The Effects of TGF-b3 Modulation on Scar Tissue Formation in the Pig

Nathan D. Schwade, PhD, James J. Fowler, MD, Joseph Leach, MD, Department of Otolaryngology - Head and Neck Surgery, University of Texas Southwestern Medical Center, Dallas, Texas

Disclosures

Wounds. 2000;12(2) 

In This Article

Discussion

This study confirms the observation that TGF-b3 plays a role in scar modulation, and verified the effect in tissue more closely resembling that of a human (porcine skin). The recent development of specific antibodies to TGF-b3 allows modification of the influence of TGF-b3 on scar formation. This work demonstrated the modulating effects of TGF-b3 and its antibody on the breaking strength of scars in the animal model. TGF-b3 antibody was able to significantly increase the breaking strength in pig wounds compared to TGF-b3 isomer and to controls. This statistically significant increase in the breaking strength was present in wounds both 7 and 14 days post injury when compared to controls. Histology confirmed the presence of more fibroplasia and inflammation in these specimens. The lowest wound breaking strength was seen in wounds injected with TGF-b3 isomer, and as expected, less fibroplasia and inflammation was noted in these wounds.

Growth factors modulate the activity of cells in a paracrine, autocrine, or endocrine fashion. After binding to surface receptors on the cell membrane, growth factors initiate a signaling cascade within regulatory proteins inside the cell which results in a biologic activity.[17] Much of the early work regarding TGF-b was derived from factor that was extracted from platelets, which contain primarily TGF-b1 and b2 with relatively little TGF-b3. Matrix molecules such as fibronectin or hyaluronic acid retain or bind growth factors and act as growth factor reservoirs.[18] TGF-b1 is produced by many cells -- especially platelets, leukocytes and bone; nearly all cells respond to it in some way. TGF-b1 acts as a potent chemoattractant for monocytes, macrophages, neutrophils, lymphocytes, and fibroblasts. TGF-b1 stimulates release of other growth factors and induces its own autoexpression.[12] TGF-b3, on the other hand, is not present in great amounts in either fluids or tissues.[19]

Since its identification in 1988, the role of TGF-b3 in scarless wound healing has been gradually delineated.[10] Levels TGF-b1 and b2 increase rapidly at day 1 post wounding, but TGF-b3 only increases a few days later when TGF-b1 levels are decreasing.[20] TGF-b3 remains in the wound until 14 days post wounding.[20] TGF-b3 downregulates the production of TGF-b1 and vice versa.[14] TGF-b3 may act with different cell membrane receptors.[14] Unlike other mammalian isomers of TGF-b, TGF-b3 reduces the monocyte and macrophage profile and blocks fibronectin, collagen I, and III deposition.[15] Application of TGF-b3 to a wound produces an improved architecture of the neodermis.[15]

It has been shown that manipulation of the ratios of TGF-b subtypes alters scarring and fibrosis.[20] If the ratio of TGF-b3 to TGF-b1 and b2 is high, wounds heal without scarring. Antibodies to TGF-b1 and b2 work to reduce scarring, but the antibodies must be given together at time of wounding or shortly thereafter.[14] It has been discovered that treatment with the antibody to TGF-b1 only marginally reduces scarring, and that treatment with antibody to TGF-b2 does not alter scarring at all. If antibodies to all three TGF-b subtypes are given, no reduction in scarring is seen.[15] Reduction in scarring therefore depends on the ratio of TGF-b3 to b1 and b2 early in the wound healing cascade. Steroids selectively increase the TGF-b3 to b1 ratio, and this may be responsible for the scar improvement effect seen with glucocorticoids.[7] Conversely, it may be expected that lowering the ratio of TGF-b3 to TGF-b1 and b2 by administration of the blocking antibody to TGF-b3 would result in increased fibrosis and scarring. Our results bear this hypothesis out.

The optimum timing and method of application of growth factors are being gradually elucidated. The timing of application may not be as critical as once thought, however. For instance, even though circulatory half life of TGF-b1 is less than five minutes, the same effect is noted whether TGF-b1 is given 24 hours before wounding, at the time of wounding, or four hours later.[21] Application of growth factors to certain wounds may not be effective due to high levels of proteases within the wounds.[20] Injection is therefore probably better than topical application. Intracutaneous injection of TGF-b3 and its antibody proved to be effective at the time of wounding in our study.

