burrow under a crusted or scabbed sur face where a moist environment is present. During their migration, epidermal cells have been observed to ingest necrotic debris they may encounter in their paths.11 Sheets of epidermal cells continue to migrate until they come into contact with other masses of epidermal cells moving across a wound from other directions. Upon finally meeting, the cells rapidly form desmosomal attachments11 and begin to resume their normal morphology and function in obedience to “ con tact inhibition.” However, where free border may still be present, a new “ ruffled membrane” will form, and migration will proceed into the remaining defect until the wound is completely covered by epidermal cells. Once this happens, the cells farthest from the wound margins assume cuboidal shapes and begin to undergo mitoses, wherewith daughter cells move upward to form the various layers of normal, resting epidermis. The placement of sutures in skin results in narrow channels that are indeed small versions of larger wounds. Epithelization of suture tracts also occurs by
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migration of epidermal cells into them. The result is small scars that may otherwise be avoided if tapes rather than sutures are used for closure of wounds. Tapes are not always practical for large wounds, but the edges of short incisions with but slight tension upon closure may be coapted by tapes especially in cosmet ically important places like the face. Alternatively, re moval of sutures within two or three days and re placement of them with tapes before epithelization has developed minimizes scarring. Normally, epitheliza tion of suture tracts is sooner or later removed by dense inflammatory infiltrates of macrophages that react against the intrusion of ordinary epithelial cells in the corium. This phenomenon may be accompanied by a foreign-body reaction with intense inflammation that is often misinterpreted as a bacterial infection or a “ stitch abscess.“ Occasionally, remnants of keratinized epithelium are trapped within the suture tracts, a complication that results in the formation of small inclusion cysts alongside the suture line. Just as migration of epidermal cells follows interrup tion of contact inhibition, so too does increase in mi totic activity follow in the tissue around wounds shortly after migration has begun. Normally, mitosis in the epidermis has a diurnal rhythm, being greatest during periods of inactivity, particularly sleep. This rhythm is lost in epidermal wounds; increased mitotic activity begins quickly and reaches a peak in 48 hours. Winter1 found that in the millimeter of epidermis adjacent to a wound in a domestic pig, there is a 17-fold increase in mitosis over that of normal, undisturbed epidermis. This increased mitotic activity declines as rapidly as epithelization proceeds so that by the time epitheliza tion is complete, mitotic activity in the wound edge falls to but three or four times the normal rate. In crease in the rate of mitosis has been attributed to decreased levels of “ epidermal chalones,’’ watersoluble, heat-labile glycoproteins first described by Bullough and Laurence12. These investigators were able to show that certain extracts of epidermis have inhibitory effects on mitosis in skin that follow typical dose-response curves. Later, chalones were found in various species of animals and these substances were further found to be tissue specific, but not species specific. Epidermal chalones were shown to be synthe sized normally by epidermal cells and that levels of them are decreased in wounds. It appears that chalones are more inhibitory in the presence of epinephrine, which explains the lesser rate of mitosis during the waking, active day and the greater rate dur ing nightly rest. It has been suggested that wounding interrupts the supply of catecholamines to the injured area, which results in decreased effectiveness of chalones and consequent increased epidermal mitoses around wounds. This hypothesis has still to be proved.
