the reinnervation and revascularisation pattern of scarless murine fetal wounds

The reinnervation and revascularisation pattern ofscarless murine fetal woundsJames Henderson,1,2 Giorgio Terenghi3 and Mark William James Ferguson4

1Department of Plastic and Reconstructive Surgery, Norfolk and Norwich University Hospital NHS Trust, Norwich, UK2Faculty of Life sciences, University of Manchester, Manchester, UK3Blond McIndoe Research Laboratories, Regenerative Biomedicine, University of Manchester, Manchester, UK4Renovo Ltd, Manchester, UK

Abstract

Fetal wounds can heal without scarring. There is evidence that the sensory nervous system plays a role in medi-

ating inflammation and healing, and that the reinnervation pattern of adult wounds differs from that of

unwounded skin. Ectoderm is required for development of the cutaneous nerve plexus in early gestation. It

was hypothesised that scarless fetal wounds might completely regenerate their neural and vascular architec-

ture. Wounds were made on mouse fetuses at embryonic day 16.5 of a 19.5-day gestation, which healed with-

out visible scars. Immunohistochemical analysis of wound sites was performed to assess reinnervation, using

antibodies to the pan neuronal marker PGP9.5 as well as to the neuropeptides calcitonin gene-related peptide

(CGRP) and substance P (SP). Staining for the endothelial marker von Willebrand factor (VWF) allowed compar-

ison of reinnervation and revascularisation. Wounds were harvested at timepoints from day 1 after wounding

to postnatal day 6. Quantification of wound reinnervation and revascularisation was performed for timepoints

up to 6 days post-wounding. Hypervascularisation of the wounds occurred within 24 h, and blood vessel den-

sity within the wounds remained significantly elevated until postnatal day 2 (4 days post- wounding), after

which VWF immunoreactivity was similar between wound and control groups. Wound nerve density returned

to a level similar to that of unwounded skin within 48 h of wounding, and PGP9.5 immunoreactive nerve fibre

density remained similar to control skin thereafter. CGRP and SP immunoreactivity followed a similar pattern

to that of PGP9.5, although wound levels did not return to those of control skin until postnatal day 1. Scarless

fetal wounds appeared to regenerate their nerve and blood vessel microanatomy perfectly after a period of

hypervascularisation.

Key words: calcitonin gene-related peptide; fetal wounds; nerves; scarless; substance P; von Willebrand factor.

Introduction

Cutaneous wounds made on fetuses before the third

trimester are reported to heal without scarring (Ferguson &

O’Kane, 2004). The age of gestation at which scarless heal-

ing occurs varies, even between individuals, and the extent

of injury is important in determining whether a wound will

scar. Even in the adult, a pinprick usually heals with no scar

(Ferguson et al. 1996). Investigation into fetal scar-free

healing has led to the development of therapeutic scar-

reducing strategies for adult wounds (Occleston et al.

2008), but there has been no investigation of the reinnerva-

tion pattern of scar-free fetal wounds, or adult wounds

treated to reduce scarring. The MRL ⁄ MpJ mouse heals ear

punch wounds without scarring, and nerve regeneration in

these wounds preceded vascularisation, in contradistinction

to dorsal skin wounds in the same animal, which heal with

a scar (Buckley et al. 2011). Scarring adult wounds are

abnormally reinnervated and hypervascular, so it is impor-

tant to clarify the reinnervation and revascularisation pat-

tern of these scar-free wounds as this may lead to future

strategies for improving wound reinnervation, as well as a

deeper understanding of the nature of scar-free healing.

Fetal wounds show less early inflammation compared

to adult wounds. The fetus is thought to be significantly

neutropenic and may not have developed self–nonself

immunological identity (Longaker et al. 1990). Fewer poly-

morphonuclear leucocytes, macrophages and lymphocytes

Correspondence

Prof Mark Ferguson, Renovo Ltd, The Manchester Incubator

Building, 48 Grafton Street, Manchester M13 9XX, UK.

T: + 44 (0)161 276 7121; F: + 44 (0)161 276 7240; E: mark.ferguson@

renovo.com

Accepted for publication 25 February 2011

Article published online 24 March 2011

ªª 2011 The AuthorsJournal of Anatomy ªª 2011 Anatomical Society of Great Britain and Ireland

J. Anat. (2011) 218, pp660–667 doi: 10.1111/j.1469-7580.2011.01366.x

Journal of Anatomy

migrate into fetal wounds, although these cells are capa-

ble of responding to inflammatory signals in the same

manner as in adult wounds (Cowin et al. 1998).

