Foso de ordeña.

Here we focus on the four phases of wound closure; 1) retraction, 2) a lag or plateau phase, 3) contraction and 4) late retraction as illustrated in Figure 2.6. However, not all of these phases are observed in any given experiment.

Retraction

Immediately following injury the wound boundary retracts, increasing the size of the wound (Billingham and Medawar, 1955). Early retraction is attributable to the im- balance in forces across the wounded and unwounded domains. Cells exert tensile stresses on their surrounding ECM. When an injury results there are no contractile cells inside the wound, and there is nothing to counter the cell traction forces and residual stresses in the tissue proximal to the wound (Silver et al., 2003), and the wound expands. McGrath and Simon (1983) report anywhere from a 30 − 60% re- traction in rat wounds, while Catty (1965) observes approximately a 30% retraction in human dermal wounds. This will be discussed further in Chapters 3 and 4.

0 10 20 30 40 50 60 0 0.5 1 1.5 2 (1) (2) (3) (4) Time (days) R elativ e Ar ea

Figure 2.6:The phases of contraction as illustrated in the experimental data obtained from McGrath and Simon (1983) for the contraction of small square wounds on rats. Arrows show the respective phases of wound contraction with (1) indicating retraction, (2) the lag phase, (3) contraction and (4) late retraction. Dashed curves help delineate the changes between retraction and the lag phase, and between the lag phase and contraction.

Plateau Phase

Following wound expansion, there is a lag phase, typically of 5 − 10 days before rapid contraction ensues, and the wound decreases in size (Grillo et al., 1958; McGrath and Simon, 1983).

Experimental results from rats and rabbits seem to indicate that, in some instances, there is no initial retraction (Abercrombie et al., 1954; Billingham and Medawar, 1955; Welch et al., 1990), or that no lag phase is observed and the wound begins contracting immediately (Abercrombie et al., 1954; Billingham and Medawar, 1955; Billingham and Russell, 1956; Doillon et al., 1987). There may be a simple possible explanation. In the experiments described by Abercrombie et al. (1954), Billingham and Medawar (1955) and Welch et al. (1990), the first wound contraction measure- ment occurs 3 − 5 days post wounding. Since the entire expansion and lag phase can be observed within this period (see for example McGrath and Hundahl (1982); McGrath and Simon (1983) and McGrath and Emery (1985)), it is not surprising that they would be absent from these results.

Alternatively, there may be some variation in the amount of initial retraction or the length of the lag phase across different species. Kennedy and Cliff (1979) observed

wound contraction in rats, rabbits and guinea pigs. Their rabbit wounds showed no retraction. Instead, a significant wound size reduction of 40% occurred over the first day post wounding. Interestingly, a lag phase followed this initial contraction, lasting approximately two days before contraction continued, although at a much slower rate. Kennedy and Cliff (1979) report that in their experiments on rat wounds they observed no early retraction, and only a minor lag phase before contraction occurred. Guinea pig wounds, on the other hand, showed about a 20% increase in wound area over the first day post wounding, before a lag phase of approximately one day which was then followed by contraction. Indeed, guinea pig wounds ap- peared to contract the slowest of the three species. Clearly there may be different mechanisms acting across different species to effect closure. Moreover, this illus- trates the difficulty in comparing wound contraction results across species, and the need for careful measurements of wound area, particularly early in the repair pro- cess.

Contraction

This feature of wound healing occurs primarily in wounds healing by second inten- tion. The exact mechanism by which contraction occurs is unknown, although sev- eral relevant features have been observed:

1. Fibrin contracts, arresting haemorrhage. Platelets or fibroblasts then contract the fibrin network further by exerting a force as they squeeze the excess fluid out of the clot.

2. Myofibroblasts, the activated form of fibroblasts, exert a contraction force on the local environment.

3. Fibroblasts themselves are able to rearrange the local collagen network, creat- ing tension and contracting the region.

These observations are consistent with wound contraction experiments by Watts and colleagues who found that contraction is “essentially a cellular process affect- ing the edge of the wound” (Watts et al., 1958; Watts, 1960).

In animals, such as mice and rats, contraction has been observed to produce approx- imately 80% of wound closure (Levenson et al., 1965; Majno and Joris, 2004). This is largely due to the flexible nature of the panniculus carnosus, the skin organ that at- taches the dermis to deep fascia or bone. However, this organ is highly specialised in human tissue, so that the tethering between the dermis and fascia is stronger, re- sulting in less flexible tissue, with wound contraction accounting for approximately a 20 − 30% reduction in wound area (Rudolph, 1979; Farahani and Kloth, 2008). Con- sequently, whilst contraction does aid in wound closure, it is not the primary means by which closure is observed in humans. Nonetheless, severe contraction is a com- mon problem in burns and around joints where it can result in excessive pulling of the skin around the wound, deformities and reduced movement (Majno and Joris, 2004).

As mentioned in Section 2.1.1, in both animal and human wounds, fibroblasts con- tinue to remodel the extracellular matrix, restoring mechanical strength to at best 70%that of normal tissue (Singer and Clark, 1999). This is an ongoing process, with only 20% of a scar’s final strength acquired during the first three weeks post injury. The rate at which tensile strength is returned is slow, relying upon continual contrac- tion and fibroblast activity (Cotran et al., 1999; Singer and Clark, 1999).

Fibroblasts and myofibroblasts together effect wound contraction. Fibroblasts pri- marily remodel the collagen network, whilst myofibroblasts exert contractile forces on the collagen fibres themselves. Grinnell and co-workers have studied the rela- tionship between fibroblasts and mechanics in fibroblast populated collagen gels extensively (Guidry and Grinnell, 1985; Grinnell, 1994, 2000; Grinnell et al., 2003; Grinnell, 2003, 2008). In some instances, lattices were shown to contract to a tenth of their original size in the absence of myofibroblasts (Guidry and Grinnell, 1985; Tamariz and Grinnell, 2002; Tomasek et al., 2002). The majority of this contraction was found to persist following inducement of fibroblast apoptosis. Guidry and Grin- nell (1985) showed that fibroblasts are able to contract the gels through physically rearranging the existing fibres, and conclude that the ability of fibroblasts to remodel the collagen architecture is critical to effecting wound closure through contraction. Indeed, Murphy et al. (2011a) developed a model to investigate wound contraction

which found that the extent of fibroblast and myofibroblast remodelling was critical in determining the magnitude of wound reduction (see Chapter 3 for a brief discus- sion).

Late Retraction

Late retraction is thought to be associated with continued remodelling of the colla- gen network, inducing a relaxation in the scar tissue. In some of the aforementioned experimental papers no late retraction was observed (Abercrombie et al., 1954; Watts et al., 1958; Kennedy and Cliff, 1979). Most experiments were terminated at about 3−4weeks following injury (Watts et al., 1958). Catty (1965) however tracked wounds up to six months post wounding in order to observe scar progress and found that late retraction had occurred. Hence, it is entirely possible that under normal cir- cumstances, all wounds experience late retraction, but that the experiments are not conducted for long enough to observe this increase in wound size.

Experimental results on rats reported by McGrath and Simon (1983) and on rabbits by Luccioli et al. (1964), however, both display late retraction within the first 30 days following injury. Catty (1965) reported that in humans this occurred sometime after the first 16 days, at which time daily wound area measurements ceased. Nonethe- less, records of the same wounds six months post-wounding revealed significant secondary retraction (this will be discussed in more detail in Chapter 4). Since scars such as hypertrophic scars may actually undergo further contraction (and therefore no late retraction), and numerous experimental results report no late expansion, it is possible that this secondary retraction is not a universal feature of wound repair.