THE ACTIVITY OF FIBROBLASTS
The dramatic metamorphosis of epidermal cells in wound healing is paralleled by equally striking mor phologic and functional changes in fibroblasts. Previ ously, the source of fibroblasts in wounds was debat able, but recent, elaborate experiments have shown them to be derived from elements within the wound itself.13 They are characterized by abundant endo plasmic reticulum, Golgi apparatus, and mitochondria.14 Fibroblasts are mobile, but, like epidermal cells, are subject to “ contact inhibition.” In wounds, fibroblasts adhere to collagen and fibrin, subject to the same type of “ contact guidance” as is seen in the migration of epidermal cells. Movement is also quite similar, being accomplished by means of “ ruffled membranes” that extend outward from the cell and become momentarily fixed to matter thereabout. Then, by contraction of those extensions cells pull themselves forward. Fibroblasts have many metabolic functions. They synthesize not only collagen but also proteoglycans and elastin and contain enzymes necessary to synthe sis of cholesterol, completion of the Kreb’s cycle, and glycolysis. Fibroblasts require vitamins B and C, oxy gen, amino acids, and trace metals in order to perform their metabolic functions. Some fibroblasts develop a rich supply of myofibrils and are referred to as “ myofibroblasts.” These elements will be discussed later in the section on wound contraction. Fibroblasts begin to appear in wounds towards the end of their inflammatory phases. Because it takes about 72 hours for fibroblasts to complete their migra tion into a wound, there is a preliminary period of wound healing up to about five days, during which collagen synthesis is not yet evident. This period is often referred to as the “ lag phase,” “ preparatory phase,” or “ substrate phase. ’’ The later period of active collagen production is referred to as the “ fibroplastic phase,” which, in sutured wounds, reaches a peak at about the sixth to seventh day. Intense synthesis and secretion of collagen and mucopolysaccharide by fi broblasts continues for about two to four weeks, after which there is a marked decrease in these activities. Despite the slow-down in collagen production, how ever, the tensile strength of wounds continues to in crease with time. This is due to the processes of “ col lagen remodelling” and “ collagen maturation.” THE REMODELLING AND MATURATION OF COLLAGEN
Young collagen fibrils consisting of polymerized tropocollagen molecules, are thin, randomly oriented and have a consistency of a gel. Such collagen is read ily soluble in neutral saline solutions, which neutralize electrostatic forces that are initially responsible for holding together molecules of tropocollagen.15 Be J. Dermatol. Surg. Oncol. 5:5 May 1979
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cause of the haphazard arrangement and fragile nature of many of these early fibrils, they are destined to serve little toward imparting ultimate tensile strength to wounds. During “ remodelling,” many of these fi brils are digested and removed by collagenases while new fibrils continue to be produced. The directions of stresses acting across wounds probably are important determinants of which fibrils are going to be broken down. In general, fibers that remain in a scar are those that are oriented parallel to lines of tension and thus impart mechanical strength to the tissue. As water and mucopolysaccharides are lost from the wound, colla gen fibrils come to be compressed, permitting closer approximation of cross-linking sites. This promotes covalent bond formation, the primary event in “ colla gen maturation.” With the formation of intermolecular cross-links, which impart greater stability to the fibril, collagen loses its saline solubility, but still can be sol ubilized by solutions of dilute acids. Later, these cova lent bonds become more numerous, and collagen fibers become completely insoluble. Histologically, mature scar tissue is composed of parallel, dense bundles of collagen containing fewer blood vessels and cells than undisturbed tissue. The degree of stress on a wound is also important in wound healing; it determines the amount of scar tissue that will ultimately be formed. More scar tissue is re quired to provide adequate tensile strength in a wound on an extremity than on less mobile places like the abdomen. Tendencies from heredity may also play a role in scar formation. Negroes and Orientals, for ex ample, are more likely than Caucasians to form keloids. NEOANGIOGENESIS At the same time that fibroblasts are actively synthe sizing collagen and mucopolysaccharides, capillaries starting as bud-like structures from nearby vessels penetrate wounds and grow into loops. Numerous anastomoses develop and create a rich, interconnect ing network of blood supply to healing wounds. Many of these new vascular channels are short-lived, be cause as synthesis of collagen decreases and high oxy gen tensions are no longer required, many of them re gress. Thus, the wound undergoes a transformation from capillary-rich, highly cellular tissue to compara tively avascular, cell-free scar composed of dense col lagen bundles. THE CONTRACTION OF WOUNDS Contraction of wounds is the process by which the area of a full-thickness, open wound is diminished by gross centripetal movement of the full thickness of sur rounding skin.15 This phenomenon should not be con fused with “ contracture” which refers to an end re 392
