burns 41 (2015) 10081016

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A histological study on the effect of pressure

therapy on the activities of myofibroblasts and
keratinocytes in hypertrophic scar tissues
after burn
Cecilia W.P. Li-Tsang a,*, Beibei Feng a,1, Lin Huang b, Xusheng Liu c,
Bin Shu c, Yvonne T.Y. Chan a, Kwok-Kuen Cheung a
a

Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hung Hom, Hong Kong
Division of Plastic, Reconstructive and Aesthetic Surgery, Department of Surgery, The Chinese University of Hong
Kong, Prince of Wales Hospital, Hong Kong
c
Burns Unit, 1st Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
b

article info

abstract

Article history:

Although pressure therapy (PT) has been widely used as the first-line treatment for

Accepted 25 November 2014

hypertrophic scars (HS), the histopathological changes involved have seldom been studied.

Keywords:

in HS. Ten scar samples were selected from six patients with HS after burn and they were

This study aimed to examine the longitudinal effect of PT on the histopathological changes
Pressure intervention

given a standardized PT intervention for 3 months while 16 scar samples were obtained on

Hypertrophic scar

those without PT. The scar biopsies were collected pre-treatment, 1 and 3 months post-

Myofibroblasts

intervention for both clinical and histopathological examinations. Clinical assessments

Keratinocytes

demonstrated significant improvement in the thickness and redness of the scars after PT.

Apoptosis

Histological examination revealed that cell density in the dermal layer was markedly
reduced in the 3-months post-pressurized scar tissues, while the arrangement of the
collagen fiber was changed from nodular to wave-like pattern. The a-smooth muscle actin
immunoreactivity was significantly decreased after 1-month pressure treatment. There was
a significant reduction of myofibroblasts population and a concomitant increase in the
apoptotic index in the dermal layer in the 3-months post-pressurized scars. A significant
negative correlation was found between the myofibroblasts population and the apoptotic
index. The keratinocyte proliferation was found inhibited after PT. Results demonstrated
that PT appeared to promote HS maturation by inhibiting the keratinocyte proliferation and
suppressing myofibroblasts population, the latter possibly via apoptosis.
# 2014 Elsevier Ltd and ISBI. All rights reserved.

* Corresponding author at: Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong.
Tel.: +852 2766 6715; fax: +852 2330 8656.
E-mail address: cecilia.li@polyu.edu.hk (Cecilia W.P. Li-Tsang).
1

Present address: Department of Rehabilitation Medicine, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
http://dx.doi.org/10.1016/j.burns.2014.11.017
0305-4179/# 2014 Elsevier Ltd and ISBI. All rights reserved.

burns 41 (2015) 10081016

1.

Introduction

Hypertrophic scarring, one of the most common complications after burns, is a pathophysiological condition involving
alterations in the cutaneous wound healing process. During
normal wound healing, the activated fibroblasts proliferate
and migrate to re-populate the damaged site. Myofibroblast, a
specialized fibroblast, possesses contractile activity that
enhances wound closure. Myofibroblasts also synthesize
and deposit collagen, which are required for re-establishing
functional structural integrity to the injured tissues [1,2]. In
hypertrophic scars (HS), however, dramatic thickening of
epidermal layers as well as an excessive and irregular
deposition of collagen that result in tissue deformation or
severe contracture over different joints is evident [3]. It was
shown that more than 70% of the Chinese population develop
HS after skin injuries [4], which may not only result in
esthetical defects, but also induce severe functional disabilities and thus inevitably undermine their quality of life and
living independence [5,6].
Pressure therapy has been regarded as the first-line
noninvasive treatment for HS, and its effects on the management of HS including its facilitation in scar maturation,
improving scar appearance, reducing the erythema and
alleviating symptoms of pain and itchiness have been
reported [3,711]. Despite the popular use of pressure garment
in the management of HS, the mechanisms of mechanical
pressure and the way pressure influences the maturation of
HS are not fully understood [1214]. It was suggested that
pressure generates an ischemic and hypoxic environment,
resulting in impaired growth of myofibroblasts [1518].
Besides, mechanical pressure has been demonstrated to
significantly inhibit the secretion of transforming growth
factor-b1 (TGF-b1), cytokine that has been implicated in
regulating fibroblast activity and stimulating ECM production
[19,20]. It has also been postulated that mechanical pressure
may contribute to the regression of granulation tissue in HS
through induction of apoptosis [20]. On the other hand, wound
healing process has been shown to involve a close interaction
between epidermis and dermis [2123]. Abnormal proliferation of keratinocytes is believed to have an impact on HS
formation and development [22,24]. However, few, if any,
studies have explored the impact of mechanical pressure on
keratinocyte activities in HS.
Although numerous studies have explored the theory
underlying pressure therapy in the in vitro models [19,20],
studies on in vivo model subjected to pressure intervention
have been rare and difficult to implement [13,14]. Costa et al.
[25] demonstrated partial restoration of the ECM organization
and reduced the myofibroblast population in HS samples from
patients subjected to pressure intervention. However, the
observations on the scar biopsies were merely recorded at a
mixed time frame (27 months) instead of a fixed time frame
after pressure intervention. Besides, the pressure treatment
regime and the pressure dosage were not described clearly.
Also of note, all the in vivo studies reported were done among
the western population; none is from Asian or Chinese
population where the prevalence of HS formation is reported
to be twice as much as the western population [11,26]. The

