Becker_subbed.qxp 21/5/09 5:22 pm Page 80
Wound Management
Figure 1: Macroscopic Appearance of the Healing Process six hours after treatment, when treated wounds appear to have less
exudate and seem to be less red and swollen (see Figure 1). The
improvement in appearance of treated incisional lesions is most
obvious on days one and two when there is little gape, redness or
swelling compared with control lesions. In addition, the rate of
outgrowth of the leading-edge keratinocytes is significantly enhanced
by Cx43-asODN treatment, which results in wounds that re-
epithelialise twice as fast. Macroscopically, the final scars appear
thinner and flatter, and histological analysis shows late-stage
granulation tissue areas to be significantly reduced. Furthermore, they
contain finer and more highly interlaced collagen bundles than
normal scars, which tend to have thick, parallel collagen bundles.
Apparently, by targeting Cx43-mediated communication at the start of
the wound-healing process, we can overcome the lag phase before
healing starts and beneficially influence the whole cascade of
subsequent events. A more detailed analysis of the cell biology
behind these beneficial effects reveals changes to almost all aspects
of wound healing, including proliferation, migration, inflammation and
granulation tissue formation. Proliferation is significantly enhanced in
the nascent epidermis and granulation tissue.
10
Decreased production
of Cx43 in fibroblasts increases both proliferation and migration in the
early stages of wound healing. The inflammatory response also
S Incision wound scores T Excision wound scores
appears to be dampened down, resulting in the recruitment of
12 12
10 10
significantly fewer neutrophils to the wound site and surrounding
8 8
tissues. Neutrophils can cause secondary damage to the host tissue
ank score
6
ank score
6
4 4
as well as clearing out infection, so a reduction in their numbers has
ound r ound r
W
2
W
2 the potential to reduce secondary damage. Macrophages – which
0 0
6h 1d 2d 1d 2d 3d
come in after the neutrophils to clean up debris in the resolving
Sense
Time-points post-wounding Time-points post-wounding wound – are also significantly reduced in number. Following the
Antisense
invasion of a wound, the neutrophils and macrophages release pro-
A–J = pairs of treated and untreated incisional and excisional wounds; A–E = macroscopic
images of pairs of control; F–J = antisense-treated; A, F = full-thickness incisional lesions on
inflammatory cytokines and chemokines, which act powerfully on all
the backs of neonatal mice at six hours (h); B, G = one day; C, H = two days (d); D, I = seven
of the different cell types in the wound to amplify the inflammatory
days; E, J = 10 days post-wounding (pw); K–R = macroscopic images of pairs of control and
antisense-treated. Full-thickness, excisional lesions on the backs of adult mice at K, O = one
response. However, in wounds that have been treated with
day; L, P = two days; M, Q = seven days; N, R = 10 days pw.
Cx43–asODN, the reduction in leukocyte numbers is associated with
The histograms show the wound rank scores of connexin (Cx)-43 antisense-treated wounds
and control wounds in the neonatal incisional model (S) and the adult excisional model (T).
significantly reduced expression of the representative chemokine
Each bar represents the mean wound rank score standard error of mean (SEM) from eight
CCL2 and cytokine tumour necrosis factor-alpha (TNF-α) at days two
animals. A significant difference was found between Cx43 antisense-treated wounds and
control wounds when these were analyzed with the Wilcoxon Signed Rank test.
and seven, respectively. It has long been suggested that a robust
*p value. Six hours (h), p=0.028; one day, p=0.001; two days, p=0.012. Scale bars in
inflammatory response is required to trigger healing, but this may not
A–J = 1mm, K–R = 0.5mm.
Figure reproduced with permission from Qiu C, et al.
9 be so. Crucially, wounds heal at a normal rate in the PU.1 knockout
mice, which lack most of the leukocyte cell lineages.
13
Cx43 is made more rapid by the application of an antisense
oligodeoxynucleotide (asODN) with a sequence that is specific to Transforming growth factor-beta-1 (TGF-β1) influences many
Cx43 messenger RNA (mRNA),
9–11
or by the conditional deletion of processes at different stages of tissue repair. Interestingly,
the Cx43 gene.
12
We have shown that a single topical treatment of a Cx43–asODN treatment results in significantly enhanced TGF-β1
mammalian wound with Pluronic™ gel containing Cx43–asODN has expression on day two after injury.
10
This elevation has been seen in
the effect of promoting both re-epithelialisation and granulation both keratinocytes and fibroblast-like cells at wound edges, and it
tissue formation and resolution, while reducing inflammation and scar may go some way towards explaining their modified behaviour
formation. Pluronic gel flows freely at 0–4°C and runs easily into the following Cx43 asODN treatment, as cell migration and proliferation
wound, but rapidly sets in place to provide sustained delivery of are both enhanced by TGF-β1. Thus, not only is re-epithelialisation
the asODN. more rapid, but granulation tissue formation is also enhanced,
because fibroblasts in treated wounds migrate more quickly and
In normal keratinocytes, Cx43 protein has a half-life of only about 1.5 proliferate more – an effect that can be replicated using cultured
hours, so it is rapidly depleted once its messenger RNA (mRNA) is fibroblasts in an in vitro scratch-wound assay. These fibroblasts not
destroyed, and virtually none can be seen in leading-edge only migrate faster, but also express more collagen I mRNA and lay
keratinocytes and wound-edge fibroblasts two hours after down more collagen, thereby enhancing granulation tissue
Cx43–asODN application. The asODN only acts locally because it is maturation.
10
Myofibroblasts appear in the granulation tissue of
rapidly broken down on contact with cells or sera (half-life 20–30 treated wounds several days ahead of controls, contract down the
minutes). However, Pluronic gel in the wound site provides a slow- granulation tissue sooner and are lost two to three days in advance
release reservoir that can sustain delivery of fresh asODN for several of controls. Another sign of advanced granulation tissue maturation
hours. The beneficial effects of Cx43–asODN can be seen as early as is an enhancement of angiogenesis by two to three days compared
80 EUROPEAN DERMATOLOGY
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