Experimental

Complementary Effects of Negative-Pressure

Wound Therapy and Pulsed Radiofrequency
Energy on Cutaneous Wound Healing in
Diabetic Mice
Bin Chen, M.D., Ph.D.
Huang-Kai Kao, M.D.
Ziqing Dong, M.D.
Zhaohua Jiang, M.D., Ph.D.
Lifei Guo, M.D., Ph.D.
Burlington, Mass.; Taoyuan, Taiwan;
and Guangzhou and Shanghai, People’s
Republic of China
Background: Negative-pressure wound therapy and pulsed radiofrequency energy
are two clinical modalities used to treat soft-tissue wounds. They are purported to
affect healing differently. The aim of this experimental study was to contrast the
two modalities at a mechanistic level and to investigate whether their combined
therapy could achieve additive and complementary effects on wound healing.
Methods: Full-thickness dorsal cutaneous wounds of diabetic, db/db, mice were
treated with either negative-pressure wound therapy, pulsed radiofrequency energy,
or combined therapies. Macroscopic healing kinetics were examined. Epidermal
regeneration (proliferation rate and length of reepithelialization) and neovascu-
larization (blood vessel density) were investigated. Messenger RNA levels indicative
of angiogenic (basic fibroblast growth factor), profibrotic (transforming growth
factor-β), epidermal proliferative (keratinocyte growth factor), and extracellular
matrix remodeling (collagen 1) processes were measured in wound tissues.
Results: All three treatment groups displayed faster wound healing. The
negative-pressure wound therapy/pulsed radiofrequency energy combined
therapy led to significantly faster healing than either the negative-pressure
wound therapy or pulsed radiofrequency energy therapy alone. Epidermal re-
generation and neovascularization were enhanced in all three groups. The two
negative-pressure wound therapy groups (alone and combined with pulsed
radiofrequency energy) demonstrated more significant increases in expression
of all assayed growth factors than the pulsed radiofrequency energy group.
Furthermore, the combined therapy exhibited a more profound elevation in
collagen 1 expression than either of the two therapies alone.
Conclusion: Combining the negative-pressure wound therapy and pulsed ra-
diofrequency energy modalities can achieve additive benefits in cutaneous
healing, and the two therapies can be easily used together to complement each
other in clinical wound treatments.  (Plast. Reconstr. Surg. 139: 105, 2017.)

