JBUR-4287; No.

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Available online at www.sciencedirect.com

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journal homepage: www.elsevier.com/locate/burns

Stromal vascular fraction improves deep partial

thickness burn wound healing

Sibel Atalay a, Atilla Coruh b,*, Kemal Deniz c

a
Private Medisu Hospital, Department of Plastic Reconstructive and Aesthetic Surgery, Antalya, Turkey
b
Erciyes University Medical Faculty, Department of Plastic Aesthetic and Reconstructive Surgery , Kayseri, Turkey
c
Erciyes University Medical Faculty, Department of Pathology, Kayseri, Turkey

article info abstract

Article history: Objective: The practice of early burn wound excision and wound closure by immediate
Accepted 23 January 2014 autologous skin or skin substitutes is the preferred treatment in extensive deep partial and
full-thickness burns. To date there is no proven definite medical treatment to decrease burn
Keywords: wound size and accelerate burn wound healing in modern clinical practice. Stromal
Deep second degree burn vascular fraction is an autologous mixture that has multiple proven beneficial effects on
Deep partial thickness burn different kinds of wounds. In our study, we investigated the effects of stromal vascular
Burn wound fraction on deep partial-thickness burn wound healing.
Stromal vascular fraction Methods: In this study, 20 Wistar albino rats were used. Inguinal adipose tissue of the rats
Adipose tissue derived stem cell was surgically removed and stromal vascular fraction was isolated. Thereafter, deep
Burn wound healing second-degree burns were performed on the back of the rats by hot water. The rats were
stem cell divided into two groups in a randomized fashion. The therapy group received stromal
vascular fraction, whereas the control group received only physiologic serum by intrader-
mal injection. Assessment of the burn wound healing between the groups was carried out by
histopathologic and immuno-histochemical data.
Results: Stromal vascular fraction increased vascular endothelial growth factor, proliferat-
ing cell nuclear antigen index, and reduced inflammation of the burn wound. Furthermore,
vascularization and fibroblastic activity were achieved earlier and observed to be at higher
levels in the stromal vascular fraction group.
Conclusions: Stromal vascular fraction improves burn wound healing by increasing cell
proliferation and vascularization, reducing inflammation, and increasing fibroblastic
activity.
# 2014 Elsevier Ltd and ISBI. All rights reserved.

most common causes of burns in general population.

1. Introduction Early tangential or full-thickness excision of severe deep
partial and full-thickness burn wounds with immediate
Burn trauma is one of the most devastating injuries that closure by auto skin grafting is the golden standard in burn
a person can experience. Scalding and flame burns are the treatment used for reducing the burn mortality, morbidity,

* Corresponding author at: Erciyes University Medical Faculty, Department of Plastic Aesthetic and Reconstructive Surgery, Turkey.
Tel.: +90 532 235 1872; fax: +90 352 437 7333.
E-mail addresses: atilla.coruh@superonline.com, atilla.coruh1955@gmail.com (A. Coruh).
http://dx.doi.org/10.1016/j.burns.2014.01.023
0305-4179/# 2014 Elsevier Ltd and ISBI. All rights reserved.

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complications and shortening the duration of therapy and

hospital stay [1,2].
Stromal vascular fraction (SVF) provides a rich source of
adipose derived stem cells (ADSCs) which differentiate into
lineages of adipocytes, osteoblasts, chondrocytes, myocytes
and neuron-like cells [3–5]. Stromal vascular fraction of
adipose tissue which acts as a source of ADSCs have anti-
apoptotic [6], anti-oxidant [7], anti-inflammatory, immune
modulatory via direct immune suppressive/tolerance indu-
cing ability [8,9]. Since angiogenesis is a critical component of
wound healing, ADSCs are angiogenic to hypoxic stimuli by
upregulating angiogenic gene expression of vascular endothe-
lial growth factor (VEGF), hepatocyte growth factor (HGF), basic
fibroblast growth factor (bFGF), platelet-derived growth factor
B (PDGFB) [10]. Adipose derived stem cells can also promote
wound healing via stimulating collagen synthesis of human
dermal fibroblasts (HDF) [11–17] and may be an alternative to
bone marrow derived mesenchymal stem cells.
Currently, there is no efficient and cost-effective treatment
that can accelerate burn wound healing, decrease the size of
burn wound excision area and reduce the tissue necrosis by
preventing conversion of deep partial thickness to full-
thickness burns. The aim of this study was to investigate
the effects of SVF on deep partial thickness burn wound
healing except epithelization.