1009

present study aimed at examining the effects of pressure

intervention on the maturation of HS on a group of Chinese
patients through an in vivo longitudinal clinical and histopathological study.

2.

Materials and methods

2.1.

Study design

A case review follow-up study design was employed to explore

the effect of pressure intervention on the maturation of postburn hypertrophic scars (HS). Subjects selected for the study
were assessed prior to the pressure therapy intervention,
followed by intervention with 1 month and 3 months
evaluation and measurement. A total of six HS patients were
recruited for pressure therapy in the study. In addition, two
groups of HS patients, one with young scar age (less than 4
months post-burn) and the other with older scar age (914
months post-burn), who had surgical excision of their scars,
contributed to the scar tissues of the non-pressurized control
group in the study. The demographic information of these
patients is shown in Table 1. Normal skin tissue sections were
obtained from a burns unit in a regional hospital in Hong Kong
for the laboratory experiments described as below.

2.2.

Patients and pressure therapy protocol

Six subjects presented with a total of 10 post-burn hypertrophic scars (Table 1) were recruited for pressure therapy at a
regional hospital in Guangzhou, China, using convenience
sampling method. The clinical diagnosis of HS was based on
the standard clinical criteria [27]. Vancouver Scar Scale (VSS)
[28] was used to score the overall scar appearance; the tissue
ultrasound palpation system [29] was used to assess the
thickness and the spectrocolorimeter [30] to assess the color of
the scar. Only active HS defined by VSS  4 and located at the
extremities with a size of 4 cm  4 cm or above were recruited
for pressure treatment. Patients with open wounds or
infection on the scar sites, or those whose scars were over
the small joints or have been given steroid or other medical
treatments were excluded.
Each patient was given pressure garments on the individual
scar regions with standardized mechanical pressure of around
15 mmHg. If needed, additional pressure padding would be
given to ensure the pressure range to be consistent. The Pliance
X system [31] was used to monitor the dosage of pressure so that
all subjects would have similar pressure applied onto the scar
tissues. Subjects were instructed to wear the pressure garment
and/or padding for 23 h per day. Clinical scar assessments on
the thickness and pigmentation of the scars were conducted at
the initial visit (the pre-pressurized sub-group), and postpressure therapy visits on 1- and 3-months (the 1-month and
3-months post-pressurized sub-group, respectively). 3 mm
punch biopsies of full thickness scar tissue were collected from
the treated area from each subject at the initial assessment,
1- and 3-months post-pressure treatments. Two subjects failed
to have their 1-month post-treatment follow-up visits, thus
their data at the 1-month assessment was missing. All the
procedures have been approved by the Ethics Committee of the

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burns 41 (2015) 10081016

Table 1 Demographic information of patients in the pressure-treated group in this study.

Patient
Pressurized group
01
02
03
04
05
06

Gender

Age (year)

Age of scar post-burn

(months)

No. of scars

Cause of injury

F
M
M
F
F
M

30
44
31
18
16
26

3.5
3
6
4
3
4

2
1
1
2
2
2

Burn
Burn
Burn
Burn
Scald
Burn

40
40
41
59
75
20
41

2
1.5
1.5
1.5
3
4
2

1
2
1
2
1
2
2

Scald
Scald
Scald
Scald
Scald
Burn
Burn

1
1
1
1
1

Burn
Scald
Scald
Burn
Burn

Non-pressurized group
Young scar age
F
01
M
02
03
M
M
04
F
05
M
06
07
M
Old scar age
M
01
M
02
F
03
F
04
F
05

7
6
13
7
6

Hong Kong Polytechnic University with written consents

obtained from all the subjects.

2.3.

