Original Research 379

Locally injected autologous

platelet-rich plasma enhanced
tissue perfusion and improved
survival of long subdermal plexus
skin flaps in dogs
M. Karayannopoulou1; L. G. Papazoglou1; P. Loukopoulos2; G. Kazakos1; A. Chantes3;
N. Giannakas1; I. Savvas1; D. Psalla2; M. Kritsepi-Konstantinou4; D. Dionyssiou3
1Department of Clinical Studies, Companion Animal Clinic, School of Veterinary Medicine, Faculty of Health Sciences,
Aristotle University of Thessaloniki, Thessaloniki, Greece; 2Laboratory of Pathology, School of Veterinary Medicine,
Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece; 3Department of Plastic Surgery,
Medical School, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece; 4Department of
Clinical Studies, Diagnostic Laboratory, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University
of Thessaloniki, Thessaloniki, Greece

Keywords logous PRP injected evenly between sutures Introduction

Dog, Laser-Doppler flowmetry, long skin flap
survival, platelet-rich plasma, subdermal
plexus skin flap
Summary
Objectives: Distal flap necrosis remains a
major complication in subdermal plexus (ran-
dom) skin flaps. Platelet-rich plasma (PRP)
has been shown to improve the survival of
ischemic random skin flaps in rats. The objec-
tive of this study was to evaluate the effect of
locally injected autologous PRP on the sur-
vival of long (5:1 length-to-width ratio) sub-
dermal plexus skin flaps in dogs.
Methods: A 2x10 cm subdermal plexus skin
flap was created bilaterally on the abdominal
wall of six Beagle dogs. One randomly
selected side received 2.5 ml of fresh auto-
Correspondence to:
Dr M. Karayannopoulou
Aristotle University of Thessaloniki
Faculty of Health Sciences
School of Veterinary Medicine
Department of Clinical Studies
Companion Animal Clinic
11 St. Voutyra str.
546 27 Thessaloniki
Greece
Phone: +30 231 099 4407
E-mail: marikara@vet.auth.gr
underneath the flap, whereas the other side
was left untreated (control). Skin flap survival
was evaluated macroscopically, histologically
and by laser-Doppler flowmetry measure-
ments of tissue perfusion.
Results: Flap percentage survival on day 10
(96.3% versus 74.5%; p = 0.046) and tissue
perfusion (p <0.036) were significantly
higher in PRP-treated flaps compared with
controls. Histologically, there was less oede-
ma in PRP-treated flaps compared to controls
(p = 0.01), whereas collagen production and
angiogenesis did not differ significantly be-
tween the two groups.
Clinical significance: The use of locally in-
jected autologous PRP increases tissue perfu-
sion and improves the survival of long sub-
dermal plexus skin flaps in dogs.
Vet Comp Orthop Traumatol 2014; 27: 379–386
http://dx.doi.org/10.3415/VCOT-14-02-0030
Received: February 18, 2014
Accepted: July 17, 2014
Epub ahead of print: August 4, 2014
Skin flaps are used in both human and vet-
erinary surgery for the closure of acute and
chronic wounds that result from trauma or
tumour excision (1, 2). In dogs, one com-
monly used type of flap is the subdermal
plexus (random pattern) flap. In dogs, the
panniculus carnosus muscle is also in-
cluded in this type of flap because its separ-
ation is almost impossible due to its ana-
tomical association with the subdermal
plexus. Subdermal plexus flaps receive
their blood supply from small vessels de-
rived from the subdermal (deep) plexus,
which consists of the terminal branches of
the direct cutaneous vessels and runs paral-
lel to the skin surface (1, 3). One major
complication of these flaps is necrosis, es-
pecially at their distal part, occurring
mainly due to insufficient vascularity and
thus inadequate blood supply for skin
metabolic requirements (1, 2, 4, 5). The
length-to-width ratio is considered to be an
important factor affecting the survival of
this type of flap (2, 6). Length-to-width ra-
tios from 1:1 to 3:1 have been suggested in
the veterinary literature (2, 7). This ratio
may be different in dogs, although specific
recommendations have not been reported
(1, 7).
Various treatments have been evaluated
and recommended to improve skin flap
survival, especially when long flaps are
needed to cover large defects (1, 2, 5). Cer-