2. Materials and methods

The study was supported by the Scientific Research Projects

Unit of Erciyes University and it was conducted in accordance
with ethical standards after approval by our Local Ethics
Fig. 1 – Diagrammatic illustration of bilaterally harvested
Committee for Laboratory Animals at Experimental and
inguinal fat pad donor sites of the rat (ventral view).
Clinical Research Center (No: 10/29; 03/10/2010).

2.1. Animals
Twenty male adult Wistar rats weighing between 187 and
380 g were used in this study. The rats were monitored within
a standard laboratory environment. All the rats were fed with
standard laboratory diet before and during the study.
2.2. Anesthesia and analgesia
The rats were anesthetized using the combination of ketamine
hydrochloride [Ketalar1, Pfizer (50 mg/kg)] and xylazine
hydrochloride [Rompun1, Bayer (5 mg/kg)] intraperitoneally.
Early post operative and five days post-burn pain management
was performed by adding 2 mg/ml paracetamol (Calpol1,
GlaxoSmithKline) in rats’ drinking water [18].
2.3. Stromal vascular fraction preparation
adipose tissues were washed extensively by phosphate buffer
solution (PBS, Sigma1) to remove the contaminating debris
and red blood cells and then minced with fine tissue scissors.
The fragmented tissues incubated with 0.1% collagenase
(collagenase from Clostridium histolyticum C0130, Sigma1)
and kept in a slow shaking water bath at 37 8C for 60 min.
Thereafter, collagenase was removed by diluting the samples
with PBS. The cell suspension was centrifuged twice at
1300 rpm (260G) for 5 min. The supernatant composed of
mature adipocytes was removed. The precipitate gathered at
the bottom was filtrated with 100 mm mesh filter and used as
SVF (Fig. 2) [3,19,20]. Viable cells were measured by adding
trypan blue to SVF and counting them by the help of Thoma
slide [4]. Approximately there were 4  106/ml viable cells. We
did not test for the ratio of viable versus non-viable cells in our
SVF.

Both inguinal regions of the rats were clipped and cleaned
with sterile 10% povidone iodine. Inguinal fat pads of 20 rats
were removed (two for each rat with 2  20 rats = 40 inguinal
fat pads) and 1  1  1 cm (1 cm3) adipose tissue was excised
from each inguinal fat pad on both sides. The incisions were
closed by 3/0 silk sutures (Fig. 1). We pooled all the cells from
the same rats to make a master batch of SVF. The excised
2.4. Creating deep partial thickness burn
A pilot study was performed to create a deep partial thickness
burn by hot water. The rats were prepared by clipping the hair
on their back skin and cleaned with sterile 10% povidone
iodine under general anesthesia. Scalds were performed with
the pet bottle opening area of 7 cm2 with a radius of 1.5 cm.

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Fig. 2 – (a) Top layer; triglyceride released from fragmented
adipose cells. (b) Middle layer; mature adipose cells and
connective tissue. (c) Bottom layer (stromal vascular
fraction): pre-adipocytes, fibroblasts, endothelial cells and
stem cells.
The bottle was held upside down with the mouth tightly
contacting with the back skin of the rat without any hot water
leakage (Fig. 3). Deep partial thickness burns were created by
applying 70 8C hot water for 30 s which were confirmed
histopathologically (Fig. 4). Burn wounds were treated by open
Fig. 3 – Creating deep partial thickness burn on the back
skin of the rat by hot water.
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3
Fig. 4 – Hematoxylin and Eosin (H&E) stained
histopathological picture of deep partial-thickness burn.
management without topical antibiotics. Created burn wound
area was approximately 2% of total body surface area (TBSA)
which was calculated by the formula defined by Gilpin [21].
The size of the burn wound area was planned enough allowing
biopsies while paying attention to make it a small burn which
may not need post-burn intravenous fluid resuscitations and
also not to cause life-threatening clinical conditions.
2.5. Application of SVF
At the same day of SVF production, deep partial thickness
burns were created by applying 70 8C hot water for 30 s on the
back skin of the rats and then they were randomized into
therapy and control groups of 10 rats each. Therapy group
group received 1cc of 0.9% NaCl intradermal injection with 26G
syringe by raising a wheal homogeneously fitting to all burn
area.
3. Evaluation
3.1. Histopathologic evaluation
Biopsies were taken sequentially from each quadrant of 7 cm
circular burn under general anesthesia at 3, 7, 10 and 14 days,
respectively. In order to decrease the inflammatory response
biopsy donor site it was closed primarily after each biopsy
(Fig. 5). Sizes of the biopsies were 5 mm  10 mm  5 mm,
which were prepared by 5m sections from the specimens
Fig. 5 – The schema of the distribution of the days and the
technique of the biopsy.
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Fig. 6 – Histopathologic evaluation. (a) Mild, (b) moderate, (c) severe vascular proliferation (T200, hematoxylin and eosin). (d)
Mild, (e) moderate, (f) severe inflammatory infiltrate mainly composed of lymphocytes (T400, hematoxylin and eosin). (g)
Mild, (h) moderate, (k) severe fibroblastic proliferation (T200, hematoxylin and eosin).