Histopathological analysis

Scar biopsies were fixed in 4% paraformaldehyde overnight for

subsequent tissue processing and embedding. Paraffin sections of 5 mm thick were prepared for routine H&E, immunohistochemistry and TUNEL analysis.

2.4.

Immunohistochemical staining

Deparaffinized tissue sections were pretreated with 0.2% Triton

X-100 and blocked with 5% normal horse serum (Vector lab)
before overnight incubation with primary antibodies at 4 8C.
Sections were subsequently incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen) and mounted with
DAPI-containing Vectashield mountant (Vector lab). Images
were captured under a fluorescent microscope (Eclipse 80i,
Nikon) using Spot Advance Software. The primary antibodies
used in this study included monoclonal antibody against a-SMA
(Sigma) and polyclonal antibody against Ki67 (Biocare Medical).
For Ki67 staining, heat-mediated antigen retrieval was required.
The a-SMA immunoreactivity for myofibroblasts was semiquantified with a 12-point weighted score by a pathologist as
previously described [32] (Table 2). The percentage of Ki67immunoreactive keratinocytes was counted by two independent researchers. A total of six views were randomly selected
from each sample and were captured at 400 magnification
for cell counting.

2.5.

TUNEL assay

Apoptosis was detected using an in situ terminal deoxynucleotidyl transferase (TdT) technique (Apoptag Kit, Millipore)

9
12
14
12
12

as previously described [32]. Sections were then hematoxylincounterstained for nuclei identification. A total of 10 views in
600 magnification were randomly selected from each sample
for cell counting. The result was expressed as apoptotic index,
which is equivalent to the number of apoptotic cells per total
number of cells in dermis per view. The cell counting was
performed by two independent researchers.

2.6.

Statistical analysis

Descriptive statistics were used to present the demographic

information of the participants in this study, including gender,
age, type of injury, age of scar. Repeated measure ANOVA
(parametric) and Friedman (nonparametric) test were
employed to find out the differences in scar conditions before
and after pressure intervention. Post hoc analysis was conducted with Bonferronis correction. Last observation carried
forward (LOCF) was used to deal with the missing data. For
histopathological parameters, Friedman test was used followed by Wilconxon Sign Rank test for comparison between
pre-pressurized, 1-month and 3-months post-pressurized

Table 2 Scoring criteria for a-SMA immunostaining.

Scoring

Population of
myofibroblasts

Intensity of a-SMA expression

++++
+++
++
+

>75%
5075%
2550%
025%
No positive cells
except in blood
vessels or glands

NAa
Intense positive staining
Faint to intense positive staining
Faint positive staining
Negative staining except in blood
vessels or glands

a
NA implies the scoring for intensity of a-SMA expression does
not include the grade of rating for ++++.

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burns 41 (2015) 10081016

groups. MannWhitney U test was used for comparison

between 3-months post-pressurized and non-pressurized
group. SPSS 17.0 statistical software was used for statistical
analysis.
For the two non-pressurized groups (young vs old scar
groups), we have first examined the histological outcome
measures (a-SMA, Ki67, TUNEL) and compared between the
two groups. No significant difference was found in any of the
histological outcome measures examined. Therefore the two
groups were combined to form the non-pressurized groups
during statistical analyses.

3.

Results

3.1.
of HS

Effect of pressure therapy on the clinical appearance

The total VSS score of scar tissues was found to be in the

decreasing trend throughout the 3-months pressure therapy
intervention. Significant reduction of scores was observed as
early as 1-month after the pressure therapy intervention.
There was a reduction of score at the 3-months pressurized
samples. Results also showed a significant reduction in scar
thickness after 1 month and 3 months pressure intervention. Significant reduction in scar redness was only observed
after 3 months of pressure intervention. No significant
differences in scar lightness and yellowness were found
before and after pressure treatment (Table 3). Fig. 1a and b
shows the progression of the appearance of HS upon
pressure therapy.

3.2.
Effect of pressure intervention on the histological
appearance of HS
Histological examination demonstrated a typically thickened
epidermis in pre-pressurized HS samples; and the dermis was
highly compact with dermal fibroblasts (Fig. 1d and g). Instead
of a regular and wavy collagen alignment as seen in the
normal skin tissues (Fig. 1c and f), nodular arrangement of
collagen fibers was found to be significantly dense in the
dermal layer of the HS tissues. After 3 months of pressure
therapy, the scar epidermis was less thickened and the
dermal cell density was decreased when compared to prepressurized scars (Fig. 2e). A more wave-like pattern of
collagen alignment was seen in the 3 months post-pressurized scar samples (Fig. 2h) when compared to pre-pressurized
scar samples.