C
hronic cutaneous wound healing remains a
challenging problem in the field of recon-
structive surgery and beyond. Many modali-
ties have been developed over the years to improve
wound healing, with some of their mechanisms of
From the Department of Plastic Surgery, Lahey Hospital
& Medical Center; the Department of Plastic and Recon-
structive Surgery, Chang Gung Memorial Hospital, Chang
Gung University; the Department of Plastic and Cosmetic
Surgery, Nanfang Hospital, Southern Medical University;
and the Department of Plastic and Reconstructive Surgery,
Shanghai Ninth People’s Hospital.
Received for publication February 4, 2016; accepted August
22, 2016.
Copyright © 2016 by the American Society of Plastic Surgeons
DOI: 10.1097/PRS.0000000000002909
action elucidated.1 Negative-pressure wound ther-
apy,2–4 among a crowded field that includes newer
Disclosure: The authors have no financial ­interest
to declare in relation to the products or devices
­mentioned in this article.
Supplemental digital content is available for
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 Plastic and Reconstructive Surgery • January 2017
generation dressings, growth factors, cell-based
therapies, pulsed radiofrequency energy,5–7 and
shockwave therapy,8 is particularly notable as an
effective clinical wound healing modality. It has
been shown in animal models to promote tissue
regeneration, neovascularization, and decreased
edema, all resulting in accelerating wound heal-
ing.2,3,9,10 Several reports have postulated that
negative-pressure wound therapy might trigger
biochemical signaling at an early stage of wound
healing, leading to cell proliferation, height-
ened inflammatory reactions and, in particular,
enhanced angiogenesis.2,4,11,12
Pulsed radiofrequency energy is a nonion-
izing energy at a shortwave radiofrequency band
of the electromagnetic spectrum, most com-
monly 27.12 MHz. Since its first introduction for
clinical application in the 1950s, numerous stud-
ies have demonstrated pulsed radiofrequency
energy therapy as an effective therapy for both
acute and chronic wound healing, and for pain
and edema.13–17 Using a cutaneous wound healing
model in diabetic mice, we have previously shown
that pulsed radiofrequency energy accelerated
healing mainly through wound contraction by
significantly increasing dermal cell proliferation,
collagen synthesis, and granulation tissue forma-
tion,5,6 a relatively late component of the cuta-
neous wound healing process. This was further
corroborated by the findings that, in our pulsed
radiofrequency energy treatment group compared
with the control group, the macroscopic wound
closure was significantly accelerated beyond day
17 after wounding,5 and that the increased der-
mal fibroblast proliferation and collagen synthesis
were also most significantly notable later in the
healing process.6
Noting the difference in timing of the most
notable effects on wound healing and the seem-
ingly different mechanisms of action involved
between negative-pressure wound therapy and
pulsed radiofrequency energy therapy, we hypoth-
esize that a combination of both therapies may
have complementary effects on cutaneous wound
healing. In particular, given the overwhelming
beneficial effects of negative-pressure wound ther-
apy on cutaneous wound healing and the relatively
late benefits of pulsed radiofrequency energy, it
would be interesting to assess whether the (appro-
priately timed) addition of pulsed radiofrequency
energy to the negative-pressure wound therapy
treatment may augment the healing process even
further. A better understanding of the two therapy
modalities has significant clinical implications in
treating difficult wounds. To that end, we would
106
Copyright © 2016 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
again resort to the well-established cutaneous
wound model using genetically engineered dia-
betic mice (db/db).5,18–20 These animals exhibit
significant delays in wound closure, with com-
promised angiogenesis and granulation tissue
formation,6,14,19,20 and are therefore ideal for the
evaluation of potentially complementary effects
of the two modalities, negative-pressure wound
therapy and pulsed radiofrequency energy, as
known for their predominant benefits on wound
healing by means of enhanced early angiogenesis
and relatively late granulation tissue formation,
respectively.
MATERIALS AND METHODS
Animals and Wound Model
The present study was conducted under the
approval by our institutional animal care and
use committee. Briefly, homozygous, genetically
diabetic male mice, B6.BKS (D)-Leprdb/J, 10 to
12 weeks of age, were purchased from The Jackson
Laboratory (Bar Harbor, Me.). On the day before
surgery, dorsal hair was clipped and depilated
(Nair; Church & Dwight Co., Princeton, N.J.). On
the day of surgery, the dorsum was disinfected with
70% alcohol, and a 1 × 1  cm2 full-thickness skin
and panniculus carnosus was excised under anes-
thesia.5,6 After digital photographing, depending
on the group, wounds were covered with a trans-
parent, semiocclusive adhesive dressing (Tega-
derm; 3M, St Paul, Minn.) or with vacuum-assisted
closure (V.A.C. Granufoam Dressing; KCI, Inc.,
San Antonio, Texas) and Tegaderm. The dress-
ings were changed twice per week.
Study Groups
A total of 40 male diabetic mice were assigned
randomly to four groups as follows (Fig.  1): (1)
control group (n = 10) (wounds were covered
with Tegaderm only); (2) pulsed radiofrequency
energy group (n = 10) [wounds were covered with
Tegaderm and treated with active fields of pulsed
radiofrequency of a Provant device (Regenesis
Biomedical Inc., Scottsdale, Ariz.) twice per day
as described5,6]; (3) negative-pressure wound
therapy group (n = 10) (wounds were treated by
a vacuum-assisted closure device set at −80 mmHg
continuous pressure; the vacuum-assisted closure
device did not affect ambulation or well-being of
the treated animals); (4) negative-pressure wound
therapy and pulsed radiofrequency energy com-
bination group (n = 10) (wounds were treated
by negative-pressure wound therapy as for the
 Volume 139, Number 1 • Cutaneous Wound Healing in Diabetic Mice

Fig. 1. Study groups and time chart of study design for each group. In the
pulsed radiofrequency energy group, wounds were treated with pulsed
radiofrequency energy twice per day for 30 minutes (at 8:30 am and
4:30  pm), whereas the animals in the negative-pressure wound therapy
group were treated with vacuum-assisted closure set at −80 mmHg con-
tinuous pressure. In the combined therapy group, wounds were treated
with vacuum-assisted closure set as negative-pressure wound therapy
group for 7 days after initial wounding, followed by pulsed radiofrequency
energy treatment as for the pulsed radiofrequency energy group in paral-
lel. The control group was sham-treated with Tegaderm only. CON, control;
NPWT, negative-pressure wound therapy; PRFE, pulsed radiofrequency
energy; VDE, negative-pressure wound therapy and pulsed radiofrequency
energy combined treatment; VAC, vacuum-assisted closure.