embedded in paraffin blocks and stained with hematoxylin–
eosin. Histologic analysis of the specimens performed under
light microscope on hematoxylin and eosin stained slides.
Tissue slides were reviewed under low power field (40
magnification) and graded for the highest scored area.
Parameters of fibroblast proliferation, vascular proliferation,
polymorphonuclear and mononuclear inflammatory infiltrate
was graded as absent (0), mild (score 1), moderate (score 2), or
marked (score 3) semiquantitatively (Fig. 6) [22]. Vascular
structures with open lumens and clustering endothelial cells
are determined as neovascularization in this study. All
histological examinations were performed by the same
pathologist, who was blinded to group allocation.
3.2. Immunostaining
The 4-mm sections were mounted on poly-L-lysine coated
slides and de-paraffinized and hydrated through graded
alcohol to water. Endogenous peroxidase activity was
removed by dipping the sections in 3% hydrogen peroxide
for 10 min. Samples were microwaved twice for 5 min in
0.1 mol/l citrate buffer of pH 6.0 before immunostaining. The

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sections were then incubated with primary antibodies to VEGF

(Thermoscientific/Lab Vision, Germany) at 1/25 dilution for
20 h, and PCNA (Thermoscientific/Lab Vision, Germany) at 1/
100 dilution for 2 h. The avidine-biotin-peroxidase procedure
was performed next. The immunoreaction was visualized
using diaminobenzidine (DAB), and sections were lightly
counterstained in Mayers hematoxylin, dehydrated, and
mounted.

3.3. Immuno-histochemical analysis

3.3.1. Vascular endothelial growth factor expression

Vascular endothelial growth factor expression was evaluated
in maximally immunostained areas at 400 magnification for
the percentage of the VEGF positive cells and the staining
intensity. The percentage of the VEGF positive cells was scored
as follows: (1) <33% of the cells, (2) 33–66% of the cells and (3)
>66% of the cells. The staining intensity of VEGF expression
was graded as (1) weak, (2) moderate and (3) strong. The sum of
these scores was calculated as the VEGF index [23].
3.3.2. Proliferating cell nuclear antigen analysis
Nuclear brown staining was recognized as positive in tissue
sections marked by proliferating cell nuclear antigen antibody.
Proliferating cell nuclear antigen expression was evaluated by
counting 100 cells in highly stained areas (hot spots) under
400 magnification. The PCNA index was determined by the
ratio of the number of positive nuclei/100 nuclei [24,25].
3.4. Statistical analysis
Statistical analysis of the study was performed with Statistical
Package for the Social Sciences for Windows (2000) (SPSS) 15.0
and Sigmastat 3.5 package programs with 95% confidence
interval. Shapiro–Wilk test was used to test whether the
variables were normally distributed or not. Mann–Whitney U
test was used to analyze two independent samples. Depen-
dent variables were evaluated by repeated measures ANOVA
and Friedman analyses. A ‘‘p’’ value of <0.05 was recognized as
statistically significant. Power index (analysis) calculation was
carried out by examining similar studies, and VEGF and PCNA
values were found to be 100% and 95%, respectively.
Fig. 7 – Comparison of inflammation in control and therapy
groups; *p < 0.05.
intensity exhibited a decline in the control group and became
(Fig. 7).
4.1.2. Vascularization
Histopathologically, there were statistically significant differ-
ences between the therapy and the control groups with regard
to the vascularization at 3, 10, and 14 days. However, no
statistically significant difference was observed at 7 days.
Vascularization was determined earlier in the therapy group.
Variance analysis investigating the distribution of vascular-
ization relative to the biopsy days revealed statistically
significant differences between the therapy and control
groups at all days (Fig. 8).
4.1.3. Fibroblastic proliferation
Histopathologic evaluation of the fibroblastic proliferation
revealed significant difference between the groups at 3 and 14
days, whereas there was no significant difference at 7 and 10
days. The fibroblastic proliferation began on day 3 in the
therapy group, on day 7 in the control group. On day 10, the
fibroblastic proliferation was similar in both groups. On day 14,
the control group exhibited a mild increase, whereas the
therapy group showed a statistically significant increase. The
variance analysis investigating the distribution of fibroblastic

4. Results

4.1. Histopathologic results

We evaluated the healing process with regard to inflamma-

tion, vascularization, and fibroblastic proliferation. Therapy
and control groups were compared with each other for each
parameter.