3.3.
Effect of pressure intervention on the basal
keratinocyte proliferation in HS
The number of Ki67 positive basal keratinocytes was significantly higher in the pre-pressurized scar samples than the
1- and 3-months post-pressurized scar samples (Fig. 1il). The
percentage of Ki67 positive cells in the basal epidermis was
dramatically decreased from 60% in the pre-pressurized
samples to 26.9% and 9.1%, in 1-month and 3-months postpressurized samples respectively ( p = 0.018 and p = 0.005)
(Fig. 1m). Significant difference between the two nonpressurized groups (old scar age vs young scar age) was not
observed. Percentage of Ki67 positive basal keratinocytes in the
non-pressurized HS samples, whether it is of old scar age or
young scar age or even combined, showed significant difference with 3-months post-pressurized groups ( p < 0.0001).

3.4.
Effect of pressure therapy on the myofibroblasts
differentiation in HS
Myofibroblasts were identified immunohistochemically
against the specific marker, a-SMA. Unlike normal skin
tissues, where a-SMA immunoreactivity was only present in
the smooth muscle layers surrounding the blood vessels and
the glands, the HS samples showed a-SMA signals all over the
dermal layer in a diffused manner (Fig. 2a and b); and intensive
signals were observed in the nodular regions. The a-SMA
immunoreactivity was found reduced in 1-month and 3months post-pressurized HS (Fig. 2c and d).
The 12-point weighted score for the myofibroblast staining
in HS samples with and without pressure intervention is
presented in Fig. 2e. Results showed that the weighted scores
for myofibroblasts were substantially reduced after 1-month
(from 10 to 5.6; p = 0.018) and 3-months pressure intervention
(from 10 to 2.2; p = 0.005). In addition, significant difference
was also found between 1-month and 3-months postpressurized HS ( p = 0.007). No significant difference was found
between the young and the old scar aged non-pressurized
groups. While the weighted scores for myofibroblasts of nonpressurized HS was similar to those of pre-pressurized HS, it
was significantly higher than the weighted scores of 3-months
post-pressurized HS ( p < 0.0001).

3.5.
Effect of pressure therapy intervention on the dermal
fibroblast apoptosis in HS
TUNEL assay was performed to detect cell apoptosis in the
dermal layer. There were very few apoptotic cells in the

Table 3 Scar clinical characteristics before and after pressure treatment.

Pre-pressurized
VSS score
Scar thickness (mm)
Scar color (AU)
Lightness
Redness
Yellowness
*

p value < 0.05.

1-Month post-pressurized
*

3-Months post-pressurized

11.2  0.42
6.28  0.93

10.6  0.52
5.43  1.39*

7.7  1.05*
4.37  1.10*

46.67  2.17
9.89  1.82
11.09  2.33

50.17  3.68
9.08  2.67
12.40  3.03

51.15  3.72
7.65  1.41*
13.22  3.30

1012

burns 41 (2015) 10081016

Fig. 1 HS upon pressure intervention. Appearance of HS samples before (a) and after (b) 3-months pressure intervention.
Histomorphology of H&E stained tissue sections of normal skin (c and f), pre-pressurized (d and g) and post-pressurized (e
and h) HS samples. Arrows (ce) indicated the epidermal layers. Larger magnification (fh) illustrated the difference in the
dermal cellularity before and after pressure intervention. Representative images of DAPI-counterstained (blue) Ki67
immunoreactive keratinocytes (pink, yellow arrows) in the epidermis of the normal skin (i), and pre-pressurized (j), 1month post-pressurized (k) and 3-months post-pressurized HS (l). Scale bar = 200 mm (for ce), 100 mm (for fh) and 50 mm
(for il). (m) The percentage of Ki67 immunoreactive keratinocytes in HS with and without pressure therapy. Error bars
represent standard error of the mean. * represented significant differences found when compared with pre-pressurized
HS; + represented significant differences found when compared with non-pressurized HS samples. (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this article.)

pre-pressurized samples but obviously more in the 3-months

post-pressurized samples (Fig. 2fi). The apoptotic index of
1.59% in 1-month pressurized samples was almost 3-fold higher
than the pre-pressurized samples, although the increment was
not statistically significant. A substantial increase in apoptotic
index was observed in 3-months post-pressurized samples
(Fig. 2j). Statistically significant difference in the apoptotic index
was found between the pre-pressurized and 3-months postpressurized ( p = 0.005), and between 1-month and 3-months
post-pressurized samples ( p = 0.013). There was also significant
difference in the apoptotic index between 3-months postpressurized HS samples and the non-pressurized samples
( p < 0.0001). Apoptotic index in the non-pressurized HS
samples were similar between groups with young and old scar
ages without significant difference. In addition, a significant
negative association between the dermal myofibroblasts and
dermal apoptosis was observed (r = 0.843; p < 0.01).