negative-pressure wound therapy group and at day
7 after wounding, pulsed radiofrequency energy
treatment, the same as for the pulsed radiofre-
quency energy group, was commenced to run par-
allel with the continued negative-pressure wound
therapy treatment).
Wound Closure Analysis
Macrophotography of the mouse wounds and a
metric ruler were photographed on days 0, 3, 7, 10,
14, 21, and 28. Time to wound closure was defined
as the time taken for the wound bed to be com-
pletely reepithelialized. Wound area was measured
and calculated as described previously using digital
planimetry (ImageJ; National Institutes of Health,
Bethesda, Md.).5 Skin samples including the wound
and 4-mm margin of surrounding skin were har-
vested at 14 days after wounding and were bisected.
Half was processed for histologic and immunohis-
tologic studies and the other half was snap-frozen
in liquid nitrogen for further real-time reverse-tran-
scriptase polymerase chain reaction studies.
Histology and Morphologic Studies
Tissues were processed as described previ-
ously.5,6 Sections from the center of paraffin-
embedded wounds were stained with hematoxylin
and eosin and were used to determine the length
of reepithelialization. This length was defined as
the distance from the advancing tip of the reep-
ithelialization lip to the site of the first hair fol-
licle at the wound margin.21,22 Measurements were
made using ImageJ software by two independent
and blinded observers.
Immunohistochemistry for Ki67 and CD31
CD31 immunostaining was used to quantify
angiogenesis and Ki67 was used to quantify cell
proliferation. Paraffin-embedded sections were
rehydrated and antigen retrieval for Ki67 and
CD31 was performed by microwaving in 10 M
sodium citrate (pH 6.0) for 10 minutes. Primary
antibodies used were as follows: Ki67 (Lab Vision,
Fremont, Calif.), platelet/endothelial cell adhe-
sion molecule-1 (or CD31) (Abcam, Cambridge,

107
Copyright © 2016 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
 Plastic and Reconstructive Surgery • January 2017
Mass.). Secondary antibody used was horseradish
peroxidase–conjugated goat anti-rabbit immu-
noglobulin G (Bio-Rad Laboratories, Hercules,
Calif.). Primary antibodies were incubated at 4°C
overnight. Following secondary antibody label-
ing, sections were incubated with Vector NovaRed
Substrate for Peroxidase (Vector Laboratories,
Burlingame, Calif.). Tissue sections were viewed
using a Nikon ECLIPSE E600 microscope (Nikon,
Melville, N.Y.).
Blood Vessel Density Quantitation
To quantify angiogenesis, blood vessel density
of the leading edge and wound bed on each tissue
slide were determined. High-power (200×) images
of stained sections were used for analysis. A total
of six digital images of CD31-stained section were
selected randomly for each sample: two from each
neodermis under the bilateral leading edges and
two from the middle part of the wound bed. The
neovascular area (CD31+ cells) was measured
using ImageJ and expressed as the percentage of
CD31+ area of the entire imaged area. Measure-
ments were obtained using ImageJ software by two
independent and blinded observers.
Quantification of Keratinocyte Proliferation
Proliferating keratinocytes were identified by
immunohistochemistry staining for the nuclear
proliferation antigen Ki67. To quantify keratino-
cyte proliferation, high-power digital images of
Ki67-stained wound sections were obtained in the
bilateral leading edges at 100× magnification. The
number of Ki67+ basal cells in a 400-μm distance
along the basement membrane from the leading
edge was counted. Two independent investigators
counting samples were blinded to the treatment
groups. The result of each sample was expressed
as an average of the numbers of positive basal cells
along the bilateral leading edges.
Quantitative Real-Time Polymerase Chain
Reaction
RNA was extracted using TRIzol reagent
(Life Technologies, Gaithersburg, Md.). RNA was
Table 1.  Primer Sequences for Real-Rime Polymerase Chain Reaction
Gene Forward
TGF-β1 5′-GGACTCTCCACCTGCAAGAC-3′
FGF-7 5′-TGTGTACCCAGCTGTTCCAA-3′
bFGF 5′-AGCGGCTCTACTGCAAGAAC-3′
α-SMA 5′-TGTGCTGGACTCTGGAGATG-3′
Col-Iα 5′-GAGCGGAGAGTACTGGATCG-3′
GAPDH 5′-ACCCAGAAGACTGTGGATGG-3′
108
Copyright © 2016 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
reverse transcribed into cDNA using the DyNAmo
cDNA Synthesis Kit (Thermo Scientific, Waltham,
Mass.). Real-time polymerase chain reaction was
performed using the DyNAmo Flash SYBR Green
qPCR Kit (Thermo Scientific) on a CFX96 real-
time polymerase chain reaction detection sys-
tem (Bio-Rad). Growth factors representative of
major cutaneous healing processes were selected
for the study, such as transforming growth fac-
tor (TGF)-β1 for fibrosis, fibroblast growth factor
(FGF)-7 for keratinocyte proliferation, and basic
fibroblast growth factor (bFGF) for angiogenesis.
Their primers designed for this study (Table  1)
were synthesized by the Massachusetts General
Hospital DNA Core facility. The amount of each
RNA sample was normalized to the housekeeping
gene GAPDH. To compare across all experimental
groups, we further normalized the results against
the values from the control group. Relative gene
expression quantification was calculated accord-
ing to the comparative threshold cycle method
(2−ΔΔCt).23
Statistical Analysis
All statistical data are presented as mean ± SD.
Data comparisons were performed by t tests and
one-way analysis of variance using SPSS Version
17.0 (SPSS, Inc., Chicago, Ill.). A value of p < 0.05
was considered significant.
RESULTS
Pulsed Radiofrequency Energy, Negative-
Pressure Wound Therapy, and Negative-Pressure
Wound Therapy with Pulsed Radiofrequency
Energy Treatments All Improve Wound Closure
Macroscopic wound closure was accelerated
in all experimental groups compared with the
control group (Fig.  2, above). (See Figure, Sup-
plemental Digital Content 1, which shows macro-
scopic images of representative wounds of each
group on postwounding days 0, 3, 7, 14, 21, and
28, http://links.lww.com/PRS/B970.) The pulsed
radiofrequency energy treatment group began
to show a gradual reduction in open wound area
Reverse
5′-GACTGGCGAGCCTTAGTTTG-3′
5′-TCGTCGCTCTTTCCAAACTG-3
5′-CCGTCCATCTTCCTTCATAG-3′
5′-GAAGGAATAGCCACGCTCAG-3′
5′-GGTTCGGGCTGATGTACCAG-3′
5′-CACATTGGGGGTAGGAACAC-3′
 Volume 139, Number 1 • Cutaneous Wound Healing in Diabetic Mice

