Fetal Wounds Have a Minimal Inflammatory Response

A major difference between fetal and adult wounds is that fetal wounds exhibit a minimal inflammatory response (McCallion and Ferguson, 1996; Yang et al., 2003; Ferguson and O'Kane, 2004), suggesting that as it matures during development, the immune system response to injury suppresses regeneration in favor of scar tissue formation. Platelets are few in number in the fetal wound. Only small numbers of platelets, neutrophils, and macrophages are present up to 18 hr after wounding (Cowin et al., 1998). All types of macrophages were eventually recruited to the fetal wound, but their numbers and persistence were much lower than in adult wounds. These observations suggest that the types and proportions of growth factors and cyto-kines associated with the inflammatory environment are different in fetal wounds compared to adult wounds.

Consistent with this idea, there is a much lower level of PDGF, TGF-pi and 2 and their receptors in unwounded and wounded fetal rat skin (Whitby and Ferguson, 1991; Sullivan et al., 1993; Durham et al., 1989; Ferguson and O'Kane, 2004; Chen et al., 2005). Conversely, TGF-P3, which is synthesized by keratino-cytes and fibroblasts, is low in adult wounds, but is high in fetal wounds (Ferguson and O'Kane, 2004; Chen et al., 2005). In adult wounds, PDGF induces the persistent expression of IL-6 by fibroblasts, which helps to maintain an environment that promotes production and deposition of fibrotic matrix. Fetal wounds also express IL-6, but this expression rapidly disappears (Liechty et al., 2000).

The negative influence of the inflammatory response on regeneration has been further established by experiments in which the molecular composition of the wound environment is manipulated. Placing pellets of bacteria in PVA sponges subcutaneously in rabbits elicited an acute inflammatory response, including recruitment of neutrophils and large numbers of leukocytes that initiated an adultlike fibrosis (Frantz et al., 1993). Adding IL-6 to fetal wounds induced scarring, whereas an ade-noviral construct overexpressing IL-10, which decreases the production of IL-6, reduced inflammation and scarring when applied to adult mouse wounds (Leichty et al., 1998, 2000). Treating fetal rat wounds with exogenous TGF-P1 induced scarring (Krummel et al., 1988). TGF-P1 decreased endogenous MMPs and increased TIMPs, which would increase collagen accumulation and scarring (Soo et al., 2001). At high concentrations, PDGF also caused a shift to a more adultlike pattern of repair in fetal skin wounds (Haynes et al., 1990). Conversely, reducing the levels of TGF-P1 and 2, but not PDGF or FGF, in vivo or in vitro by application of neutralizing antibodies reduced scarring in adult rat wounds (Shah et al., 1994, 1995; Houghton et al., 1995). Reduction in scarring was enhanced only by applying the antibodies immediately after injury. This suggests that TGF-P1 and 2 exert early inflammatory effects in adult wounds that carry through the whole molecular cascade of wound repair events and that intervention must be done prior to the point where this cascade is easily able to withstand perturbation (O'Kane and Ferguson, 1997; Shah, 2000; Ferguson and O'Kane, 2004).

Addition of exogenous TGF-P3 to adult wounds at levels found in fetal wounds resulted in reduced or absent scarring (Shah et al., 1995) (figure 2.7). Wounds in the skin of fetal TGF-P3 null mice showed the adult repair response. Fetal fibroblasts of these mice exhibited slower migration in collagen gels, an effect that could be rescued by treatment with exogenous TGF-P3, but not by TGF-P1 or 2 (Ferguson and O'Kane, 2004). These results indicate that the proportion of TGF-P3/ P1/P2 is critical to determining whether dermal tissue will regenerate or scar in response to injury. Finally, fetal wounds may contain inhibitors of TGF-P, such as decorin and a2-macroglobulin (Yamaguchi et al., 1990; Danielpour and Sporn, 1990).

There may be species and locational differences in the composition of the fetal versus adult wound environment and in the environments of adult wounds. For example, Longaker et al. (1994) reported that macrophages were recruited into wounds in fetal sheep skin and that the concentration TGF-P2 was higher in the fetal wound than in the adult wound. Interestingly, adult mammalian saliva contains high levels of TGF-P2, yet oral lesions in adults heal with little scarring, suggesting that inhibitors of TGF-P could play a role in minimizing scarring.

Most studies comparing adult and fetal wounds have been on incisional wounds. However, studies on fetal sheep indicate that deep dermal burn wounds heal without scarring as well, whereas they heal with scarring in lambs (Fraser et al., 2005). This difference is associated with only a slight increase in TGF-P1 after wounding in the fetus, but a massive increase in the lamb that does not begin to fall off until 21 days postinjury and has not reached the control value even by day 60.

Some studies suggest that, in addition to differences in the inflammatory response, there are intrinsic differences between fetal and adult fibroblasts that are the result of differentiation independent of the maturation of the immune system. Secondary fibroblasts derived from primary cultures of human dermal fibroblasts fall into seven types, based on morphology and patterns of protein synthesis, that are a function of population doubling number and time kept in secondary culture

Wound Culture Normal Values

FIGURE 2.7 Effect of treating an adult rat deep (1 cm) incisional wound with TGF-P3, viewed 84 days post-wounding. (a) surface view (top) and histology (bottom) of placebo-treated wound. Scar tissue is the result (arrows). (b) Surface view (top) and histology (bottom) of TGF-P3 treated wound. No scar is visible in the surface view and dermal histological organization is similar to that of normal skin. Reproduced with permission from Ferguson and O'Kane, Scar-free healing: from embryonic mechanisms to adult therapeutic intervention. Phil Trans R Soc Lond B 359:839-850. Copyright 2004, The Royal Society.