4.

Discussion

In this study, we have conducted a longitudinal study to reveal

the effect of mechanical pressure on HS obtained from a group
of Chinese patients who are more predisposed to formation of
HS when compared to the Caucasian population. Their scar
maturation process seemed to be prolonged and with
complications. Their responses toward pressure therapy
may also be different. The results in this study could shed
light to our future work to address the challenges of severe
scar problems among the Chinese population.
Previous clinical studies conducted by our research team
had shown that patients responded positively upon pressure
therapy intervention [7]. The improvements were documented using our clinical scar evaluation system, which included
the measurement of scar thickness and pigmentation using

burns 41 (2015) 10081016

1013

Fig. 2 Changes in myofibroblasts and apoptosis in HS before and after pressure therapy. Representative images of DAPIcounterstained (blue) sections showing a-SMA immunoreactive myofibroblasts (red, yellow arrows) in the dermal layer of
the normal skin (a), pre-pressurized (b), 1-month post-pressurized (c) and 3-months post-pressurized (d) HS. (e) The
weighted score for a-SMA immunoreactive myofibroblasts in HS with and without pressure therapy. Representative
images of hematoxylin (blue) counterstained sections showed apoptotic nuclei in the dermal layer of the normal skin (f),
pre-pressurized (g), 1-month post-pressurized (h) and 3-months post-pressurized (i) HS. Apoptotic nuclei framed in the
small rectangular inserts were shown in higher magnification at the bottom right corner of each image. Arrows indicated
more apoptotic nuclei. (j) Bar chart summarizing the apoptotic index in HS with or without pressure therapy. Scale
bar = 50 mm. Error bars represent standard error of the mean. * represented significant differences found when compared
with pre-pressurized HS; + represented significant differences found when compared with non-pressurized HS samples;
# represented significant differences found when compared with 1-month post-pressurized HS samples. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)

the spectrocolorimeter. At the same time, our results also

found significant histopathological changes in the first 3
months of intervention. It was shown that myofibroblasts,
which were shown to be accountable for the pathological

formation of HS, were absent in the normal tissues but were

abundantly present in all of our pre-pressurized and nonpressurized HS tissues. Our pressure therapy protocol
appeared to be effective in inducing scar maturation as shown

1014

burns 41 (2015) 10081016

in our clinical assessment. The mechanical pressure applied

has been shown to significantly suppress the myofibroblasts
population, which was consistent with the decrease in
cellularity by H&E staining. Myofibroblasts, unlike in normal
wound healing which were transiently present for deposition
of collagen and wound closure but disappeared via apoptosis,
persistently existed in the HS tissues that contributed to scar
contracture and collagen overproduction [33]. Myofibroblasts
were also found to be negatively related to the occurrence of
apoptosis in the dermal layer. It appeared that the mechanical
pressure intervention not only suppressed the myofibroblasts
population but also promoted apoptosis. Instead of the two
independent events, it appeared that the mechanical pressure
induced myofibroblasts to undergo apoptosis, resulting in a
reduction in cellular content and improvement in collagen
fiber alignment in our pressurized samples, though such
hypothesis requires further examination. An in vitro study
showed a significant increase in apoptosis in HS tissues
subjected to mechanical compression [20], which was in
accord with our present findings. It will be more evident to
compare the effects of pressure therapy intervention by
comparing the results of the 1-month post- and 3-months
post-pressurized treatment. It was also noted that when
compared with the pressure therapy samples with the nonpressurized samples, significant reduction in myofibroblasts
population and marked increase in apoptosis were both
observed. Considering the possibility of endothelial cells or
glands undergoing apoptosis during pressure intervention, we
intentionally excluded those cell types when calculating the
apoptotic index. Thus, the results of the apoptotic index would
most likely represent the degree of apoptosis in myofibroblasts
(and/or fibroblasts), which are the predominant cell types in
dermal layers. It was suggested that mechanical pressure
promoted secretion of TNF-a and IL-1b that are responsible for
induction of apoptosis in hypertrophic scar tissues in vitro [20].
On the other hand, TNF-a has also been demonstrated to
suppress a-SMA expression in myofibroblasts culture [34].
Further investigation should be done to examine the changes
of TNF-a level upon pressure intervention in vivo. Although
the myofibroblasts population was significantly reduced in
3-months post-pressurized scar samples, reduced level of
a-SMA immunoreactivity was evident as early as 1 month
after pressure treatment. The down-regulation of the a-SMA
expression in dermal myofibroblasts after pressure intervention might to some extent be related to a weakened contractile
ability of myofibroblasts which would possibly reduce the
chance of scar contracture occurrence.
For the non-pressurized HS samples acquired, the weighted scores of a-SMA staining of these scars were significantly
higher than those of the 3-months post-pressurized HS, with a
presentation of large population of myofibroblasts and a
rather intense a-SMA expression. These results echoed that of
the previous study, which demonstrated a significant reduction of myofibroblasts in pressure-treated scar tissues compared with those without pressure intervention [25]. Although
the two groups of non-pressurized samples obtained have
distinctive discrepancy in terms of scar ages, both groups
showed similar histological parameters particularly in apoptotic indices as well as myofibroblast population, suggesting
that abundant myofibroblasts were the characteristics of the