Fig. 2. Macroscopic changes in wound healing. (Above) Time-course

changes of raw surface revealed highly significant differences between the
control group and the pulsed radiofrequency energy group after 14 days
(*p < 0.05); however, both negative-pressure wound therapy–only and

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 Plastic and Reconstructive Surgery • January 2017
from day 3 on, reaching statistical difference by
day 14 (p < 0.05), whereas both negative-pressure
wound therapy treatment and negative-pressure
wound therapy combined with later addition of
pulsed radiofrequency energy treatment hastened
the rates of wound closure in diabetic mice even
more significantly, reaching statistical significance
by day 3 (p < 0.05) and continued this more sig-
nificant difference throughout the experimental
period (from day 14 to day 28; p < 0.01) (Fig. 2,
above).
Combined Pulsed Radiofrequency Energy and
Negative-Pressure Wound Therapy Improves
Wound Healing Further Than Either Therapy
Alone
To determine whether the later addition of
pulsed radiofrequency energy treatment would
affect wound healing after 7 days of negative-
pressure wound therapy treatment, the changes in
wound closure in all three treatment groups were
normalized to their respective averaged day-7 val-
ues to achieve a leveled comparison starting at day
7, when there were no more modality changes to
any of the groups. This strategy was necessitated to
accommodate the unexpected animal death from
anesthesia, in congruence with the expectation
that pulsed radiofrequency energy is a relatively
late effector on wound healing compared with
negative-pressure wound therapy. When the two
Fig. 2. (Continued). negative-pressure wound therapy and pulsed
radiofrequency energy combined treatment (VFP) groups exhib-
ited significantly faster wound closure compared with the control
group after day 3 (#p < 0.05, ##p < 0.01), and the negative-pres-
sure wound therapy and pulsed radiofrequency energy com-
bined treatment group had statistically significant wound closure
after day 10 compared with the pulsed radiofrequency energy
group (&p < 0.05, &&p < 0.01). (Center) Based on the normalized
wound closure, animals subjected to negative-pressure wound
therapy and pulsed radiofrequency energy combined treatment
produced significantly faster wound closure compared with the
group treated with negative-pressure wound therapy, especially
10 days after pulsed radiofrequency energy treatment was added
(*p < 0.05). (Below) Normalized wound closure at day 7 to account
for the lead bias from the beneficial effects of negative-pressure
wound therapy during the first 7 days, the combined treatment
group displayed significantly faster wound closure than those
treated with pulsed radiofrequency energy alone (*p < 0.05,
**p < 0.01). CON, control; NPWT, negative-pressure wound ther-
apy; PRFE, pulsed radiofrequency energy; VDE, negative-pressure
wound therapy with pulsed radiofrequency energy combined
treatment. For macroscopic images of representative wounds of
each group on postwounding days 0, 3, 7, 14, 21, and 28, see Sup-
plemental Digital Content 1, http://links.lww.com/PRS/B970.
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Copyright © 2016 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
normalized wound closure curves were compared,
negative-pressure wound therapy combined with
pulsed radiofrequency energy treatment produced
a significantly faster rate of wound closure than the
group treated with only negative-pressure wound
therapy, reaching statistical significance by day 17,
or 10 days after the addition of pulsed radiofre-
quency energy (p < 0.05) (Fig. 2, center). Similarly,
when the comparison between the pulsed radio-
frequency energy and the combined treatment
groups was made, even with normalization at day
7 to account for the lead bias from the beneficial
effects of negative-pressure wound therapy dur-
ing the first 7 days, the combined therapy group
displayed significantly faster wound closure than
those treated with pulsed radiofrequency energy
alone (Fig. 2, below).
Pulsed Radiofrequency Energy, Negative-Pressure
Wound Therapy, and Negative-Pressure Wound
Therapy Combined with Pulsed Radiofrequency
Energy Treatment Enhance Reepithelialization by
Up-Regulating Keratinocyte Proliferation
Epidermal healing was analyzed in terms of
the length of reepithelialization and keratinocyte
proliferation in the bilateral leading edges of each
wound on day 14 (Fig. 3, above).21,22 Compared with
the control group, lengths of the newly formed
epithelium were increased in mice treated with
pulsed radiofrequency energy, negative-pressure
wound therapy, or negative-pressure wound ther-
apy combined with pulsed radiofrequency energy
on day 14 after wounding (p < 0.05) (Fig. 3, below,
left). Furthermore, proliferating Ki67+ basal kera-
tinocytes of all treatment groups were significantly
increased (p < 0.05) (Fig. 3, below, right).
Pulsed Radiofrequency Energy, Negative-Pressure
Wound Therapy, and Negative-Pressure Wound
Therapy Combined with Pulsed Radiofrequency
Energy Treatment Up-Regulate Angiogenesis
in Wound Beds and the Combined Treatment
Displayed Additional Beneficial Effects
Angiogenesis was determined by quantification
of blood vessel density using CD31 immunohisto-
chemical staining (Fig.  4, above). Rates of CD31+
areas of both the wound edge and the wound bed
were increased in all treatment groups on day 14
compared with the control group, but only the
increases from the wound bed were statistically sig-
nificant (p < 0.05, p < 0.01). Both the negative-pres-
sure wound therapy and the combined therapy
groups demonstrated more robust angiogenesis
compared with the pulsed radiofrequency energy
 Volume 139, Number 1 • Cutaneous Wound Healing in Diabetic Mice