4.1.1. Inflammation
According to the analyzed tissue specimens, both groups had
almost equal severity of inflammation at post burn third and
seventh days. On day 10, inflammation intensity was observed
to be markedly increased in the control group, while it was
decreased in the therapy group and the difference between the Fig. 8 – Comparison of vascularization scores in control and
groups was statistically significant. On day 14, inflammation therapy groups; *p < 0.05.

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Fig. 11 – Comparison of PCNA index in control and therapy

Fig. 9 – Comparison of fibroblastic proliferation in control groups; *p < 0.05.
and therapy groups; *p < 0.05.

values between the groups showed that there were statisti-

proliferation relative to the biopsy days in control group
revealed that there were differences at 3, 7 and 10 days,
whereas no difference was determined at 14 days. In the
therapy group, there was statistically significant increase in all
days (Fig. 9).
4.2. PCNA index
Stromal cells including endothelial cells and fibroblasts
showed nuclear positivity for PCNA and these positive cells
were counted to find the PCNA index. The comparison of PCNA
cally significant differences at 3, 10 and 14 days (Fig. 10a and b).
The variance analysis concerning the distribution of PCNA
index relative to the biopsy days revealed that there were
statistically significant difference between day 3 and other
days in the control group. The therapy group showed
statistically significant difference between all days (Fig. 11).
4.3. VEGF expression score
Vascular endothelial growth factor expression was seen in
stromal cells including endothelial cells inflammatory cells

Fig. 10 – Nuclear PCNA staining in fibroblasts and in endothelial cells (arrows): (a) in control group and (b) in therapy group at
the 10th day (T400). Cytoplasmic VEGF staining in fibroblasts and in endothelial cells (c) in control group and (d) in therapy
group at the 10th day (T200).