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sult, which in some instances may be attributable in part to the process of contraction. The forces produc ing wound contraction reside in the granulation tissue that fills the wound. Within this tissue, modified fibro blasts containing contractile proteins are found and likely play an important role in wound contraction. In lower mammals, contraction is the major process by which surface wounds are healed. These animals possess a well-developed layer of subcutaneous stri ated muscle, the panniculus camosus, which enables the skin to move easily over the underlying fascia. This muscle and the lack of substantial attachment of the skin to underlying structures permit contraction to occur to its fullest extent without mechanical interfer ence from underlying structures. Man has but vestiges of a panniculus camosus, and dermal mobility is less than in lower animals because the human integument is more firmly attached to underlying fat and superficial fascia, which, in turn, is attached to musculature, bone, and other deep structures. Unless skin is mobile and can be stretched over the wound, simple contrac tion will not accomplish closure. In anatomical areas of cutaneous laxity, contraction of wounds may be of great practical advantage. Such is the case in the treatment of large decubitus ulcers on the buttocks, where areas requiring skin grafting may be greatly re duced in size by allowing wound closure to proceed by contraction for a number of weeks before grafting is undertaken. The same process occurring over a joint may result in a flexion contracture. On the face, where the skin is attached to structures such as the eyelid, nose or lip, wound contraction may result in distortion of these structures with resultant ectropion, retraction of the alae nasi or retraction of the oral lips. Because contraction develops as a result of move ment of tissue around the edges of wounds, thinning, stretching, and tautness may be expected to operate in and around contracting wounds. In compensation for these effects epithelial and mesenchymal thickening, termed “ intussusceptive growth,” develops and serves to restore original dimensions. Contraction of wounds ceases when countervailing force in surround ing skin begins to exceed the force of contraction. Therefore, the mobility of skin around wounds deter mines how much contraction will occur. In animal experiments, it has been shown that con traction proceeds at a fairly uniform rate of 0.6 to 0.75 mm per day, no matter what the size of the wound.15 The shape of a full-thickness defect, however, influ ences rate of contraction. Round wounds do not con tract as quickly or completely as do rectangular or stellate defects. Although the mechanism of wound contraction has yet to be fully elucidated, there are certain principles that have been determined. Most important of these is
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the fact that collagen is not essential in the contraction of wounds. This has been shown by experimentation in scorbutic guinea-pigs in whom wound contraction pro ceeded normally, despite the fact that resultant scars contain merely 15% as much collagen as do wounds in healthy animals.16 It has been further shown that a special contractile cell appears in contracting wounds. These cells, called “ myofibroblasts,” have charac teristics of both fibroblasts and smooth-muscle cells in that they exhibit both collagen synthesis and contrac tility. Myofibroblasts are common in granulating wounds, but are not found in sutured wounds. It is not yet known what determines whether a precursor cell will develop into a myofibroblast or into an ordinary fibroblast. Myofibroblasts have many characteristics that dif ferentiate them from fibroblasts of normal tissues.17 Morphological differences are numerous indentations in the nucleus and fibrillar protein within the cyto plasm of myofibroblasts. The fibrils measure approxi mately 40-80 angstroms in width and they lie parallel to the long axis of the cell. They may be selectively labeled with anti-actin antibodies derived from pa tients with chronic hepatitis who form such an tibodies.18 Myofibroblasts also develop cell-to-cell and cell-to-stroma connections. They contract when stimu lated with various agents that excite smooth muscle. When treated with smooth-muscle relaxants, their con traction is inhibited.19 It is likely that myofibroblasts are responsible for wound contraction in humans.
REFERENCES 1. Winter, G. D. Epidermal regeneration studied in the domestic pig. In: Maibach, H. I., and Rovee, D. T., eds. Epidermal Wound Healing. Chicago, Year Book Medical Publishers, 1972. 2. Grillo, H. C., and Gross, J. Collagenolytic activity and epithelial-mesenchymal interaction in healing mammalian
wounds. J. Cell Biol. 23:39A, 1964. 3. Eisen, A. Z., and Gross, J. The role of epithelium and mesen chyme in the production of a collagenolytic enzyme and a hyaluronidase in the anuran tadpole. Dev. Biol. 12:408, 1965. 4. Brown, S. I., and Weller, C. A. Cell origin of collagenase in normal and wounded corneas. Arch. Ophthalmol. 83:74, 1970. 5. Grillo, H. C., and Gross, J. Collagenolytic activity during mammalian wound repair. Dev. Biol. 15:300, 1967. 6. Leibovich, S. J., and Ross, R. The role of the macrophage in wound repair: a study with hydrocortisone and antimac rophage serum. Am. J. Pathol. 78:71, 1975. 7. Ödland, G., and Ross, R. Human wound repair. I. Epidermal regeneration. J. Cell Biol. 39:135, 1968. 8. Krawczyk, W. S. A pattern of epidermal cell migration during wound healing. J. Cell Biol. 49:247, 1971. 9. Gabbiani, G., and Ryan, G. B. Development of a contractile apparatus in epithelial cells during epidermal and liver regener ation. J. Submicr. Cytol. 6:143, 1974. 10. Weiss, P. Specificity in growth control. In: Butler, E. G., ed. Biological Specificity and Growth. Princeton, Princeton Univ. Press, 1955, p. 195. 11. Martinez, I. R. Fine structural studies of migrating epithelial cells following incision wounds. In: Maibach, H. I., and Rovee, D. T., eds. Epidermal Wound Healing. Chicago, Year Book Medical Publishers, 1972, pp. 323-342. 12. Bullough, W. S., and Laurence, E. B. Mitotic control by inter nal secretion: the role of the chalone-adrenaline complex. Exp. Cell Res. 33:176, 1964. 13. Van Winkle, W., Jr. The fibroblast in wound healing. Surg. Gynecol. Obstet. 124:369, 1967. 14. Ross, R. The fibroblast and wound repair. Biol. Rev. 43:51, 1968. 15. Peacock, E. E., and Van Winkle, W. Wound Repair. Philadel phia, W. B. Saunders Co., 1976. 16. Abercrombie, M., Flint, M. H., and James, D. W. Wound con traction in relation to collagen formation in scorbutic guineapigs. J. Embryol. Exp. Morphol. 4:167, 1956. 17. Ryan, G. B., Cliff, W. J., Gabbiani, G., et al. Myofibroblasts in human granulation tissue. Hum. Pathol. 5:55, 1974. 18. Hirschei, B. J., Gabbiani, G., Ryan, G. B., and Majno, G. Fibroblasts of granulation tissue: immunofluorescent staining with antismooth muscle serum. Proc. Soc. Exp. Biol. Med. 138:466, 1971. 19. Majno, G., Gabbiani, G., Hirschei, B. J., Ryan, G. B., and Statkov, P. R. Contraction of granulation tissue in vitro: simi larity to smooth muscle. Science 173:548, 1971.