Fig. 3. Reepithelialization in wound healing tissues. (Above) Ki67 staining of epidermal leading edge in each group for basal kerati-
nocyte proliferation on day 14 (original magnification, × 100). (Below, left) The length of reepithelialization was measured and sta-
tistical analyses were performed for each group. Compared with the control group, lengths of the newly formed epithelium were
increased in mice treated with pulsed radiofrequency energy, negative-pressure wound therapy, or negative-pressure wound
therapy combined with pulsed radiofrequency energy treatment on day 14 after wounding (p < 0.05), but there was no significant
difference between groups (p > 0.05). (Below, right) The number of Ki67+ basal keratinocytes in 400-μm distances along the base-
ment membrane from the leading edge was counted for each group. The results showed that lengths of the newly formed epithe-
lium were increased in all three treated groups compared with the control group (p < 0.05), but with no significant difference when
compared with each other (p > 0.05). CON, control; NPWT, negative-pressure wound therapy; PRFE, pulsed radiofrequency energy;
VDE, negative-pressure wound therapy combined with pulsed radiofrequency energy treatment.

group (p < 0.05) (Fig.  4, below). In addition, the or pulsed radiofrequency energy treatment alone,
combined therapy group appeared to have stron- although statistical significance was reached only
ger angiogenesis in both the wound edge and bed in the combined therapy and pulsed radiofre-
than just either negative-pressure wound therapy quency energy comparison in the wound bed.

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 Plastic and Reconstructive Surgery • January 2017

Fig. 4. Angiogenesis in wound tissues. (Above) Wound bed tissues were stained with CD31 for vascularity in
each group on day 14 (original magnification, × 100). (Below) Compared with the control group, the rates of
CD31+ area of wound bed of groups treated with pulsed radiofrequency energy, negative-pressure wound
therapy, or negative-pressure wound therapy combined with pulsed radiofrequency energy treatment were
significantly increased on day 14 after wounding (*p < 0.05, **p < 0.01). The negative-pressure wound therapy
and combined therapy groups both expressed higher angiogenesis compared with the pulsed radiofrequency
energy group (#p < 0.05). Although the pulsed radiofrequency energy, negative-pressure wound therapy, and
combined therapy groups expressed an increased CD31+ area in the neodermis under leading edges compared
with the control group, there was no significance. CON, control; NPWT, negative-pressure wound therapy; PRFE,
pulsed radiofrequency energy; VDE, negative-pressure wound therapy combined with pulsed radiofrequency
energy treatment.

