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Skin Research and Technology 2008; 14: 436439 r 2008 The Authors

Printed in Singapore  All rights reserved Journal compilation r 2008 Blackwell Munksgaard
doi: 10.1111/j.1600-0846.2008.00308.x Skin Research and Technology

Changes of human skin in subepidermal wound healing

Keiichi Sugata, Takashi Kitahara and Yoshinori Takema
Biological Science Laboratories, Kao Corporation, Tochigi, Japan

Background/purpose: The wound healing process involves In two out of six subjects, fibrous tissues were observed in
unexplained mechanisms. An aberration in this process is the upper dermis, whereas in one other subject, melano-
known to cause dermal disorders such as keloid or hyper- cyte-like dendritic cells were observed in the epidermis
trophic scars, but the mechanism by which these scars are dermis border in later phases of wound healing.
formed remains to be elucidated. Here we examined the Conclusion: This non-invasive method using in vivo LCSM
usefulness of a non-invasive optical imaging device to revealed chronological changes in the dermis and epidermis
clarify mechanisms of wound healing and of scar formation. during wound healing. In addition, although a scar was not
Methods: An 8 mm experimental wound was made in formed in any of study subjects, this microscopy revealed
the forearms of six subjects by a suction blister method. aspects similar to the fibrous tissue overgrowth or to mela-
To observe chronological changes associated with wound nocyte migration, both of which may relate to wound healing.
healing, horizontal cross-sectional images were non-inva- These results indicate the usefulness of this non-invasive
sively obtained of the wounded area from the skin surface method in studies of wound healing and of scar formation.
down to 129 mm below at 21.5 mm intervals using in vivo
laser confocal scanning microscopy (LCSM). Key words: wound healing scar in vivo confocal laser
Results: The wounds were covered with a new epidermis by scanning microscopy
week 2, at which time the dermal papilla count decreased
while the thickness from the skin surface to the apex of the & Blackwell Munksgaard, 2008
dermal papilla increased. The count and the thickness Accepted for publication 29 December 2007
returned to the initial levels when the wound was healed.

D ERMAL WOUND healing requires continuous

modification of the complex cellular sup-
port matrix. The mechanism has not been clar-
non-invasive method in understanding the me-
chanism and variations of wound healing.

ified yet, but it is believed that an imbalance

during wound healing causes fibrous tissue over- Materials and Methods
growth known as keloid or hypertrophic scars. Subjects
Besides aesthetic problems, these disorders tend The subjects used in this study were six healthy
to cause irritation or even pain. The mechanism males (mean age, 34 years). After explaining the
of these dermal disorders is likewise unknown test to all subjects, their written consent was
due to the lack of proper animal models. In obtained, and adequate consideration was given
particular, there is no treatment for keloid that to their human rights and safety throughout the
is considered to be effective in all patients due to study.
the fact that an excision of the affected region of a
patient may induce the formation of similar or
even larger scars. Experimental wound
In this study, we non-invasively observed the An experimental superficial wound was created
chronological changes in healing of superficial by the suction blister method (3, 4). Firstly, the
wounds using in vivo laser confocal scanning blistering device with a 6-mm diameter syringe
microscopy (LCSM), which provides us an image induced a blister using a vacuum pump (DA-
of the human skin at any depth (1, 2). The aim of 30D, ULVAC Inc., Kanagawa, Japan) for appro-
this study was to assess the usefulness of this ximately 1.5 h. Secondly, the 6-mm diameter

Subepidermal wound healing process

syringe was replaced by an 8-mm diameter syr- These procedures were repeated at weeks 1, 2,
inge in order to raise the blister. Finally, the blister 4, 6 and 8.
roof (the epidermis) was removed from the arm
skin, after which the wound was covered with a
transparent dressing (3M Co. Ltd., St. Paul, MN,
USA). Figure 2 shows the representative photographs at
each depth, taken before inducing the suction
blister wound and during the wound healing
Analysis of internal skin structure (1) Before inducing the suction blister wound: Der-
As shown in Fig. 1, 10 visual fields (2.7  0.8 mm) mal papillae were observed (21.5 mm). Fine
were fixed in the central-outer area sof each fibrous elements of the reticular layer were
wound. Using LCSM (Vivascope 1000 , Lucid observed (64.5 mm).
Inc., Rochester, NY, USA), a horizontal image (2) Two weeks after creation of the suction blister
was taken in each visual field from the skin wound: The smooth shiny epidermis appeared
surface to a depth of 129 mm at 21.5 mm intervals. after a blood clot soughed (0 mm). Large
A surface image of the wound
was photographed keratinocytes similar in size to granular cells
using a Minolta a 707 Si with 50 mm macro lens occupied the epidermis (43 mm). Vague out-
(Konica Minolta Holdings Inc., Osaka, Japan). lines of dermal papillae appeared (64.5 mm).
(3) Four weeks: Outlines of the dermal papillae
were refined (43 mm).
(4) Eight weeks: The dermal papillae recovered to
initial levels (21.5 mm).
It was inferred that the observed chronological
change of dermal papillae may relate to wound
healing. To confirm this hypothesis, we counted
the number of dermal papillae (Fig. 3a) and then
measured the thickness between the skin surface
and the apex of dermal papillae (Fig. 3b). Results
were expressed as the mean value of each week
compared with the mean reference value of 100%
at week 0 (Fig. 3). The dermal papilla count
sharply decreased approximately 80% by week
Fig. 1. Representative image of the skin surface 2 weeks after removal 1 and recovered to its initial level by week 8.
of the epidermis. Confocal images were captured from 10 fields The thickness doubled by week 1, and gradually
(2.7  0.8 mm) within the suction blister wound (dashed line). recovered to its initial level by week 8.

