-
Collagen & the Wound Healing Process
A wound is a trauma to any of the tissues of the body by either direct injury or as a result of surgical incisions. Wound healing is a complex continuous process that may be divided into several overlapping stages. Collagen is a unique material common to all of these stages.
Collagen proteins are the most abundant family of proteins found in the human body. The function of most major body systems and organs in some way incorporates collagenous structures. Furthermore, approximately 70% of the dry weight of the skin is collagen. As a result, use of collagen in wound healing has drawn tremendous interest from scientist in recent years. Due to the complexity of collagen’s involvement and interaction with many chemical and biological factors, research on collagen’s role in wound healing is incomplete. Conclusions must be drawn from a number of sources.
Collagen Helps in Hemostasis
Hemostasis (stoppage of bleeding) is the first step in the wound process. Blood platelets and soluble clotting factors play a major role as intravascular hemostatic factors (Shoshan, 1981). Platelets not only initiate hemostasis, they also release a number of biologically active substances including extracellular matrix molecules such as fibronectin, fibrinogen, thrombhospondin and some growth factors (Clark, 1988). Collagen is a very efficient hemostatic agent because platelets adhere to collagen, swell and release substances that initiate hemostasis (Hovig, 1968; Zucker & Borrelli, 1962). Furthermore, collagen can provide both positive and negative active polar sites. Collagen is also a molecule of sufficient size for platelet aggregation (Wilner, et al., 1971). The platelets adhere to collagen more closely than they adhere to the undigested subendothelial surface (Baumgartner, et al., 1980). Various other studies have demonstrated that collagen is an effective hemostatic agent (Abbott, 1974, Beachey et al., 1979, Sanborn, 1993).
Hemostasis is followed by vasoconstriction and vasodilatation (Gogia, 1992). Vasoconstriction occurs for about 5-10 minutes after injury and slows down blood loss in the affected area (Gogia, 1992). During vasodilatation, the non-injured vessels become more permeable and leak hormones, plasma proteins, electrolytes, antibodies, fluid, and polymorph nuclear leukocytes (Bryant, 1987). Vasodilatation continues for approximately one hour after injury.
Collagen Assists Wound Debridement
Vasoconstriction and vasodilatation are followed by debridement or cleaning of the wound. During this stage, polymorph nuclear leukocytes and macrophages rapidly accumulate at the site of injury. Neutrophils infiltrate the wound and rid it of bacteria (Clark 1988). Monocytes, as macrophages, phagocytize the wound and scavenge tissue debris (Albritton, 1991; Clark, 1988; Mosiello, et al., 1994). A reduction in the total number of macrophages present at the wound site was found to delay debridement (Leibovich & Ross, 1975). Debridement is an essential prerequisite to fibroblast proliferation (Leibovich and Ross, 1975).
Collagen Enhances Granulation and Angiogenesis
The wound begins to rebuild once the debridement process has concluded. Macrophages release cytokines and hydrolytic enzymes further modifying the growth factors at the wound site (Kirsner and Eaglestein, 1993). This may result in the formation of granular tissue. With the release of various chemical substances from the macrophages, there is a rapid outburst of fibroplasia and angiogenesis (Knighton, et al., 1983). Granulation tissue consists of an array of macrophages, fibroblasts, and neovasculature embedded in a matrix of collagen, fibronectin and hyaluronic acid (Clark, 1988).
Angiogenesis into the wound bed occurs concomitantly with the ingrowth of fibroblasts and deposition of extracellular matrix (ECM) (Clark, 1988). During angiogenesis, new capillary buds grow on the preexisting small vessels (Kirsner & Eaglestein, 1993). If a wound is closed, the new capillaries meet similar cells from the other side and a network is formed across the entire wound (Hunt, 1988). If the wound is open, the new capillaries form granulation tissue (Schoefl, 1963). The growth of new capillaries depends on the support available at the wound site (migration of fibroblasts and production of new collagen). For their survival, the synthesis of new tissues depends on the nutrition available, and hence on the new capillaries present. A very delicate balance exists between the formation of a fibroblastic network and growth of new capillaries. It is unclear if external collagen plays a direct role in the growth of new vessels, but collagen does play an instrumental role in providing the necessary support.