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380 M. Karayannopoulou et al.: Platelet-rich plasma and skin flap survival in dogs
tain growth factors implicated in neovascu-
larisation, including vascular endothelial
growth factor (VEGF), basic fibroblast
growth factor (bFGF), platelet-derived
growth factor (PDGF) and transforming
growth factor-beta (TGF-β), have demon-
strated ability to improve the microcircu-
lation and thus the viability of a skin flap
(5, 6, 8–11). It has been suggested that the
combined use of growth factors may be
more effective than the use of a single
growth factor for improving wound healing
(12, 13).
The platelet α-granules contain many of
the aforementioned growth factors that are
released in response to tissue injury and
after platelet aggregation. Platelet-rich
plasma (PRP) contains a high concen-
tration of platelets and is, therefore, rich in
growth factors (14–17). To date, PRP appli-
cations have been reported in both experi-
mental and clinical studies, mostly in the
fields of orthopaedic surgery (bone, tendon
or ligament trauma), plastic and maxillofa-
cial surgery, soft tissue surgery (chronic
wounds such as diabetic ulcers), and den-
tistry (16–19). Platelet-rich plasma also im-
proved the survival of ischemic random
skin flaps in rats (20, 21). Our hypothesis
was that PRP may be useful in improving
the viability of skin flaps in dogs as well.
The purpose of this experimental study
was to evaluate whether the use of locally
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Figure 1
Creation of one 2x10
cm subdermal plexus
skin flap on the
abdominal wall of a
dog. Note the expan-
sion of the wound
margins (left). Apposi-
tion of the flap back to
the recipient bed per-
formed in a two-layer
fashion by simple con-
tinuous sutures. Note
the sites of platelet-
rich plasma injections
(arrows), underneath
the flap and between
sutures (right).
injected autologous PRP improves the sur-
vival of long (5:1 length-to-width ratio)
subdermal plexus skin flaps in dogs.
Materials and methods
Animals
This study was approved by the Hellenic
State Veterinary Authorities on Animal
Care and Use. Six purpose-bred Beagle
dogs, three castrated males and three
spayed females, aged three to four years
and weighing 12–15 kg were used. Prior to
the experiment, each animal underwent a
complete physical examination, haematol-
ogy, serum biochemical analyses and uri-
nalysis, in order to ensure good health. The
dogs were housed in individual indoor
runs and had outdoor access and food
(commercial dry maintenance diets) twice
daily and water ad libitum.
Preparation of PRP
To prepare the PRP solution, 53 ml of
whole blood (3.5–4.4 ml/kg of body
weight) were collected via jugular veni-
puncture one hour before flap creation.
One ml of this blood volume was used for
complete baseline haematology performed
by an automated haematology analyzera.
The rest of the blood was used for PRP
preparation by using a platelet separator
systemb. Eight millilitres of acid citrate dex-
trose type A (an anticoagulant that pre-
serves the integrity of platelet membrane)
were drawn into a 60 ml syringe, followed
by 52 ml of blood according to the separ-
ator system’s instructions, in order to ob-
tain the adequate PRP quantity with a pla-
telet concentration at least five times higher
than that in blood. A 1/2 ml of PRP was
used for counting the platelets. The rest of
it was drawn into insulin syringes and used
for flap treatment within an hour after its
preparation, although it can be kept for up
to eight hours at room temperature until its
use (22, 23).
Subdermal plexus flap creation, PRP
treatment and postoperative care
On the day of flap creation (day 0), each
dog was pre-medicated with acepromazine
(0.05 mg/kg of body weight) and butorpha-
nol (0.1 mg/kg) administered intravenously
(IV). Anaesthesia was induced with so-
dium thiopental 2.5% (8 mg/kg IV) and
maintained with isoflurane in oxygen. Lac-
tated Ringer’s solution was administered
(10 ml/kg/h IV) throughout anaesthesia.
The dorsolateral area of the trunk from the
cranial thoracic to coxofemoral region bi-
laterally was clipped and prepared for asep-
tic surgery. Just before surgery, a single
dose of cefuroximec (20 mg/kg of body
weight IV) was administered to each dog.
With the animal in lateral recumbency,
one 2 cm wide x 10 cm long skin flap,
upper based and with its longitudinal axis
perpendicular to the dorsal midline, was
marked on the abdominal wall near the last
rib on either side consecutively. Each flap,
including the skin and subcutaneous tissue
with the panniculus carnosus muscle, was
totally elevated by blunt dissection.
Haemostasis was performed, if necessary,
by light pressure using sterile gauze. Ap-
position of the flap back to the recipient
bed was performed in a two-layer fashion
by simple continuous sutures using metric
1.5 poliglecaprone for subcutaneous tissue
a Advia 120: Siemens, Munich, Germany
b Magellan® Autologous Platelet Separator system:
Medtronic Inc., Minneapolis, MN, USA
c Zinacef®: GlaxoSmithKline, Brentford, UK
© Schattauer 2014