FIGURE 2.7 Effect of treating an adult rat deep (1 cm) incisional wound with TGF-P3, viewed 84 days post-wounding. (a) surface view (top) and histology (bottom) of placebo-treated wound. Scar tissue is the result (arrows). (b) Surface view (top) and histology (bottom) of TGF-P3 treated wound. No scar is visible in the surface view and dermal histological organization is similar to that of normal skin. Reproduced with permission from Ferguson and O'Kane, Scar-free healing: from embryonic mechanisms to adult therapeutic intervention. Phil Trans R Soc Lond B 359:839-850. Copyright 2004, The Royal Society.

(Bayreuther et al., 1988). This heterogeneity suggests the existence of a fibroblastic stem cell that gives rise to a seven-stage terminal cell lineage. It is possible that earlier parts of the lineage are capable of responding to injury with a regenerative response. Fetal fibroblasts may be those that comprise the earlier part of the lineage. The transition from fetal to the adult pattern of wound healing might then be the result of differentiation of the majority of these cells to a terminal cell type that is unable to respond in a regenerative way.

Fetal fibroblasts were reported to have a unique phe-notype that differs from that of the adult fibroblast, including production of and response to growth factors, synthesis of matrix molecules, pericellular HA coats and antigen determinants (Moriarty et al., 1996; Ellis et al., 1997; Gosiewska et al., 2001). They maintained their regenerative response when grafted subcutane-ously into adult athymic mice, even though these mice heal by scarring (Lorenz et al., 1992; Lin et al., 1994). The transition from regeneration to scarring in inci-sional wounds of cultured 14-day mouse limb buds takes place autonomously, in the absence of immune cells and systemic factors (Chopra et al., 1997), and this presumably would involve transition of fibroblast molecular phenotype.

Nevertheless, the fact that changing the concentrations of growth factors and cytokines associated with inflammation can shift the nature of the injury response in both fetal and adult dermis strongly suggests that the wound environment is the major determining factor in the response. There may indeed be intrinsic differences between fetal and adult skin fibroblasts, but it appears that these differences can either be largely eliminated in either direction by the appropriate signaling molecules, or by the recruitment of fibroblasts that give a regenerative response. One type of experiment that could be done to investigate these possibilities is to examine the molecular changes in phenotype that might occur following treatment of fetal or adult fibroblasts in vivo or in vitro with the appropriate growth factor and cytokine combinations.

Other data lend support to these conclusions. Observations on tattoos show that the adult human dermis can respond to injury either by regeneration or scar formation, depending on the extent of the injury (Ferguson and O'Kane, 2004). Tatoo artists use needles to deposit patterns of ink particles in the dermis, which are then phagocytosed by fibroblasts. Despite the large total area injured, tattooed dermis heals without scarring, because each individual injury from the needle is small and does not evoke an inflammatory response. Tatoos can be removed by lysing the fibroblasts containing the pigment particles, which again creates numerous microwounds in the skin. These wounds, however, exhibit a minimal inflammatory response and heal without scarring. The microwounds of tattooing

FIGURE 2.8 Healing of incisional skin wounds in wild-type (a) and PU.1 null neonatal mice (b), 2-3 days post-wounding. The wild-type wound is covered by epidermis migrating beneath the scab. Below the epidermis is disorganized connective tissue with many inflammatory cells. This wound will form a scar. The PU.1 wound has a much better formed epidermis beneath the scab, and dermal tissue that appears similar to that of unwounded skin. Reproduced with permission from Redd et al., Wound healing and inflammation: embryos reveal the way to perfect repair. Phil Trans R Soc Lond B 359:777-784. Copyright 2004, The Royal Society.

FIGURE 2.8 Healing of incisional skin wounds in wild-type (a) and PU.1 null neonatal mice (b), 2-3 days post-wounding. The wild-type wound is covered by epidermis migrating beneath the scab. Below the epidermis is disorganized connective tissue with many inflammatory cells. This wound will form a scar. The PU.1 wound has a much better formed epidermis beneath the scab, and dermal tissue that appears similar to that of unwounded skin. Reproduced with permission from Redd et al., Wound healing and inflammation: embryos reveal the way to perfect repair. Phil Trans R Soc Lond B 359:777-784. Copyright 2004, The Royal Society.

regenerate, whereas incisional or excisional wounds of equal total volume heal by fibrosis! Tatoo wounds have been shown to have low levels of TGF-pi and higher levels of TGF-p3 (Ferguson and O'Kane, 2004).

PU.1 null mice lack a hematopoietic lineage transcription factor that results in the absence of both macrophages and neutrophils. These mice die within 24 hr after birth unless they are given a wild-type bone marrow transplant because they are prone to infection, showing the importance of these cells for phagocytic and bactericidal functions (Dovi et al., 2004). Antibiotics can be used to prolong the lives of neonate PU.1 mice. Martin et al. (2003) investigated excisional wound repair in PU.1 mouse neonates maintained on antibiotics. Not only were excisional wounds repaired in antibiotic-maintained PU.1-null mice at the same rate as wounds in wild-type mice, they were repaired by regeneration, not scar (Martin et al., 2003; Redd et al., 2004 figure 2.8.) Regeneration was associated with greatly reduced expression of IL-6 and TGF-P mRNA (Redd et al., 2004). These results suggest that PU.l-null mice do not undergo the transition from the fetal to adult wound healing response! Whether the absence of macrophages in these mice is causal to the failure to shift to the adult healing response is unknown. It would be interesting to compare wounded versus unwounded PU.1 null mice to learn how gene activity differs between the two in the absence of macrophages (Martin et al., 2003). We will encounter differences between fetal and adult wound healing in other chapters as well, some of which have been correlated with the absence or presence of an inflammatory response.

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