Fig. 2. Representative confocal images of internal skin after removal of the epidermis. Image size: 400  540 mm.

Sugata et al.

In two out of six subjects, fibrous tissues were with the fibrous tissues, the dermal papilla count
observed at week 8 between the depths of 64.5 was apparently smaller than the initial number.
and 129 mm. These tissues were not determined In one other subject, many bright dendritic
before the suction blister inducement (Fig. 4) and cells were observed around the epidermisder-
were found to be irreversible. In those subjects mis border at week 2 (Fig. 5a). Hyperpigmenta-
tions were also seen in the surface image of this
subject (Fig. 5b).

Wound healing is known to involve interactions
between the epidermis and the dermis. These
interactions are well organized, as if these layers
are communicating each other (5). In this study,
although the epidermis was carefully removed
from the dermis without damaging it, a promi-
nent change was observed in this undamaged
layer. The dermal papillae disappeared after in-
duction of the suction blister wound, followed
by a slow recovery period of more than 6 weeks.
The changes in the dermal layer seem to be an
event associated with, or contributing to the
recovery of the upper epidermal layer.
Previously, Suetake et al. detected a parameter
for evaluating the proliferative changes of the
dermis in scars. They confirmed that transepider-
mal water loss in the stratum corneum reflects the
abnormal conditions of the dermis in various
scars (6). In our study, fibrous tissues were
observed in two subjects. These tissues were
Fig. 3. Chronological changes in the number of dermal papillae: (a) irreversible for the duration of this study, and
chronological changes in the thickness between the skin surface and the
looked similar to a mild level scar. Interestingly,
apex of dermal papilla; (b) values were determined from the mean of
six subjects but at weeks 1 and 2, four subjects and one subject, the dermal papilla count was apparently smaller
respectively, were excluded due to the difficulty in obtaining an image in areas with fibrous tissues. Our result suggests
through crusta. that the dermal papilla count may likewise reflect

Fig. 4. Confocal images of dermis taken at depths of 64.5 mm: (a) 86 mm (b) and 129 mm (c) before and 8 weeks after removal of the epidermis. *Dermal
papillae. Image size: 400  540 mm.

Subepidermal wound healing process

dermis during the wound healing. We could not

confirm the migration of melanocytes and its
contribution to scar formation, and thus further
investigation using an optical device with higher
resolution will be required. In vivo two-photon
excited fluorescence microscopy that permits the
identification of substances seems to be the most
promising tool for this investigation (8, 9). At any
rate, an optical device has the potential to the
primary tool in the study of dermal disorders,
especially in the absence of suitable animal models.

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Fig. 5. (a) A confocal image that shows dendritic cells. Image size: 6. Suetake T, Sasai S, Zhen YX, Ohi T, Tagami H. Functional
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melanocytes relate to scar formation. In this lution and picosecond time resolution. J Biomed Opt
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Yamashita et al. (7) had previously determined 253.
that melanocytes appear bright in a LCSM image,
and thus these bright cells are assumed to be Address:
Keiichi Sugata
melanocytes that might have migrated from the Biological Science Laboratories
peri-wound margin or from hair follicles. LCSM Kao Corporation
cannot however, fully differentiate melanocytes 2606, Akabane
from other cells; another image taken from the Haga-gun
surface of this wound shows hyperpigmentations Tochigi 321-3497
supporting our assumption. Japan
In summary, LCSM enabled us to non-inva- Tel: 181 285 687491
sively observe changes, presumably controlled Fax: 181 285 687360
by interactions between the epidermis and e-mail: sugata.keiichi@kao.co.jp