Experimental reduction of transforming growth factor

beta (TGF-b)1 levels in adult wounds using a neutralising

antibody markedly improves scarring (Shah et al. 1994). By

contrast, fetal wounds contain high levels of TGF-b3 (Fergu-

son & O’Kane, 2004) and addition of human recombinant

TGF-b3 reduces scarring in experimental animals (Shah et al.

1995) and man (Ferguson et al. 2009; Bush et al. 2010).

Cutaneous nerve fibres are seen in dorsal mouse skin at

embryonic day 15 (e15) of a 19.5-day gestation, and by

day e16, some fibres appear to be associated with develop-

ing hair follicles (Peters et al. 2002). These may be the pre-

cursor of the follicular neural network seen in adult skin. At

e18, some nerves were seen in the epidermis. Calcitonin

gene-related peptide (CGRP) and substance P (SP) were

detected only at postnatal day 1 (p1) in subcutaneous and

dermal nerve fibres of the dorsal skin, although SP immuno-

reactivity has been found in mouse cranial nerve nuclei at

day e13, and in facial skin and mucosa at days e16–17

(Mohamed & Atkinson, 1982). A similar pattern of feather

innervation was seen in chicks (Saxod et al. 1996). Ablation

of chick ectoderm at embryonic day 4 prevented cutaneous

nerve plexus formation. It is suggested that embryonic skin

may trigger divergence of nerve branches and plexus devel-

opment by secretion of trophic factors (Lumsden & Davies,

1986; Martin et al. 1989).

Human fetal cutaneous innervation follows a similar

sequence of events to that found in mice; nerve plexuses

were detected with antibodies to PGP 9.5 from 10 weeks

whilst the unequivocal presence of CGRP and small

amounts of SP were not detected until 17 weeks (Terenghi

et al. 1993).

Adult murine wounds become reinnervated (Rajan et al.

2003) and hypervascular (Henderson et al. 2006) during the

healing process, and the pattern of cutaneous innervation

of adult wounds is altered (Zhang & Laato, 2001; Liang

et al. 2004; Henderson et al. 2006). Neonatal wounds have

been found to be hyperinnervated by capsaicin-sensitive (C

and Ad) nerve fibres (Reynolds & Fitzgerald, 1995), but the

reinnervation and revascularisation pattern of nonscarring

fetal wounds are unknown.

We hypothesised that fetal wounds healing without scar-

ring would completely regenerate their cutaneous nerve

and vascular plexuses, possibly after transient hyperinnerva-

tion and hypervascularisation. We aimed to assess quantita-

tively wound revascularisation and reinnervation to test this

hypothesis.

Materials and methods

All procedures were performed under Home Office licence and

in accordance with the UK Animal Act (1986). Fetal wounds

were performed using an operating microscope and microsurgi-

cal instruments. A pregnant female CD1 mouse of 16.5 days’

gestation (the day of finding a vaginal plug being taken as

day 0) was anaesthetised with oxygen, nitrous oxide and isoflu-

orane. The mouse was placed supine, and the lower abdomen

shaved and cleaned with 70% ethanol. A sterile drape was used

and the procedure was performed using aseptic techniques. A

low midline laparotomy was performed. The skin and abdomi-

nal wall musculature were divided. Fetuses were identified

through the translucent wall of the elongated bicornate uterus

and were orientated to place an incision over the body wall of

the fetus. A uterotomy incision allowed the fetal body wall

(flank) to be wounded with a small cup-shaped sharp forceps

(Aesculap). Wound sites were marked to allow wounds to be

identified postnatally with two 9 ⁄ 0 sutures. Control skin was

harvested from the contralateral flank of each animal at the

time of wound harvest.

The gestation period of the CD1 mice was a consistent

19.5 days. Fetal wounds were harvested at embryonic days e17.5

and e18.5, as well as postnatal days p1, p2, p3 and p6. Day p1 is

the day of birth, equivalent to e19.5. Six embryos were

wounded for each timepoint (n = 36 in total), but not all were

available for analysis, due to loss of some marker sutures and

maternal cannibalism. Table 1 shows the final numbers of

wounds available for analysis. Data from the second postnatal

day and the sixth postnatal day, where the numbers of wounds

analysed were small (one or two tissue samples in most cases)

should be regarded as provisional findings. The data are

Table 1 Numbers of wounds analysed at each timepoint after fetal wounding. Three sections from each wound were analysed.