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The self-repairing capability o f the skin may be studied experimentally in both humans and small animals. A great deal o f work has been done with animal models and the findings therefrom are important to dermatologic surgeons. In this paper some aspects o f the biology o f wound healing are reviewed and in subsequent papers several other aspects o f the phenomenon o f wound heal ing will be reviewed in the same way.
F o l l o w i n g d i s r u p t i v e i n j u r y to the skin by acci dent or surgical design, a predictable, orderly se quence of biological events occurs. At the moment of the injury, blood enters the wound, bringing with it not only cellular elements but also various proteins, of which fibrinogen is very important. A fibrin network is formed from these precursor molecules and serves not only in hemostasis but, in the case of incisions, also to unite weakly wound edges. Subsequently, that net work of fibrin is destined to play a role in both epithelization and in fibroblast migration, as will be seen later. Within a few hours, the wound surface becomes dry due to clotting of blood and evaporation of moisture. A scab forms on the surface. Winter1 found that in the domestic pig, drying of the exposed dermal tissue be gins immediately after removal of epidermis. The sur face of the dermis continues to dry out for about 18 hours and a “ water table” is established approxi mately 0.3 mm below the surface. As a result, the most superficial portion of the wound that is able to support cellular life lies just beneath the desiccated region. Consequently, epidermal migration often takes place not across the surface of the wound, but through the fibrous protein of the superficial corium either from adnexal epithelium if left or from the nests of epithelial cells from the edges. It is likely that migrating epider mal cells secrete a collagenolytic enzyme that helps
Dr. Pollack is Clinical Instructor in Dermatology, New York Uni versity School of Medicine, New York, New York. Address reprint requests to Dr. Sheldon Pollack, Chemosurgery Section, Skin and Cancer Unit, New York University Medical Center, 566 First Avenue, New York, New York 10016.
them in this difficult transit.2 Collagenases have been found in skins of amphibians,3 in human corneal epithelium,4 and in mammalian skin5. If external fac tors are such that much tissue is excessively dehy drated, epidermal cells may have to reach deeper into the dermis in order to obtain a sustaining milieu. This results in slower wound healing because of the longer time required for complete epithelization. Proper dress ing of wounds, a subject to be discussed in a later article, is important in this regard. Following blood-clot formation, and likely as a re sult of the release of vaso-active substances from in jured tissue, nearby intact blood vessels become “ leaky.” This results in increased blood flow to the wound, leading to the availability of substances and nutrients that will later be required in the healing pro cess. In addition, the exudate that oozes into a wounded area contains albumin, globulin, and im munoglobulins. Such a fluid also provides an environ ment that enables immigrating leukocytes to debride wounds and ingest bacteria. Polymorphonuclear leukocytes appear first, within six hours, and engulf any bacteria that may be present. Most of them move towards the surface and become trapped in the upper portion of the wound where dehydration is greatest and brings death to them. They are readily identifiable there in biopsy specimens even after many days, shrunken in cytoplasm but in the main not autolysed. It is likely too that they serve as a barrier against bacte rial contamination during the early phase of wound healing. Polymorphonuclear leukocytes have a short life span, but within five days a second kind of leuko
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cyte, the macrophage, appears and becomes predomi nant. Tissue macrophages derive from monocytes that enter the wound via the bloodstream. Unlike neu trophils, they have a long life span and are demonstra ble in healing wounds into late phases of the woundhealing process. Macrophages have the important function of de bridement by phagocytosis of necrotic tissue, foreign material, and dead cells. They also appear to have some other duties in wound healing6 and if they are absent, wound repair suffers. Macrophages play a metabolic role by digesting ingested material and by excreting products of that digestion into the surround ing environment. This “ recycling” of useful substrate materials is an efficient way of supplying necessary building blocks such as amino acids and simple sugars required for wound repair. Macrophages also release chemotactic factors that attract more of them to the area and stimulatory substances that cause multiplica tion of fibroblasts and neoangiogenesis. It has been shown that if macrophages are eliminated from newly made incisions by specific anti-serum, wound repair tends to be inhibited and development of wound strength to be poor.6 After a wound has been debrided and a supportive environment created, the stage is set for elimination of the tissue defect. This requires progressive coopera tion of epidermal cells to resurface the heeding wound in step with fibroblasts as they reconstitute collagen. EPITHELIZATION