Collagen Enhances Fibroblastic Activity
The fibroblastic phase provides strength to the wound and lasts approximately four days to 20 days in duration (Bryant, 1987). Fibroblasts are the primary cell type involved in the fibroblastic phase. The origin of fibroblasts is not yet fully understood. Fibroblasts may originate from surrounding connective tissue, from the keratinocytes, from the perivascular sheaths surrounding the blood vessels (Hadfield, 1963), or from the blood monocytes undergoing metaplastic transformation (Helpap & Cremer, 1972). Whatever the origin, wound fibroblasts proliferate and migrate during the healing process. Active migration of fibroblasts has been shown in both in vitro studies and in vivo studies (Postlethjwaite et al., 1978). The activity of fibroblasts governs the restoration of tissue continuity and the strengthening of ensuing repair tissue (Ross, 1968). Fibroblasts adhere effectively to collagen (Elsdale & Bard, 1972; Kleinman, et al., 1978; Pearlstein, 1976). It has been shown that fibroblasts possess membrane receptors with specific binding sites for collagen (Chiang, et al., 1978; Engvall, et al., 1977). Chemotactic attraction of fibroblasts to type I, II, and III collagen and collagen-derived peptides, and binding of chemotactic collagen-derived peptides to fibroblasts has also been demonstrated (Chiang, et al., 1978; Postlethwaite, et al., 1978). It has been reported that collagen attracts fibroblasts in cell culture and causes directed migration of cells (Dunn & Ebendal, 1978; Tomaseck, et al., 1982). Therefore, collagen is involved in one of the major events of the wound healing process, the migration of fibroblasts.
Collagen also supports fibroblast activity due to its ability to interact with fibronectin. Fibroblast replication, adhesion, spreading and migration are reported to be stimulated by fibronectin (Clark, et al., 1982; Doillon, et al., 1984; Doillon, et al., 1986). Fibronectins act as adhesive proteins that bind cells to other cells or to substrata (Yamada, et al., 1976). The ability of fibronectin to mediate cell attachment depends on its interaction with other molecules (Ruoslahti & Engvall, 1980). The binding of fibronectin to native collagen and the crosslinking of fibronectin to collagen has been demonstrated (Kleinman, et al., 1978; Mosher, et al., 1979; Ruoslahti & Engvall, 1974; Vaheri & Mosher, 1978). The binding results in a matrix that can support cell adhesion and provide tissue integrity (Kleinman, et al., 1978). Fibronectin’s ability to bind to collagen may ultimately influence the length and width of collagen fibers since fibronectin influences the rate of fibrillogenesis (Grinnell, et al., 1981; Kleinman, et al., 1981).
Collagen, along with fibronectin, is closely involved in cell adhesion and attachment under both normal and fibrotic conditions (Hewitt, et al., 1980; Kleinman, et al., 1979 Vaheri & Mosher, 1978; Varheri, et al., 1978). Collagen has specific sites for cell attachment proteins (Kleinman, et al., 1976; Kleinman, et al., 1978).
Collagen Assists Reepithelialization
When skin integrity is disrupted, reepithelialization begins within hours of the injury (Clark, 1988). Epithelial cells proliferate at the wound edges and migrate across the wound bed (Alvarez, 1989; Bryant, 1987). Wound debris, eschar, and blood clots may obstruct the epithelialization process (Bryant, 1987). Two important factors in the re-establishment of skin integrity following an injury are the cohesion of epithelial cells (keratinocytes) and the adhesion of the epidermis to the dermis (Shoshan, 1981). Collagen sheets were shown to support keratinocyte growth during in vitro studies and in vivo studies (Morykwas, et al., 1989). One study has reported a selective adherence of basal cells to a collagen substrate (Stanley, et al., 1968). Another study has reported attachment and differentiation of epidermal cells on collagen (Murray et al., 1979). Mammary epithelial cells cultured on collagen membranes in a medium containing insulin, hydrocortisone, and prolactin were found to maintain differentiation through one month in culture (Emerman and Pitelka, 1977). Conversely, cell cultures on plastic, glass or collagen gels formed a confluent epithelial sheet but lost secretory and myoepithelial specializations (Emerman and Pitelka, 1977). The effects of collagen-coated substrates in tissue culture on cell adherence, differentiation, and protein synthesis are further indications that collagen is an important factor in epithelial cell migration (Emerman and Pitelka, 1977, Morykwas, 1989; Reddi, 1976). Fibronectin and fibrin have been found to provide a provisional matrix for epidermal cell migration during wound reepithelialization (Clark, et al., 1982). Collagen supports differentiation and migration of epidermal cells and may also play a role in reepithelialization because of its ability to bind with fibronectin.