Timepoint

Marker

PGP9.5 VWF CGRP SP

Wound Control Wound Control Wound Control Wound Control

e16.5 n ⁄ a 6 n ⁄ a 6 n ⁄ a 6 n ⁄ a 5

e17.5 4 5 4 5 4 5 2 5

e18.5 4 5 3 5 4 6 3 5

e19.5 = p1 6 6 6 6 6 6 6 5

p2 1 4 1 2 1 2 1 4

p3 3 3 3 3 0 3 3 5

p6 2 2 1 2 2 2 2 2


Reinnervation & revascularisation of fetal wounds, J. Henderson et al. 661

included because they are consistent with the findings from

other timepoints, and add to the overall picture. For each

wound, three sections were analysed.

Tissue processing

The harvested tissue from all experiments was immediately

placed into cold Zamboni’s solution and stored at 4 �C for 24 h

before being transferred to 15% sucrose in phosphate-buffered

saline (PBS), which was changed daily until the tissue was satu-

rated. Whole tissue samples were frozen in optimum cutting

temperature (OCT) embedding matrix (Cellpath, Powys, UK) over

liquid nitrogen. Samples were then stored at )80 �C until analy-

sis. A cryostat (CM3050; Leica, Nussloch, Germany) was used to

cut 14-lm sections, which were collected sequentially onto slides

that had been coated with poly-L-lysine. Toluidine blue stain

(Sigma) along with the marker sutures was used to confirm

wound location in the sections. The slides were dried overnight

at 37 �C before immunohistochemical or simple staining.

Immunohistochemistry

Sections were permeabilised in 0.2% Triton detergent for

60 min, followed by washes (2 · 3 min) in PBS at pH 7.4. To

decrease background autofluorescence, the sections were placed

in a PBS solution containing 10% pontamine sky blue (BDH;

Poole, Dorset, UK) and 10% dimethylsulphoxide (DMSO). After

two more washes in PBS, sections were incubated with primary

antibodies for 20 h at 4 �C in PBS with 1% sodium azide preser-

vative and 5% goat serum as a blocking agent. Primary antibod-

ies were rabbit anti-human protein gene product 9.5 (PGP9.5)

(diluted 1 : 500; Affiniti, Exeter, UK), which stains all nerve

tissue; rabbit anti-human von Willebrand factor (VWF) (diluted

1 : 2000; Abcam, Cambridge, UK), or rabbit anti-rat calcitonin

gene-related peptide (CGRP) (diluted 1 : 3000; Affiniti) or rabbit

anti-cow Substance P (SP) antibodies (diluted 1 : 5000; Affiniti).

Following more washes in PBS (2 · 6 min) the sections were

incubated with a fluorescein conjugated polyclonal goat anti-

rabbit secondary antibody (diluted 1 : 100; Vector Laboratories,

Burlingame, CA, USA) at room temperature for 1 h. After final

washes in PBS (2 · 6 min) sections were mounted with Vecta-

shield� (Vector). The slides were stored in the dark at 4 �C to

avoid fading of fluorescence, and analysed within 48 h.

Microscopy

A Leica DMRB microscope was used to view the images under

fluorescent light. Images were captured at 20· magnification

using a digital camera (Diagnostic Instruments, Sterling Heights,

MI, USA) from three adjacent sections of each wound. Images

were analysed using an automated method of quantifying the

area of positive staining in each field of view (Image Pro-Plus;

Media Cybernetics, Silver Spring, MD, USA).

Masson’s trichrome staining was carried out on separate

sections of all wounds, allowing wound architecture to be com-

pared with wound area measurements and immunohistochemi-

cal findings.

Statistical analysis was performed between timepoints using

analysis of variance, and between wounded and unwounded

tissue at the same timepoint with a non-parametric t-test,

assuming unequal variance between groups.

Results

Fetal wounds healed extremely well, and by the day of

birth (e19.5, also called p1) 3 days’ post-wounding, the

wounds were invisible to the naked eye except for some

residual erythema. Masson’s stained sections of the wounds

showed how the histological architecture of the healing

wounds changed over time, becoming virtually indistin-

guishable by postnatal day 6, by which time the wound has

become almost impossible to distinguish from the surround-

ing skin. Although an increase in cellularity is apparent, it is

difficult to be certain of the exact wound margins (Fig. 1).