Within 12 hours of wounding, the normal orderly ar rangement of adjacent epidermis is disrupted by changes in cellular morphology and function. Epider mal cells adjacent to the wound, both basal and suprabasalar, become somewhat flattened, lose many of their junctional complexes, and develop “ ruffled bor ders” in the form of pseudopod-like extensions of their cytoplasms. Other cellular changes are appearances of inclusion bodies that most likely are phago lysosomes,7 prominent rough endoplasmic reticulum, and well-developed Golgi complexes. A “ cortical band” of filaments, 40-80 angstroms in diameter, also develops8 at the periphery of migrating epithelial cells and selectively fixes anti-actin antibodies. This con tractile protein is not demonstrable in normal epithelium adjacent to the wound and disappears from the epithelium after healing.9 This phenomenon sug gests that epidermal-cell migration during wound heal ing is accomplished by means of a newly formed con tractile microfilamentous apparatus. Having undergone these physical changes, epider mal cells no longer carry on their previous function of keratin formation and enter into different functions of migration, and multiplication by division. The mecha 390
J. Dermatol. Surg. Oncol. 5:5 May 1979
nism by which migration of epithelial cells is activated is not known precisely, but many investigators conjec ture that it is a result of loss of whatever makes for “ contact inhibition.” In his “ co-aptation theory,” Weiss10 suggested that cells have specific stereochemi cal bonds (templates) which become saturated when contact with homologous cells occurs. When these templates are saturated an equilibrium develops that is disturbed when a wound is created and then cells re sume their inherent propensity to move. Epidermal cells migrate in sheets. Desmosomes be tween cells are present within these sheets, but they are smaller and fewer in number than those seen in resting epithelial cells. Their migration seems to com bine ameboid motion of individual cells and mass movement of the sheets of them.11 In wounds that ex tend wide and deep in the corium, epithelization must develop from the periphery alone, but in more superfi cial injury it proceeds from the epidermal appendages (hair follicles and sweat glands). As a result, in superfi cial abrasions, second-degree burns, donor sites of split-thickness skin grafts, and dermabraded areas, migration of cells is as short as are the distances be tween appendages, and epithelization is therefore rapid. In deep, broad defects, such as occur in thirddegree burns, extensive Mohs’ surgery, and decubitus ulcers, epithelization developing from the edges of such defects takes a much longer time. Epidermal cells are guided in their migration by the network of fibrin strands that function as a scaffolding over which the cells creep onto a wound.12 This is termed “ contact guidance.” As mentioned earlier, if the surface of the wound is too desiccated, epidermal cells have to burrow under a crusted or scabbed sur face where a moist environment is present. During their migration, epidermal cells have been observed to ingest necrotic debris they may encounter in their paths.11 Sheets of epidermal cells continue to migrate until they come into contact with other masses of epidermal cells moving across a wound from other directions. Upon finally meeting, the cells rapidly form desmosomal attachments11 and begin to resume their normal morphology and function in obedience to “ con tact inhibition.” However, where free border may still be present, a new “ ruffled membrane” will form, and migration will proceed into the remaining defect until the wound is completely covered by epidermal cells. Once this happens, the cells farthest from the wound margins assume cuboidal shapes and begin to undergo mitoses, wherewith daughter cells move upward to form the various layers of normal, resting epidermis. The placement of sutures in skin results in narrow channels that are indeed small versions of larger wounds. Epithelization of suture tracts also occurs by
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migration of epidermal cells into them. The result is small scars that may otherwise be avoided if tapes rather than sutures are used for closure of wounds. Tapes are not always practical for large wounds, but the edges of short incisions with but slight tension upon closure may be coapted by tapes especially in cosmet ically important places like the face. Alternatively, re moval of sutures within two or three days and re placement of them with tapes before epithelization has developed minimizes scarring. Normally, epitheliza tion of suture tracts is sooner or later removed by dense inflammatory infiltrates of macrophages that react against the intrusion of ordinary epithelial cells in the corium. This phenomenon may be accompanied by a foreign-body reaction with intense inflammation that is often misinterpreted as a bacterial infection or a “ stitch abscess.