Scar related activities occur during the maturation or remodeling phase of wound healing. It begins approximately 20 days after the injury and frequently occurs for more than a year (Bryant, 1987). In an ideal case of wound healing, lost or damaged tissue completely regenerates with no scar formation. Scarring results from the accumulation of collagen at the wound site and is dependant on the rate of collagen synthesis as well as the rate of collagen degradation. Electron microscopy revealed that collagen in rat wounds was in the form of irregular masses 100 days after an injury (Forrester, et al., 1969).
Collagen has an ability to bind collagenase inhibitors (Vater, et al., 1978; 1979) reducing the amount of collagen degradation. Keratinocytes grown on type I collagen showed a significantly higher amount of collagenase as compared to keratinocytes grown on basement membrane proteins (Sudbeck, et al., 1994). Keratinocytes themselves play a major role in the degradation of extracellular matrix (Saarialho-Kere, 1993). Keratinocytes migrate on type I collagen and can be grown on collagen sheets (Morykwas, et al., 1989; Saarialho-Kere, 1993; Sudbeck, et al., 1994). Thus, collagen can regulate degradation of the extracellular matrix by controlling collagenase activity and by helping in growth and differentiation of keratinocyte cells.
Studies performed on white guinea pigs showed the amount of collagen in the wound increased rapidly from five days to eight days, but decreased thereafter (Grillo & Gross, 1967). Removal of excessive collagen is initiated by collagenase liberated from granulocytes (Lazarus, et al., 1968), macrophages (Robertson, et al., 1972), or from epithelium and mesenchyme (Grillo & Gross, 1967). Collagenase is specific to the collagen molecule and is essential to successful remodeling (Parks, 1995). Collagenase cleaves collagen molecules into two specific pieces, making collagen susceptible to degradation by other proteases (Cortivo, 1995; Jefferey, 1995; Lazarus, 1968; Robertson, et al., 1972). Cleavage is preceded by activation of the collagenase by other proteases (Vater, et al., 1978).
Collagen’s interaction with collagenase inhibitors is thought to be responsible for a reduced scar size following third degree burns in guinea pigs as well as for reduced fibrous adhesions (excess scar tissue) following tendon surgery in chickens (Porat, et al., 1980). It may be noted that presence of a scar may be a result of lack of organization of collagen within the wound and not due to a lack of collagen formation.
Conclusions: Collagen as a Biomaterial
Research indicates that collagen dressings are effective as wound healing agents because they (i) stop bleeding by interacting with blood platelets, (ii) debride the wound by attracting monocytes, (iii) deposit new fibers by helping in production, adhesion, spreading and migration of fibroblasts, (iv) reepithelialize the wound by supporting growth, attachment and differentiation of epidermal cells, and (v) reduce scar by size by controlling collagenase activity and by playing a role in orientation and organization of fibers.
Collagen’s biological and physical properties and its role in each phase of the wound healing process indicate specific advantages over traditional dressing methods, growth hormones, or biological coverings.
Stoop (1970) summarized his experiences using collagen sponges in the treatment of pressure sores. The following was observed after collagen sponge application:
- 1. Wounds were clean and bacterial infection was retarded.
- 2. Wound secretion and drainage were reduced.
- 3. Improvement in the formation of new granulation tissue was noted.
- 4. Undermined edges of the pressure sores were closed.
- 5. Increased production of granulation tissue.
- 6. No contractures in closed wounds.
- 7. Moist pressure sore fissures showing no tendency to heal were closed.
- 8. No immunological reactions toward collagen were noted.
- 9. General patient condition improved.
A number of other researchers found collagen to be a good biomaterial for the following reasons:
A significant increase in the production of fibroblasts was reported when collagen sponge dressings were used with hyaluronic acid and fibronectin (Doillon & Silver, 1986; Doillon, et al., 1988). Collagen sponges have a large capacity to bind fluid and this hydrophilic property may be important in encouraging fibroblast permeation since hydrophobic materials resist invasion (Burton, et al., 1978). Morphological studies suggest that collagen-based wound dressings enhance the deposition of oriented, organized fibers by attracting fibroblasts and causing a directed migration of cells (Doillon, et al., 1984). Collagen dressings were found to have firm adhesion to wounds, aid in the uptake and bio-availability of fibronectin, help preserve leukocytes, macrophages, fibroblasts, and epithelial cells between collagen fibrils, and assist in the maintenance of the chemical and thermostatic microenvironment of the wound (Palmieri, 1992).
In conclusion, only collagen serves as the most important common element in all stages of wound healing. Collagen plays a key role in regulating the arrival and activity of multiple types of cells involved in tissue repair, regeneration and wound healing.