A C

B D

Fig. 1 Masson’s stained section through a

wound at embryonic days 17.5 (A), postnatal

day 1 (B), postnatal day 3 (C), and postnatal

day 6 (D). The scale bar is 100 lm in each,

and the red arrows show the edges of the

wound. By postnatal day 6, the wound has

become almost impossible to distinguish from

the surrounding skin. Although an increase in

cellularity is apparent, it is difficult to be

certain of the exact wound margins.


Reinnervation & revascularisation of fetal wounds, J. Henderson et al.662

Not all of the wounds created could be used for analysis,

either because the marker sutures had come off or been

removed by the mother, or due to maternal cannibalism.

Without marker sutures being present, it was not possible

reliably to identify the wound sites in cut sections. The

result of this problem is that fewer wounds were available

for analysis, particularly at later timepoints. The number of

wounds and control skin specimens finally used for analysis

are shown in Table 1. Some of the timepoints are only rep-

resented by one wound, in which case analysis was based

on three sections through the one available wound.

Unwounded fetal skin showed a changing pattern of

innervation as the fetuses matured. PGP9.5 immunostaining

was present from day e16.5, showing the presence of a

developing nerve plexus (Fig. 2A) although SP was only

present in very small amounts, and CGRP was not detected

until e17.5 and only in small amounts until day p1 (Fig. 3).

VWF immunoreactivity demonstrated the presence of a vas-

cular plexus from day e16.5 (Fig. 4A).

Wound reinnervation during healing appeared to be by

both collateral sprouting from intact nerves in the base of

the wound and by regeneration of divided axons at the

wound peripheries (Fig. 2B) (classified by Griffin et al.

2010). Revascularisation was also seen to occur from the

wound edge (Fig. 4B).

A reduction in cutaneous nerve fibre density was seen in

unwounded fetal skin at day e18.5 (P < 0.05 compared to

e17.5) (Fig. 5). The overall reinnervation pattern demon-

strated by PGP9.5 immunofluorescence showed that reinn-

ervation of the wounds made at day 16.5 occurred over

2 days (Figs 2 and 5). Although the innervation density of

wounds 1 day after wounding (day e17.5) was significantly

less than in the control skin, nerve fibre density was similar

in control and wound groups by day e18.5, and remained

so for the duration of the study period.

The reinnervation of the wound by nerve fibres immuno-

positive for CGRP occurred over 4 days after wounding. The

levels of CGRP in wounds were significantly lower than in

unwounded skin at days e17.5 and e18.5. By day p1, CGRP

levels in healing wounds were lower than in controls

(P = 0.12) and there was no significant difference between

wounded and unwounded skin CGRP density thereafter

(Fig. 6).

Very little substance P could be detected in wounds until

day p1, at which point the levels were higher than in con-

trol skin, although not significantly so. At all subsequent

timepoints, there was no difference between the density of

control skin and wound SP innervation (Fig. 7).

Fetal wounds showed dramatic hypervascularisation in

the 3 days after wounding (Figs 4 and 8), although the den-

sity of VWF staining returned to levels similar to those in

control wounds by day p2 (4 days after wounding) and

remained so thereafter. Revascularisation preceded reinner-

vation.

A B

Fig. 2 Immunostaining for the pan neuronal marker PGP9.5 in green. (A) Unwounded fetal skin from day e16.5. A cutaneous nervous plexus is

present (white arrows). SP and CGRP immunoreactivity were not present at this stage of development. (B) A fetal wound at postnatal day 1,

3 days after wounding. PGP9.5 immunostaining shows nerve fibres in green (white arrow). In this wound they appear to be regenerating from the

adjacent skin. The blue arrows indicate the wound edges. Scale bar: 100 lm.

Fig. 3 Unwounded mouse skin from the day of birth (e19.5, which is

the same as p1) showing immunostaining for CGRP (White arrows)

that was only detected in very small amounts before this time. Scale

bar: 100 lm.



A B

Fig. 4 Immunostaining for the vascular marker von Willebrand factor (VWF). (A) Unwounded fetal skin at day e16.5. A vascular plexus is seen

(white arrows). (B) VWF immunostaining (green) in a fetal wound at postnatal day 1 (white arrows). The epithelium is counterstained red. The blue

arrows show the wound margins, one of which is right at the edge of the field of view. Vessels appear to be regenerating from the wound edge

at the right-hand side. Scale bar: 200 lm.