hypertrophic scars and would very likely be existing for long

time before the scar becoming mature. Considering that the
scar ages of the non-pressurized HS were either comparable to
or longer than that of the pressurized group, the reduced
population of myofibroblasts observed in pressurized HS was
unlikely to be due to natural maturation. Instead, our results
strongly suggested that pressure therapy intervention accelerated the scar maturation in the HS patients.
Previous study showed that actively proliferating keratinocytes were abundant in young HS and also in those scars
remained hypertrophic for 12 months but reduced when scars
became more mature [24]. In our study, inhibition in the
proliferative capacity of keratinocytes has already been
observed in the first month of pressure treatment, further
supporting the theory that mechanical pressure accelerated
the HS maturation. Reno` et al. [35] showed that mechanical
pressure up-regulated MMP-28 expression in vitro, which was
supposed to be low in hypertrophic keratinocytes but
increased when scars matured, also supporting the notion.
It has long been shown that conditioned medium from
keratinocyte culture induced a significant increase in fibroblast replication [36], suggesting that keratinocytes influenced
fibroblast proliferation through cytokines. Using a tissueengineered, reconstructed human skin model composed of
either normal or hypertrophic keratinocytes on top of the
dermis, Bellemare et al. [21] have demonstrated that significantly thicker dermis formation was always observed when
the epidermis was engineered with hypertrophic keratinocytes than with normal keratinocytes. In addition, they also
found that the hypertrophic keratinocytes also induced an
increase in collagen secretion and a reduction in collagenase
synthesis. Recent findings have demonstrated that suprabasal
keratinocytes synthesized and released latent TGF-b1 that
liberated into the dermal layer and activated the expression of
a-SMA in the fibroblasts, resulting in the induction of
myofibroblast differentiation [37]. However, the interactions
between keratinocytes and fibroblasts/myofibroblasts are not
unidirectional. In fact, studies have also shown that inhibition
of a-SMA expression could enhance epidermal differentiation
that attenuated the proliferative capacity of keratinocytes [38],
suggesting the existence of feedback mechanisms between
the dermal and epidermal activities. Our results showed that
simultaneous alterations in the proliferative capacity of
keratinocytes and myofibroblasts differentiation occurred
upon pressure intervention, but whether mechanical pressure
acted primarily on either keratinocytes or myofibroblasts or
both remained unclear.
Although this study only managed to recruit 10 samples of
scar tissues with 3 months of pressure therapy intervention,
results appeared to show consistency in both the clinical and
histopathological changes after pressure intervention. The
mechanism of histopathological changes observed in our
Chinese patients appears to be comparable with the previous
studies on Caucasian population, though the scar tissues
appear to be quite hypertrophic already prior to intervention.
As this study aimed to find out the effect of pressure
therapy on HS, it is essential to control the magnitude of
pressure during the 3 months of intervention. It is our first
attempt to monitor the pressure intervention through a
standardized pressure measurement system. A pressure

burns 41 (2015) 10081016

magnitude of around 15 mmHg generated by pressure

garments was applied on to the scar samples. The Pliance X
system [31] was used to accurately monitor the extent of
pressure exerted during intervention. Given the advantage of
this system, modification of the pressure garment could
therefore be made regularly to ensure that the desired
pressure range was achieved.

5.