112
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 Volume 139, Number 1 • Cutaneous Wound Healing in Diabetic Mice
Negative-Pressure Wound Therapy and Negative-
Pressure Wound Therapy Combined with Pulsed
Radiofrequency Energy Treatment, but Not
Pulsed Radiofrequency Energy Treatment, Up-
Regulate Growth Factor mRNA Expression in
Wounded Skin Tissues
Quantitative real-time polymerase chain reac-
tion analyses of tissues harvested from wound beds
were performed to assess growth factor expres-
sion. At 14 days after wounding, mRNA expres-
sion of TGF-β1, FGF-7, and bFGF was higher in
animals from the negative-pressure wound ther-
apy and combined therapy groups than those
in the control group (p < 0.05). By contrast, the
expression levels of these growth factors were not
significantly different between the pulsed radio-
frequency energy and control groups (Fig.  5,
above). In addition, compared with the pulsed
radiofrequency energy group, only bFGF expres-
sion was significantly increased in the combined
treatment group (p < 0.05).
Pulsed Radiofrequency Energy, Negative-
Pressure Wound Therapy, and Negative-
Pressure Wound Therapy Combined with Pulsed
Radiofrequency Energy Treatment Up-Regulate
Col-Iα and α-SMA mRNA Expression in Wounded
Skin Tissues
Fourteen days after wounding, α-SMA and Col-
Iα expression levels were significantly increased
in the pulsed radiofrequency energy, negative-
pressure wound therapy, and combined treatment
groups compared with the control group (p < 0.05,
p < 0.01) (Fig.  5, below). The increases in Col-Iα
among the treatment groups were more even
remarkable than those in α-SMA. Furthermore,
Col-Iα expression in the combined therapy group
was notably higher than either pulsed radiofre-
quency energy or negative-pressure wound ther-
apy alone, reaching statistical significance against
the pulsed radiofrequency energy group (Fig.  5,
below) (p < 0.05).
DISCUSSION
Negative-pressure wound therapy and pulse
radiofrequency energy are two clinically well-
established treatment modalities for wounds.24,25
Using the db/db mouse model that has been
widely used and validated to investigate chronic
cutaneous wound healing,20,26,27 several stud-
ies have demonstrated the beneficial effects of
either negative-pressure wound therapy or pulsed
radiofrequency energy on wound healing.28,29
This, however, is the first study investigating the
Copyright © 2016 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
potential complementary relationship between
negative-pressure wound therapy and pulsed
radiofrequency energy in diabetic cutaneous
wounds. Although negative-pressure wound ther-
apy is widely used in the treatment of a variety of
soft-tissue wounds,30 its optimal application is still
being explored.31–33
In the present study, all wounds treated with
negative-pressure wound therapy (negative-pres-
sure wound therapy–only and combined therapy
groups) demonstrated significantly faster wound
closure compared with those treated without neg-
ative-pressure wound therapy (control and pulsed
radiofrequency energy–only groups) at every time
point, whereas the pulsed radiofrequency energy
group exhibited significantly faster closure than
the control group (Fig.  2). Furthermore, there
appeared to be a complementary effect of the
pulsed radiofrequency energy treatment which,
when added to the negative-pressure wound ther-
apy treatment, produced an even more robust
cutaneous healing than the negative-pressure
wound therapy treatment alone in a diabetic
mouse wound healing model.
One of the original studies on negative-pres-
sure wound therapy in a pig wound healing model
pointed to its significant physiologic effects with
increasing granulation tissue formation, decreas-
ing bacterial loads, and increasing tissue sur-
vival, all seemingly perpetuated by an immediate
increase of blood flow to the wound site.34 In a rat
wound model, faster wound closure rates were evi-
dent with negative-pressure wound therapy at all
time points; interestingly, whereas the expression
of vascular endothelial growth factor and bFGF,
both proangiogenic factors, increased early on,
they dramatically decreased by day 7.35
Collagens are the most abundant proteins
in animal and constitute the main structural ele-
ment of extracellular matrix.36 Miller et al. found
that although immature collagen (a precursor
marker for cutaneous healing) had significantly
increased in negative-pressure wound therapy–
treated wounds at day 9, there was no difference
early on at days 4 and 7 compared with the con-
trol group.37 Likewise, in a study by Jacobs et al.,
all negative-pressure wound therapy–treated
wounds exhibited amounts of collagen content
comparable to those in the control wounds.35 By
contrast, pulsed radiofrequency energy therapy
accelerated macroscopic wound closure in the
late phase of healing in our previous studies, with
increased granulation tissue formation and colla-
gen deposition not significant until 17 days after
wounding.5,6
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 Plastic and Reconstructive Surgery • January 2017