Fig. 5 Histogram showing mean ± SEM

reinnervation density of fetal wounds and

control skin as indicated by PGP9.5

immunostaining at times after wounding.

Wounds were made at day e16.5. A

significant reduction in innervation density in

control skin was seen between days e17.5

and e18.5 (*P < 0.05). Nerve density was

significantly less in wounds 1 day after

wounding (e17.5), but at all other timepoints,

the density of nerve fibres was similar

between wounds and control skin. Nerve

density was measured as percentage

immunofluorescence per high-power field

after artefacts were excluded.



control skin as indicated by CGRP


Wounds were made at day e16.5, at which

time no CGRP was detected in the fetal skin.

A reduction in innervation density in control

skin was seen between days p1 and p2. The

CGRP innervation density of wounds was

significantly lower than that of control skin

for the first 2 days after wound creation;

thereafter, CGRP density between control and

wounded skin was similar. Nerve fibre density

was measured as percentage

immunofluorescence per high-power field

after artefacts were excluded.



Conclusions

Murine fetal wounds made at e16.5 healed without scarring

and also appeared to regenerate completely their cutane-

ous neurovascular structures along with the rest of the

cutaneous architecture by postnatal day 2. Wounds were

hypervascularised during the healing process, but no hyper-

innervation occurred.

Discussion

We found that unwounded fetal skin contains nerve fibres

from e16.5, CGRP from day e17.5 and SP from day p1, the

same sequence and similar time course as found by others

(Peters et al. 2002). The levels of SP and CGRP immunoreac-

tivity are low in comparison with that of PGP9.5. Wounds

made at e16.5 on CD1 mouse fetuses were found to regen-

erate their cutaneous nerve and vascular plexuses after a

period of hypervascularisation.

Given that nerve fibres were only just becoming detect-

able in the murine skin at the time of wounding in these

experiments, it might be interesting to wound fetuses at

earlier times in gestation to see whether there is a time

before which damage to the surface prevents innervation.

Developing skin provides trophic factors for its own inner-

vation, and removal of ectoderm from chick hindlimb pre-

vents normal cutaneous innervation of that limb (Honig

et al. 2004). It was suggested that embryonic skin may trig-

ger divergence of nerve branches and plexus development

by secretion of trophic factors (Martin et al. 1989), and so

the effects of fetal wounding on wound reinnervation may

be dependent on the timing and nature of the injury

inflicted.

Fetal skin reinnervation after wounding differed from

adult wound reinnervation. Although adult skin nerve

density was not elevated overall (Rajan et al. 2003), SP

and GGRP levels were found to be elevated during the

healing process and SP levels remained elevated (Hender-

son et al. 2006). Initial adult wound hyperinnervation fol-

lowed by a return in nerve density to that of unwounded

skin has been found in guinea pig burn wounds (Kishimoto,

1984) and superficial wounds in the rat (Aldskogius et al.



control skin as indicated by SP


Wounds were made at day e16.5. A

significant reduction in innervation density in

control skin was seen between day e17.5 and

day e18.5 (*P < 0.05. Very little SP was

detected in the fetal skin until the first

postnatal day. SP levels were decreased in

wounds on the first day. Otherwise, there

was no difference in the density of SP in the

wounds and control tissue at any timepoint.

Fig. 8 Histogram showing mean ± SEM for

fetal wound revascularisation and control skin

as indicated by density of VWF

immunoreactivity at times after wounding.

Wounds were made at day e16.5. The

hypervascularisation of wounds seen at

days e17.5, e18.5 and p1 is statistically

significant (*P < 0.05). VWF density was

measured as percentage immunofluorescence

per high-power field after artefacts were

excluded.



1987). This study found no hyperinnervation during or

after healing of scarless fetal wounds. Nerves have been

identified growing into adult wounds either from the

adjacent skin edges (regeneration) or as branches from

intact deeper nerves (collateral sprouting; Rajan et al.

2003). Our results suggest that wound reinnervation in

this model also appeared to be by mechanisms of both

collateral sprouting from intact nerves in the base of the

wound as well as by regeneration of divided axons at the

wound peripheries (Fig. 2D). However, the fetal model

differs from those of adult murine nerve regeneration

(Griffin et al. 2010) because the nerve plexuses are also

developing de novo in the fetal skin at and around the

time of experimental wounding. This may make the dis-

tinction between repair by regeneration of injured axons

or collateral sprouting from uninjured deep nerves diffi-

cult to differentiate from axonal growth and sprouting

that would occur in an uninjured fetus.