“ Occasionally, remnants of keratinized epithelium are trapped within the suture tracts, a complication that results in the formation of small inclusion cysts alongside the suture line. Just as migration of epidermal cells follows interrup tion of contact inhibition, so too does increase in mi totic activity follow in the tissue around wounds shortly after migration has begun. Normally, mitosis in the epidermis has a diurnal rhythm, being greatest during periods of inactivity, particularly sleep. This rhythm is lost in epidermal wounds; increased mitotic activity begins quickly and reaches a peak in 48 hours. Winter1 found that in the millimeter of epidermis adjacent to a wound in a domestic pig, there is a 17-fold increase in mitosis over that of normal, undisturbed epidermis. This increased mitotic activity declines as rapidly as epithelization proceeds so that by the time epitheliza tion is complete, mitotic activity in the wound edge falls to but three or four times the normal rate. In crease in the rate of mitosis has been attributed to decreased levels of “ epidermal chalones,’’ watersoluble, heat-labile glycoproteins first described by Bullough and Laurence12. These investigators were able to show that certain extracts of epidermis have inhibitory effects on mitosis in skin that follow typical dose-response curves. Later, chalones were found in various species of animals and these substances were further found to be tissue specific, but not species specific. Epidermal chalones were shown to be synthe sized normally by epidermal cells and that levels of them are decreased in wounds. It appears that chalones are more inhibitory in the presence of epinephrine, which explains the lesser rate of mitosis during the waking, active day and the greater rate dur ing nightly rest. It has been suggested that wounding interrupts the supply of catecholamines to the injured area, which results in decreased effectiveness of chalones and consequent increased epidermal mitoses around wounds. This hypothesis has still to be proved.
THE ACTIVITY OF FIBROBLASTS
The dramatic metamorphosis of epidermal cells in wound healing is paralleled by equally striking mor phologic and functional changes in fibroblasts. Previ ously, the source of fibroblasts in wounds was debat able, but recent, elaborate experiments have shown them to be derived from elements within the wound itself.13 They are characterized by abundant endo plasmic reticulum, Golgi apparatus, and mitochondria.14 Fibroblasts are mobile, but, like epidermal cells, are subject to “ contact inhibition.” In wounds, fibroblasts adhere to collagen and fibrin, subject to the same type of “ contact guidance” as is seen in the migration of epidermal cells. Movement is also quite similar, being accomplished by means of “ ruffled membranes” that extend outward from the cell and become momentarily fixed to matter thereabout. Then, by contraction of those extensions cells pull themselves forward. Fibroblasts have many metabolic functions. They synthesize not only collagen but also proteoglycans and elastin and contain enzymes necessary to synthe sis of cholesterol, completion of the Kreb’s cycle, and glycolysis. Fibroblasts require vitamins B and C, oxy gen, amino acids, and trace metals in order to perform their metabolic functions. Some fibroblasts develop a rich supply of myofibrils and are referred to as “ myofibroblasts.” These elements will be discussed later in the section on wound contraction. Fibroblasts begin to appear in wounds towards the end of their inflammatory phases. Because it takes about 72 hours for fibroblasts to complete their migra tion into a wound, there is a preliminary period of wound healing up to about five days, during which collagen synthesis is not yet evident. This period is often referred to as the “ lag phase,” “ preparatory phase,” or “ substrate phase. ’’ The later period of active collagen production is referred to as the “ fibroplastic phase,” which, in sutured wounds, reaches a peak at about the sixth to seventh day. Intense synthesis and secretion of collagen and mucopolysaccharide by fi broblasts continues for about two to four weeks, after which there is a marked decrease in these activities. Despite the slow-down in collagen production, how ever, the tensile strength of wounds continues to in crease with time. This is due to the processes of “ col lagen remodelling” and “ collagen maturation.” THE REMODELLING AND MATURATION OF COLLAGEN
Young collagen fibrils consisting of polymerized tropocollagen molecules, are thin, randomly oriented and have a consistency of a gel. Such collagen is read ily soluble in neutral saline solutions, which neutralize electrostatic forces that are initially responsible for holding together molecules of tropocollagen.15 Be J. Dermatol. Surg. Oncol. 5:5 May 1979
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cause of the haphazard arrangement and fragile nature of many of these early fibrils, they are destined to serve little toward imparting ultimate tensile strength to wounds. During “ remodelling,” many of these fi brils are digested and removed by collagenases while new fibrils continue to be produced. The directions of stresses acting across wounds probably are important determinants of which fibrils are going to be broken down. In general, fibers that remain in a scar are those that are oriented parallel to lines of tension and thus impart mechanical strength to the tissue. As water and mucopolysaccharides are lost from the wound, colla gen fibrils come to be compressed, permitting closer approximation of cross-linking sites. This promotes covalent bond formation, the primary event in “ colla gen maturation.” With the formation of intermolecular cross-links, which impart greater stability to the fibril, collagen loses its saline solubility, but still can be sol ubilized by solutions of dilute acids. Later, these cova lent bonds become more numerous, and collagen fibers become completely insoluble. Histologically, mature scar tissue is composed of parallel, dense bundles of collagen containing fewer blood vessels and cells than undisturbed tissue. The degree of stress on a wound is also important in wound healing; it determines the amount of scar tissue that will ultimately be formed. More scar tissue is re quired to provide adequate tensile strength in a wound on an extremity than on