Conclusion

This is the first systematic in vivo pilot study to investigate the

early histopathological responses of HS in Chinese patients
with burn scar upon well-controlled pressure therapy intervention, which was examined in a longitudinal manner.
Significant improvements in both the clinical and histopathological characteristics of scar tissues were observed from the
sampled subjects with early pressure intervention. It showed
positive result on HS maturation through suppressing dermal
myofibroblasts possibly by induction of apoptosis, resulting in
reduced collagen overproduction and improved scar appearance. Pressure therapy intervention appeared to show
improvement in HS by inhibiting the proliferative capacity
of keratinocyte. Whether the actions of pressure intervention
act primarily on either keratinocytes or myofibroblasts or both
requires further investigation.

Conflict of interest
The authors declare no conflicting interests in preparing this
manuscript.

Acknowledgements
Our research team would like to thank the participants who
kindly contributed scar tissue samples for our study. This
work was supported by the General Research Fund provided by
Research Grants Council of the Hong Kong SAR Government
(PolyU 5630/12M). The authors would also like to sincerely
thank Dr. Raymond Chung for his advice in statistical analysis
and other research team members who have contributed their
effort to the project. Finally, without the patient subjects, the
project could not be commenced. The authors would like to
thank all the participants who kindly consent to have the skin
biopsy for this study.

references

[1] Diegelmann RF, Evans MC. Wound healing: an overview of

acute, fibrotic and delayed healing. Front Biosci 2004;9:2839.
[2] Eastwood M, McGrouther DA, Brown RA. Fibroblast
responses to mechanical forces. Proc Inst Mech Eng H
1998;212:8592.
[3] Wienert V. Compression treatment after burns. Wien Med
Wochenschr 1999;149:5812.
[4] Li-Tsang CW, Lau JC, Chan CC. Prevalence of hypertrophic
scar formation and its characteristics among the Chinese
population. Burns 2005;31:6106.

1015

[5] Bombaro KM, Engrav LH, Carrougher GJ, Wiechman SA,

Faucher L, Costa BA, et al. What is the prevalence of
hypertrophic scarring following burns. Burns 2003;29:
299302.
[6] Falder S, Browne A, Edgar D, Staples E, Fong J, Rea S, et al.
Core outcomes for adult burn survivors: a clinical overview.
Burns 2009;35:61841.
[7] Candy LH, Cecilia LT, Ping ZY. Effect of different pressure
magnitudes on hypertrophic scar in a Chinese population.
Burns 2010;36:123441.
[8] Cheng JC, Evans JH, Leung KS, Clark JA, Choy TT, Leung PC.
Pressure therapy in the treatment of post-burn
hypertrophic scar: a critical look into its usefulness and
fallacies by pressure monitoring. Burns Incl Therm Inj
1984;10:15463.
[9] Engrav LH, Heimbach DM, Rivara FP, Moore ML, Wang J,
Carrougher GJ, et al. 12-Year within-wound study of the
effectiveness of custom pressure garment therapy. Burns
2010;36:97583.
[10] Juckett G, Hartman-Adams H. Management of keloids and
hypertrophic scars. Am Fam Physician 2009;80:25360.
[11] Van den Kerckhove E, Stappaerts K, Fieuws S, Laperre J,
Massage P, Flour M, et al. The assessment of erythema and
thickness on burn related scars during pressure garment
therapy as a preventive measure for hypertrophic scarring.
Burns 2005;31:696702.
[12] Alster TS, Tanzi EL. Hypertrophic scars and keloids: etiology
and management. Am J Clin Dermatol 2003;4:23543.
[13] Anzarut A, Olson J, Singh P, Rowe BH, Tredget EE. The
effectiveness of pressure garment therapy for the
prevention of abnormal scarring after burn injury: a metaanalysis. J Plast Reconstr Aesthet Surg 2009;62:7784.
[14] Atiyeh BS. Nonsurgical management of hypertrophic scars:
evidence-based therapies, standard practices, and
emerging methods. Aesthetic Plast Surg 2007;31:46892
[discussion 493494].
[15] Hambleton J, Shakespeare PG, Pratt BJ. The progress of
hypertrophic scars monitored by ultrasound
measurements of thickness. Burns 1992;18:3017.
[16] Kischer CW, Shetlar MR. Microvasculature in hypertrophic
scars and the effects of pressure. J Trauma 1979;19:75764.
[17] Macintyre L, Baird M. Pressure garments for use in the
treatment of hypertrophic scars a review of the problems
associated with their use. Burns 2006;32:105.
[18] Reid WH, Evans JH, Naismith RS, Tully AE, Sherwin S.
Hypertrophic scarring and pressure therapy. Burns Incl
Therm Inj 1987;13(Suppl.):S2932.
[19] Chang LW, Deng WP, Yeong EK, Wu CY, Yeh SW. Pressure
effects on the growth of human scar fibroblasts. J Burn Care
Res 2008;29:83541.
[20] Reno` F, Sabbatini M, Lombardi F, Stella M, Pezzuto C,
Magliacani G, et al. In vitro mechanical compression
induces apoptosis and regulates cytokines release in
hypertrophic scars. Wound Repair Regen 2003;11:3316.
[21] Bellemare J, Roberge CJ, Bergeron D, Lopez-Valle CA, Roy M,
Moulin VJ. Epidermis promotes dermal fibrosis: role in the
pathogenesis of hypertrophic scars. J Pathol 2005;206:18.
[22] Koskela A, Engstrom K, Hakelius M, Nowinski D, Ivarsson
M. Regulation of fibroblast gene expression by
keratinocytes in organotypic skin culture provides possible
mechanisms for the antifibrotic effect of
reepithelialization. Wound Repair Regen 2010;18:4529.
[23] Zhang GY, Li X, Chen XL, Li ZJ, Yu Q, Jiang LF, et al.
Contribution of epidermal stem cells to hypertrophic scars
pathogenesis. Med Hypotheses 2009;73:3323.
[24] Andriessen MP, Niessen FB, Van de Kerkhof PC, Schalkwijk
J. Hypertrophic scarring is associated with epidermal
abnormalities: an immunohistochemical study. J Pathol
1998;186:192200.