Cytokines acting in wound healing have actions on

nerve growth and regeneration, particularly nerve growth

factor (NGF). NGF is produced by keratinocytes (Yaar et al.

1991), possibly more so after they have been injured

(Taherzadeh et al. 2003). Keratinocytes cause hyperexcit-

ability and chronic pain when interacting with peripheral

nerves in an injury model (Radtke et al. 2010). NGF mRNA

and then protein are increased after cutaneous injury in

adult rats. NGF preferentially stimulates the growth of

sensory neurons that express CGRP and SP (Terenghi,

1999; Micera et al. 2003), leading to the selective survival

of C and Ad fibres (Hari et al. 2004), which mediate pain,

temperature and pruritis. NGF is produced by keratino-

cytes (Yaar et al. 1991), Mast cells (Artuc et al. 1999) and

by injured tissue (Hasan et al. 2000; Cruise et al. 2004)

and denervated skin (Diamond et al. 1992). Dorsal root

ganglia in vitro have been found to grow preferentially

towards injured rather than uninjured skin (Taherzadeh

et al. 2003).

It would be interesting to compare NGF mRNA and pep-

tide levels in adult and fetal wounds to see if these corre-

late with reinnervation patterns.

We found transient marked hypervascularisation of the

mouse fetal scarless wounds. This is consistent with the find-

ing of a two-fold increase in vessel counts in scar-free rat

fetal wounds (Colwell et al. 2005), which was associated

with an increase in vascular endothelial growth factor

(VEGF) expression. Our finding that revascularisation pre-

ceded reinnervation is different to the scarless healing seen

following punch wounds to the MRL ⁄ MpJ mouse ear, in

which nerve regeneration preceded vascularisation (Buckley

et al. 2011). Increased vessel counts were found in scarring

fetal wounds, but without the same transient increase in

VEGF (Colwell et al. 2005). VEGF is expressed in normal

human fetal endothelial cells and is likely to have a role in

normal developmental differentiation and angiogenesis,

which in the context of fetal injury is part of the mechanism

by which tissue regeneration occurs instead of scarring

(Peters et al. 1993). It would be of interest to quantify

VEGF.

Human adult scars appear to have variable innervation

patterns reflecting the wide range of sensory symptoms

experienced by patients. Electron microscopic examina-

tion of human punch biopsy scars found small unmyeli-

nated fibres growing into the neodermis and epidermis

of the scar but almost no myelinated epidermal fibres

(Mihara, 1984). Hypertrophic human scars have been

found to contain greater levels of SP, CGRP and NPY

compared with normal skin, although normotrophic scars

were not found to contain any neuropeptides (Crowe

et al. 1994). Only SP and CGRP immunopositive fibres

were found to penetrate into painful human hypertro-

phic scars and it was suggested that SP antagonism

might reduce scar hypertrophy (Parkhouse et al. 1992).

Normotrophic scars in humans were found to contain

less SP, CGRP, vasoactive intestinal polypeptide (VIP)

and neuropeptide Y (NPY) than control skin at 7 months

(Altun et al. 2001).

Clinical evaluation of keloid scars found that 86% were

pruritic and 46% painful, suggesting abnormalities of

small fibre innervation within keloid scars (Lee et al. 2004).

Limited immunohistochemical studies of keloid innervation

have confirmed abnormal nerve morphology within the

scar (Zhang & Laato, 2001).

Our findings that fetal wounds that heal without scar-

ring also appear to regenerate their cutaneous innervation

is reassuring for the development of scar-reducing treat-

ments for human use. It might be extrapolated that scar

reduction would encourage more normal cutaneous reinn-

ervation than the limited unmyelinated pattern observed

in adult wounds, although clearly this will need to be

investigated.

Acknowledgements

We are grateful to Kelly Middleton for tuition in the fetal

wounding technique, and to Renovo Ltd and The Royal College

of Surgeons of Edinburgh for financial assistance.

Authors’ contributions

The experiment was conceived by all three authors. J.H. carried

out the experimental work, which was supervised by G.T. and

M.W.J.F. All authors were involved in writing the paper.

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the reinnervation and revascularisation pattern of scarless murine fetal wounds

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