less mobile places like the abdomen. Tendencies from heredity may also play a role in scar formation. Negroes and Orientals, for ex ample, are more likely than Caucasians to form keloids. NEOANGIOGENESIS At the same time that fibroblasts are actively synthe sizing collagen and mucopolysaccharides, capillaries starting as bud-like structures from nearby vessels penetrate wounds and grow into loops. Numerous anastomoses develop and create a rich, interconnect ing network of blood supply to healing wounds. Many of these new vascular channels are short-lived, be cause as synthesis of collagen decreases and high oxy gen tensions are no longer required, many of them re gress. Thus, the wound undergoes a transformation from capillary-rich, highly cellular tissue to compara tively avascular, cell-free scar composed of dense col lagen bundles. THE CONTRACTION OF WOUNDS Contraction of wounds is the process by which the area of a full-thickness, open wound is diminished by gross centripetal movement of the full thickness of sur rounding skin.15 This phenomenon should not be con fused with “ contracture” which refers to an end re 392
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sult, which in some instances may be attributable in part to the process of contraction. The forces produc ing wound contraction reside in the granulation tissue that fills the wound. Within this tissue, modified fibro blasts containing contractile proteins are found and likely play an important role in wound contraction. In lower mammals, contraction is the major process by which surface wounds are healed. These animals possess a well-developed layer of subcutaneous stri ated muscle, the panniculus camosus, which enables the skin to move easily over the underlying fascia. This muscle and the lack of substantial attachment of the skin to underlying structures permit contraction to occur to its fullest extent without mechanical interfer ence from underlying structures. Man has but vestiges of a panniculus camosus, and dermal mobility is less than in lower animals because the human integument is more firmly attached to underlying fat and superficial fascia, which, in turn, is attached to musculature, bone, and other deep structures. Unless skin is mobile and can be stretched over the wound, simple contrac tion will not accomplish closure. In anatomical areas of cutaneous laxity, contraction of wounds may be of great practical advantage. Such is the case in the treatment of large decubitus ulcers on the buttocks, where areas requiring skin grafting may be greatly re duced in size by allowing wound closure to proceed by contraction for a number of weeks before grafting is undertaken. The same process occurring over a joint may result in a flexion contracture. On the face, where the skin is attached to structures such as the eyelid, nose or lip, wound contraction may result in distortion of these structures with resultant ectropion, retraction of the alae nasi or retraction of the oral lips. Because contraction develops as a result of move ment of tissue around the edges of wounds, thinning, stretching, and tautness may be expected to operate in and around contracting wounds. In compensation for these effects epithelial and mesenchymal thickening, termed “ intussusceptive growth,” develops and serves to restore original dimensions. Contraction of wounds ceases when countervailing force in surround ing skin begins to exceed the force of contraction. Therefore, the mobility of skin around wounds deter mines how much contraction will occur. In animal experiments, it has been shown that con traction proceeds at a fairly uniform rate of 0.6 to 0.75 mm per day, no matter what the size of the wound.15 The shape of a full-thickness defect, however, influ ences rate of contraction. Round wounds do not con tract as quickly or completely as do rectangular or stellate defects. Although the mechanism of wound contraction has yet to be fully elucidated, there are certain principles that have been determined. Most important of these is
PO LLACK
the fact that collagen is not essential in the contraction of wounds. This has been shown by experimentation in scorbutic guinea-pigs in whom wound contraction pro ceeded normally, despite the fact that resultant scars contain merely 15% as much collagen as do wounds in healthy animals.16 It has been further shown that a special contractile cell appears in contracting wounds. These cells, called “ myofibroblasts,” have charac teristics of both fibroblasts and smooth-muscle cells in that they exhibit both collagen synthesis and contrac tility. Myofibroblasts are common in granulating wounds, but are not found in sutured wounds. It is not yet known what determines whether a precursor cell will develop into a myofibroblast or into an ordinary fibroblast. Myofibroblasts have many characteristics that dif ferentiate them from fibroblasts of normal tissues.17 Morphological differences are numerous indentations in the nucleus and fibrillar protein within the cyto plasm of myofibroblasts. The fibrils measure approxi mately 40-80 angstroms in width and they lie parallel to the long axis of the cell. They may be selectively labeled with anti-actin antibodies derived from pa tients with chronic hepatitis who form such an tibodies.18 Myofibroblasts also develop cell-to-cell and cell-to-stroma connections. They contract when stimu lated with various agents that excite smooth muscle. When treated with smooth-muscle relaxants, their con traction is inhibited.19 It is likely that myofibroblasts are responsible for wound contraction in humans.