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Fig. 12 – Comparison of VEGF scores in control and therapy
groups; *p < 0.05.
and fibroblasts. The VEGF values were statistically significant
between the therapy and control groups at 3 and 10 days
(Fig. 10c and d). The therapy group had a VEGF expression
score that was almost twice the value of the control group on
day 3. The VEGF expression scores continued to be higher in
the therapy group than in the control group until day 10. On
day 14, both groups had similar VEGF expression scores. The
variance analysis concerning the distribution of VEGF expres-
sion scores relative to the biopsy days revealed that there were
statistically significant difference between day 3 and other
days in both therapy and control groups (Fig. 12).
5. Discussion
We preferred scalding to create deep second-degree burns on
rats’ back skin which induces homogenous burns with the
same depth. In the literature, various temperatures and
durations have been reported creating deep second-degree
burn models [26–29]. We used bottles with the mouth opening
area of 7 cm2 which created approximately 2% TBSA burn. We
could have used the immersion method as well, but this would
cause leakage of hot water and provides less control over the
size of the created burn surface (Fig. 3). We applied SVF
intradermally below the necrotic tissue since when it is
applied topically, it might leak through the necrotic tissue
barrier and fail to induce the expected effects on the burn
wound. Stromal vascular fraction was applied immediately
after the creation of burns, because it is a well-known fact that
conversion of burn wound may be prevented by performing
appropriate therapies within the first 72 h [30,31].
The presence of proliferating cells is an indicator of wound
healing. Alemdaroglu et al. [26] used bromodeoxyuridine in
the detection of proliferating cells in burn wound. This is an
indirect measurement test for proliferating cells. Souza et al.
[32] preferred PCNA method for the evaluation of third degree
burns. We preferred PCNA method which allows direct
detection of DNA-producing cells during replication and
repair phases. In our study, vascularization was evaluated
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7
by revealing tubular structures in histologic examinations and
angiogenesis was evaluated by VEGF expression of the control
and the therapy groups. We preferred the semi-quantitative
method defined by Li et al. [23] for the evaluation of VEGF
expression by immuno-histochemical analysis. Increased
vascularization by SVF was evidenced by both histological
analysis showing the increase in tubular vascular structures
and immuno-histochemical analysis exhibiting raised VEGF
levels. Rehman et al. [6] showed that the angiogenic impact of
stromal cells were associated with the proliferation of
endothelial cells and raised VEGF release.
Wound healing was monitored and evaluated for 14 days.
On day 14, the epithelization was found to be incomplete in
both groups. Khorasani et al. [27] detected epithelization at 25
days, whereas Baoyong et al. [33] observed formation of new
epidermal cells at 14 days and completion of epithelization at
21 days. In our study, monitoring period should be longer than
14 days in order to evaluate epithelialization. Baoyong et al.
[33] also monitored the healing of second degree burn wounds
by performing biopsies at 3, 5, 7, 14 and 21 day, whereas
Alemdaroglu et al. [26] performed biopsies at 3, 7, and 14 days.
Our pilot study revealed that burn depth and distinction of
viable cells became clear only at post-burn 3 days. Therefore,
we started to perform biopsies on day 3. In consideration of
wound healing phases, biopsies aiming to evaluate cell
proliferation were performed at 7 and 10 days. Several full-
thickness excisional biopsies, like ours at different times in the
same burn wound had the same amount of influence, because
of the same excisional wounds were carried out in both control
and therapy groups [26,33].
Fibroblast density was found to be significantly higher in
SVF group than the control group according to the biopsy
results at 3 days. This finding shows that fibroblasts are
protected and their proliferation is induced in SVF group. This
favorable effect is believed to be associated with the direct
transfer of fibroblasts into the damaged area as well as the
protective influence ADSCs on fibroblasts. Kim et al. [17]
reported that ADSCs increase fibroblast proliferation by their
paracrine effects. These data are consistent with the increased
fibroblast levels in our study. Adipose derived stem cells
accelerated wound healing in an ischemic wound model by
Rehman et al. [6], a full-thickness excisional wound model by
Kim et al. [7] and radiation and non-radiation groups by
Ebrahimian et al. [15] which their findings were also consistent
with those of our SVF therapy group.
Inflammation did not increase after 3 days in the burn
wounds treated with SVF, whereas the control group exhibited
progressively increased inflammation until day 10. Gonzales
et al. [9] showed that ADSCs reduced autoimmune response
and inflammation by immune modulation. Local injection of
ADSCs reduced the synthesis of various inflammatory
cytokines and chemokines, while increasing the release of
interleukin-10, which is an anti-inflammatory cytokine. Kim
et al. [7] studied full-thickness excisional wound healing and
showed that dermal inflammatory cell density was lower in
the ADSC group. These data are consistent with the results of
our study, as well.
The role of stem cells in burn wound healing has been
investigated by various studies. Rea et al. [34] used bone
marrow-derived stem cells and showed that stem cells
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differentiated into inflammatory cells, fibroblasts and kerati-
nocytes. Rasulov et al. [35] demonstrated in their clinical study
that the bone marrow-derived stem cells accelerated wound
healing in burns. Obtaining stem cells from bone marrow is
difficult than obtaining them from adipose tissue. Adipose
tissue derived stem cells are a rich source of autograft which can
be obtained easily [36]. Furthermore, recent basic research and
preclinical studies have revealed that the use of ADSCs in
regenerative medicine is not limited to mesodermal tissue but
extends to both ectodermal and endodermal tissues and organs,
although ADSCs originate from mesodermal lineages [5].
The efficacy of purified ADSCs have a greater potential for
in vitro differentiation as compared with that of SVF
composed of heterogenous cell mixture [4] However, ADSC
isolation from SVF requires more equipment, longer time, and
higher expenditure. In cases requiring immediate medical
intervention such as in burns, the use of freshly obtained SVF
will be more appropriate. Since adipose tissue is obtained from
patient’s own tissues, it does not cause allogeneic reaction or
pose an ethical problem. Acquisition of SVF by lipoaspiration
is an easy-to-apply method that does not cause marked
increases in morbidity.
6. Conclusion
We showed that applying SVF intradermally into the deep
partial thickness burns will help and accelerate burn wound
healing and may reduce the need for skin grafting, decrease the
length of hospital stay, morbidity, mortality and expenditure of
burn therapy by increasing cell proliferation, vascularization,
fibroblastic activity and reducing inflammation.
Conflict of interest statement
All authors of this manuscript did not receive any funding for
this study from any organization except ‘‘Scientific Research
Projects Unit of Erciyes University’’.
Funding
No funding received for this study from any organization except
‘‘Scientific Research Projects Unit of Erciyes University’’.
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