1016

burns 41 (2015) 10081016

[25] Costa AM, Peyrol S, Porto LC, Comparin JP, Foyatier JL,
Desmoulie`re A. Mechanical forces induce scar remodeling.
Study in non-pressure-treated versus pressure-treated
hypertrophic scars. Am J Pathol 1999;155:16719.
[26] Santucci M, Borgognoni L, Reali UM, Gabbiani G. Keloids
and hypertrophic scars of Caucasians show distinctive
morphologic and immunophenotypic profiles. Virchows
Arch 2001;438:45763.
[27] Sahl Jr WJ, Clever H. Cutaneous scars: part I. Int J Dermatol
1994;33:68191.
[28] Baryza MJ, Baryza GA. The Vancouver Scar Scale: an
administration tool and its interrater reliability. J Burn Care
Rehabil 1995;16:5358.
[29] Lau JC, Li-Tsang CW, Zheng YP. Application of tissue
ultrasound palpation system (TUPS) in objective scar
evaluation. Burns 2005;31:44552.
[30] Li-Tsang CW, Lau JC, Liu SK. Validation of an objective scar
pigmentation measurement by using a spectrocolorimeter.
Burns 2003;29:77984.
[31] Lai CH, Li-Tsang CW. Validation of the Pliance X system in
measuring interface pressure generated by pressure
garment. Burns 2009;35:84551.
[32] Chan WY, Cheung KK, Schorge JO, Huang LW, Welch WR,
Bell DA, et al. Bcl-2 and p53 protein expression, apoptosis,

[33]
[34]

[35]

[36]
[37]

[38]

and p53 mutation in human epithelial ovarian cancers. Am

J Pathol 2000;156:40917.
Hinz B. Formation and function of the myofibroblast during
tissue repair. J Invest Dermatol 2007;127:52637.
Goldberg MT, Han YP, Yan C, Shaw MC, Garner WL.
TNF-alpha suppresses alpha-smooth muscle actin
expression in human dermal fibroblasts: an implication
for abnormal wound healing. J Invest Dermatol
2007;127:264555.
Reno` F, Sabbatini M, Stella M, Magliacani G, Cannas M.
Effect of in vitro mechanical compression on Epilysin
(matrix metalloproteinase-28) expression in hypertrophic
scars. Wound Repair Regen 2005;13:25561.
Garner WL. Epidermal regulation of dermal fibroblast
activity. Plast Reconstr Surg 1998;102:1359.
Hata S, Okamura K, Hatta M, Ishikawa H, Yamazaki J.
Proteolytic and non-proteolytic activation of ketatinocytederived latent TGF-b1 induces fibroblast differentiation in a
wound-healing model using rat skin. J Pharmacol Sci
2014;124:23043.
Yang L, Hashimoto K, Tohyama M, Okazaki H, Dai X,
Hanakawa Y, et al. Interactions between myofibroblast
differentiation and epidermogenesis in constructing
human living skin equivalents. J Dermatol Sci 2012;65:507.