Fig. 5. Quantitative real-time polymerase chain reaction of the mRNA

expression of wound healing tissues at 14 days after wounding. Data
were standardized by the expression level of GAPDH in each sample and
presented as the relative quantitation. (Above) Growth factors relative to
wound healing (TGF-β1, FGF-7, and bFGF). Levels of mRNA expression
of TGF-β1, FGF-7, and bFGF were higher in animals from the negative-
pressure wound therapy and the combined therapy group than those in
the control group (*p < 0.05). Compared with the pulsed radiofrequency
energy group, only bFGF expression was significantly increased in the com-
bined treatment group (#p < 0.05). By contrast, the expression of TGF-β1,
FGF-7 and bFGF was not significantly different between the pulsed radio-
frequency energy group and the control group (p > 0.05). (Below) Collagen
Iα (Col-Iα) and α-smooth muscle actin (α-SMA). The expression of collagen
Iα was significantly increased in wounds treated with pulsed radiofre-
quency energy, negative-pressure wound therapy, and negative-pressure
wound therapy combined with pulsed radiofrequency energy treatment
compared with the control group, and α-SMA gene expression was signifi-
cantly increased in both the negative-pressure wound therapy group and
the combined therapy group (*p < 0.05, **p < 0.05). The increasing trend of
Col-Iα mRNA of the combined therapy group was remarkable and was sig-
nificantly higher than in the pulsed radiofrequency energy treatment–only
group (#p < 0.05). CON, control; NPWT, negative-pressure wound therapy;
PRFE, pulsed radiofrequency energy; VDE, negative-pressure wound ther-
apy combined with pulsed radiofrequency energy treatment.