REFERENCES 1. Winter, G. D. Epidermal regeneration studied in the domestic pig. In: Maibach, H. I., and Rovee, D. T., eds. Epidermal Wound Healing. Chicago, Year Book Medical Publishers, 1972. 2. Grillo, H. C., and Gross, J. Collagenolytic activity and epithelial-mesenchymal interaction in healing mammalian
wounds. J. Cell Biol. 23:39A, 1964. 3. Eisen, A. Z., and Gross, J. The role of epithelium and mesen chyme in the production of a collagenolytic enzyme and a hyaluronidase in the anuran tadpole. Dev. Biol. 12:408, 1965. 4. Brown, S. I., and Weller, C. A. Cell origin of collagenase in normal and wounded corneas. Arch. Ophthalmol. 83:74, 1970. 5. Grillo, H. C., and Gross, J. Collagenolytic activity during mammalian wound repair. Dev. Biol. 15:300, 1967. 6. Leibovich, S. J., and Ross, R. The role of the macrophage in wound repair: a study with hydrocortisone and antimac rophage serum. Am. J. Pathol. 78:71, 1975. 7. Ödland, G., and Ross, R. Human wound repair. I. Epidermal regeneration. J. Cell Biol. 39:135, 1968. 8. Krawczyk, W. S. A pattern of epidermal cell migration during wound healing. J. Cell Biol. 49:247, 1971. 9. Gabbiani, G., and Ryan, G. B. Development of a contractile apparatus in epithelial cells during epidermal and liver regener ation. J. Submicr. Cytol. 6:143, 1974. 10. Weiss, P. Specificity in growth control. In: Butler, E. G., ed. Biological Specificity and Growth. Princeton, Princeton Univ. Press, 1955, p. 195. 11. Martinez, I. R. Fine structural studies of migrating epithelial cells following incision wounds. In: Maibach, H. I., and Rovee, D. T., eds. Epidermal Wound Healing. Chicago, Year Book Medical Publishers, 1972, pp. 323-342. 12. Bullough, W. S., and Laurence, E. B. Mitotic control by inter nal secretion: the role of the chalone-adrenaline complex. Exp. Cell Res. 33:176, 1964. 13. Van Winkle, W., Jr. The fibroblast in wound healing. Surg. Gynecol. Obstet. 124:369, 1967. 14. Ross, R. The fibroblast and wound repair. Biol. Rev. 43:51, 1968. 15. Peacock, E. E., and Van Winkle, W. Wound Repair. Philadel phia, W. B. Saunders Co., 1976. 16. Abercrombie, M., Flint, M. H., and James, D. W. Wound con traction in relation to collagen formation in scorbutic guineapigs. J. Embryol. Exp. Morphol. 4:167, 1956. 17. Ryan, G. B., Cliff, W. J., Gabbiani, G., et al. Myofibroblasts in human granulation tissue. Hum. Pathol. 5:55, 1974. 18. Hirschei, B. J., Gabbiani, G., Ryan, G. B., and Majno, G. Fibroblasts of granulation tissue: immunofluorescent staining with antismooth muscle serum. Proc. Soc. Exp. Biol. Med. 138:466, 1971. 19. Majno, G., Gabbiani, G., Hirschei, B. J., Ryan, G. B., and Statkov, P. R. Contraction of granulation tissue in vitro: simi larity to smooth muscle. Science 173:548, 1971.
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