Of the three phases of wound healing (substrate, proliferative, and remodeling), TGF-b seems to produce its most important effects during the first or substrate phase. The substrate phase lasts three to four days, and is so named because at this time cellular interactions within the wound milieu prepare for other events. This is a time for migration of monocytes, fibroblasts, and keratinocytes into the wound.[22] During this phase, TGF-b enhances extracellular matrix synthesis, reduces collagenase production, and inhibits cellular proliferation.[23] Monocytes are attracted into the wound by a number of different molecules, among them TGF-b. TGF-b stimulates fibroblast proliferation and collagen synthesis.[24]

The second or proliferative phase of wound healing begins during the third or fourth days and lasts 10 to 14 days. During this time, fibroblasts move into the wound along a fibronectin network. In the proliferative phase, TGF-b increases the formation of fibronectin, collagen, and protease inhibitors.[24] Once the monocytes have evolved into activated macrophages, they perform a number of functions, the foremost of which is wound debridement.[25] The macrophages themselves begin to secrete growth factors (including TGF-b) that stimulate neovascularization and fibroblast activation and chemotaxis.[26] Granulation tissue, consisting of new blood vessels, macrophages, fibroblasts, and loose connective tissue, begins forming at four days post wounding.[18,27] After two to three weeks, the total amount of collagen reaches a maximum within the wound, and the third, or remodeling phase begins. During this time, the original collagen gel is replaced by a basket weave type of matrix.[28] This causes a progressive increase in breaking strength in the wound for up to a year.[25] It is unclear what role the subtypes of TGF-b play in the second and third phases of wound healing. It is also true that wound healing occurs more quickly in lower mammals than in man. In the rat, for instance, wounds are often invisible by two weeks. In our study, wounding appeared to be complete in the pig by 14 days.

Cutaneous healing may be problematic for many reasons. Poor wound healing is not only associated with displeasing aesthetic results, but also with functional disabilities and increased healthcare costs. Unregulated or overabundant scarring may produce contractures, keloids, or hypertrophic scars. Keloids are a widespread occurrence in populations of African, Asian, or Native American heritage. Keloid fibroblasts have been noted in vitro to produce 12 times more collagen when stimulated with TGF-b than normal fibroblasts and four times more than hypertrophic scar fibroblasts.[3] The ratio of type I to type III collagen is elevated from a normal of 3:1 in normal tissue to 18:1 in keloidal tissue.[29] Blocking the activity of TGF-b1 and b2 while enhancing the activity of TGF-b3 may represent effective ways of controlling the growth of keloids and hypertrophic scars.

Other wounds may heal slowly or not at all, as evidenced in the chemotherapy patient, the patient post-radiation therapy, or the patient with venous stasis disease. Macrophage and fibroblast functions decline with age.[30] Impaired wound healing in the elderly is hypothesized to result either from lack of growth factors or to a decrease in the response of tissue to growth factors. Radiation causes significant reductions in wound bursting strength, presumably by its effect on macrophages and fibroblasts.[31] Chronic wounds may either lack growth factors or contain inhibitory substances.[17] In laboratory animals, growth factors have improved wound breaking strength and wound healing time.[32,33,34,35] In general, TGF-b is particularly effective at improving wound contraction and breaking strength. Supplemental TGF-b has been shown to reverse much of the healing deficit seen in rats treated with adriamycin.[9] TGF-b in low doses can increase wound strength in irradiated skin.[31] Antibodies to TGF-b3, by lowering the ratio of TGF-b3 to TGF-b1 and b2, might be an effective method for improving the healing of chronic wounds.

Research has recently focused on methods to alter the hormonal milieu in order to minimize scarring without sacrificing wound strength. Indeed, abundant scarring and fibroplasia do not necessarily connote high breaking strength -- a mature scar is only about 80 percent as strong as the original tissue.[25] It has been demonstrated that adult wounds heal with scar tissue formation while fetal wounds can heal without any observable scar. TGF-b is largely absent from fetal wounds.[36] Paradoxically, as the fetus develops, the number of TGF-b receptors on fibroblast surfaces decreases.[37] It is unclear which TGF-b isomers are active in fetal tissue and what response they induce. Although scarless fetal healing has many advantages, it may be that the fibrotic scarring seen in adults represents the best adaptation of the organism to a contaminated, less controlled environment.

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