114
Copyright © 2016 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
 Volume 139, Number 1 • Cutaneous Wound Healing in Diabetic Mice
The difference in timing of those augmenta-
tive effects by negative-pressure wound therapy
and pulsed radiofrequency energy on cutaneous
wound healing suggests complementary mecha-
nisms. Studies have shown that negative-pressure
wound therapy facilitates immediate wound clo-
sure by vacuum-induced macrodeformation and
further subjects impacted tissues to simultane-
ous microdeformational changes that give rise
to increased angiogenesis and tissue prolifera-
tion.3,10,12,30 It is generally accepted that subat-
mospheric pressure–induced mechanical tissue
deformation accelerates wound healing by activat-
ing signal transduction and promoting cell prolif-
eration.2,11,12,35,38 Pulsed radiofrequency energy, in
contrast, has been known to augment cutaneous
wound healing by means of relatively late effects.
In previous studies, using a 1-cm2 full-thickness
skin wound in a genetically diabetic mouse (db/db)
cutaneous healing model, we demonstrated that
pulsed radiofrequency energy accelerated cuta-
neous wound healing by increasing collagen pro-
duction and deposition, an effect not manifested
significantly until more than 2 weeks after wound-
ing.5,6 This late effect is also confirmed in the pres-
ent study. Similarly, a study by Callaghan et al.,
using a 0.5-cm-diameter circular cutaneous wound
in both db/db and wild-type mice, also found a sig-
nificant reduction in wound area after day 7.14
These reproducible results suggest that pulsed
radiofrequency energy therapy produces a rela-
tively late-onset action in the acceleration of mac-
roscopic wound closure. It is therefore interesting
to note from the current study in wounds treated
with negative-pressure wound therapy from day 1
that the addition of pulsed radiofrequency energy
treatment at day 7 (as in the combined therapy
group) would further enhance wound healing
over negative-pressure wound therapy alone, an
improvement that became statistically signifi-
cant by day 10 (day 17 after wounding) (Fig.  2)
(p < 0.05).
Wound healing represents an intricate and
dynamic process of angiogenesis, cell prolifera-
tion, extracellular matrix deposition, and wound
contraction.39–41 Derailment of any of these steps
can lead to impaired healing or abnormal scar
formation.39,40 Ultimately, wound closure is man-
ifested as extracellular matrix deposition and
reepithelialization potentiated initially through
healthy neoangiogenesis.36,42
In our previous study,6 we showed proliferation
of dermal cells following treatment with pulsed
radiofrequency energy, and we now show that kera-
tinocytes also undergo heightened proliferation
Copyright © 2016 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
under pulsed radiofrequency energy. This finding
is supported by the length of reepithelialization
14 days after wounding. Negative-pressure wound
therapy produces robust increases in epidermal
proliferation,43 which was again demonstrated by
the current study. However, although we showed
that the keratinocyte proliferation and conse-
quent reepithelialization increased significantly
in all experimental groups treated with negative-
pressure wound therapy, pulsed radiofrequency
energy, and their combination compared with the
control group (Fig. 3) (p < 0.05), we did not detect
any significant added benefits of the combination
therapy. This would likely suggest that there may
not be a diverging pathway to affect keratinocyte
proliferation by either pulsed radiofrequency
energy or negative-pressure wound therapy.
Robust angiogenesis is a hallmark effect of
negative-pressure wound therapy on cutaneous
wound healing. It is a relatively early component
of macroscopic healing, on which the rest of the
wound healing process is predicated.44–46 Inter-
estingly, bFGF, a proangiogenic factor, has been
shown to increase significantly early in wound
healing in response to negative-pressure wound
therapy but to decrease dramatically by day 7.35
Pulsed radiofrequency energy, in contrast, did not
appear to show an overwhelming effect on neo-
vascularization when examined over the whole
extent of the wound in our prior study at the
mRNA level.6 However, in our present study, we
further refined the area of focus on neovascular-
ization into that of the wound edge and wound
bed when determining the extent of CD31-pos-
itivity, the vascular marker. Our results showed
that although angiogenesis in the three treatment
groups was increased compared with the control
group, only those of the wound bed reached sig-
nificant difference, with the two negative-pressure
wound therapy groups showing more profound
increases. Again, we witnessed marginal added
benefits on neovascularization by combining
pulsed radiofrequency energy and negative-pres-
sure wound therapy, underscoring the predomi-
nant neoangiogenic effects of negative-pressure
wound therapy on wound healing and that the
complementarity exerted on cutaneous wound
healing by pulsed radiofrequency energy and
negative-pressure wound therapy probably does
not arise from their effects on neovascularization
alone.
Likewise, although all increased at the mRNA
expression level at day 14 compared with the control
group (with those of the negative-pressure wound
therapy groups reaching statistical significance),
115
 Plastic and Reconstructive Surgery • January 2017
none of the major growth factors for profibrosis,
epidermal proliferation, and angiogenesis showed
any additive effects of pulsed radiofrequency
energy and negative-pressure wound therapy. We
presented here representative results from TGF-β,
FGF-7, and bFGF, respectively. These results fur-
ther support the notion that not one single growth
factor–related process was likely to give rise to the
complementary effects that pulsed radiofrequency
energy and negative-pressure wound therapy
have on wound healing. Alternatively, in the case
of bFGF, given our seeming lack of a significant
increase in expression at the mRNA level on pulsed
radiofrequency energy treatment and the well-sup-
ported findings of profound increases of angiogen-
esis, mostly at the histologic47 and protein levels,14,48
the possibility of additional posttranscriptional
modulation of these growth factors during wound
healing on various treatment modalities cannot be
ignored and should be the focus of future studies.
It has been demonstrated that collagen pro-
duction increases as a relatively late response to
either pulsed radiofrequency energy6 or negative-
pressure wound35,49 therapy, and we have also
validated these findings in our present study.
Interestingly, we noted that collagen expression
has significantly increased when the pulsed radio-
frequency energy and negative-pressure wound
therapy therapies were combined compared
with either therapy alone. This is important, as
increases in collagen production and granula-
tion formation are hallmarks of improved wound
healing effected by either pulsed radiofrequency
energy or negative-pressure wound therapy ther-
apy. The synergy at the molecular level of collagen
expression exerted by both of those therapies can
seemingly lead to the added benefits in cutaneous
wound healing at the macroscopic level.
The totality of our results seems to suggest
that negative-pressure wound therapy provides an
early boost to healing by means of its profound
effects on neovascularization, that both negative-
pressure wound therapy and pulsed radiofre-
quency energy therapies result in heightened cell
proliferation, and that relatively late effects caused
by negative-pressure wound therapy and pulsed
radiofrequency energy during collagen expres-
sion and presumably production may explain the
complementary effects of those two therapies on
cutaneous wound healing. Further experiments
may be needed to delineate the precise molecu-
lar pathways of these complementary actions, but
what we have presented here could become the
basis for combination therapies in the clinical
wound healing arena.
116
Copyright © 2016 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
Lifei Guo, M.D., Ph.D.
Department of Plastic Surgery
Lahey Hospital & Medical Center
41 Mall Road, 6W
Burlington, Mass. 01805
lifei.guo@lahey.org
Zhaohua Jiang, M.D., Ph.D.
Department of Plastic and Reconstructive Surgery
Shanghai Ninth People’s Hospital
Shanghai Jiao Tong University School of Medicine
Shanghai, People’s Republic of China
zhhjiang@hotmail.com
acknowledgment
This study was funded through a grant to Lahey
Hospital & Medical Center by Regenesis Biomedical,
Inc. (Scottsdale, Ariz.), and the Robert E. Weiss Foun-
dation (Burlington, Mass.).
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