and metric 2 nylon for the skin (▶ Figure
1). In each dog, one randomly selected flap
received PRP treatment following the sur-
gical procedure, whereas the flap on the
other side was left untreated and served as
a control. The prepared PRP (2.5 ml) was
injected between the flap and the recipient
bed, in eight equally distributed sites be-
tween sutures (arrows in ▶ Figure 1) and at
equal doses (approximately 0.3 ml). Each
flap was covered with a sterile non-adher-
ent dressingd. Cotton roll gauzee was
wrapped around the trunk and secured in
place with an adhesive tapef. Elizabethan
collars were also used. Bandages were
changed every two days until the removal
of sutures on day 10 post-surgery. Anal-
gesia was provided for the initial 24 hours
post-surgery by the administration of
butorphanol (0.2 mg/kg IV) at four hour
intervals.
Evaluation of skin flap survival
Macroscopic evaluation
Macroscopic evaluation was based on vis-
ual inspection and palpation every two
days during bandage changes and on calcu-
lation of the percentage of the viable part of
the flap on day 10 (sutures removal). Viable
skin was determined grossly based on ap-
pearance, colour, and texture. The distal
flap areas that were dark in colour, hard in
texture, or covered with eschar were con-
sidered as necrotic. On day 10, the dimen-
sions (length x width) of total and necrotic
areas of each flap were measured and the
surviving area (total area minus necrotic
area) was calculated in cm2. The percen-
tage flap survival was then determined by
dividing the surviving area by the initial
flap area x 100 (24, 25).
Laser-Doppler flowmetry
For the measurement of tissue perfusion by
laser-Doppler flowmetry (LDF), an objec-
tive and non-invasive way to evaluate cut-
aneous microcirculation, the dogs were ac-
climatized to the study room (temperature
of 20°-22°C) at least half an hour before an-
d Mellolin: Smith and Nephew, London, England
e Velbad: Smith and Nephew, London, England
f Peha-haft: Hartmann, Heidenheim, Germany
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M. Karayannopoulou et al.: Platelet-rich plasma and skin flap survival in dogs
Figure 2
Day 10, sutures
removal: The flap on
the left (platelet-rich
plasma-treated) is
almost completely
healed, whereas the
flap on the right
(control) has a necrotic
area at its distal part
(note the necrotic
eschar, the formation
of granulation tissue,
and the amount of
wound exudate).
aesthesia (26, 27). A laser-Doppler vel-
ocimeterg with a standard right angle probe
was used. On each flap, LDF was perform-
ed at three different sites, near its base
(proximal), the centre (mid), and the distal
viable (distal) part of the flap, on days 0
(just before and immediately after flap cre-
ation) 2, 4, 6 and 10 post-surgery. At each
site, four separate readings at 15 second in-
tervals were recorded and a mean value
was obtained. Values were expressed in
perfusion units (PU).
Histological evaluation
Specimens for histological evaluation were
obtained with the dogs under general an-
aesthesia, on days 4 and 10 post-surgery
and after the LDF procedure. Each time,
two full-thickness skin specimens were ob-
tained, one from the proximal and one
from the distal viable part of the flap dia-
gonally, using a 3 mm biopsy punch. Each
specimen was fixed in neutral-buffered for-
malin and labelled so that the pathologist
was aware of the part of the flap, the animal
and the day, but not aware of the group
corresponding to the specimen (PRP-
treated or control). The specimens were
routinely processed, embedded in paraffin,
sectioned at 5 μm and stained with hae-
matoxylin and eosin. Sections were then
g Laserflo model BPM: Vasamedics Inc., St. Paul, MN
381
evaluated microscopically to assess and
quantify the degree of inflammatory cell
infiltration, oedema, mesenchymal cell
proliferation, and collagen production
based on a scoring system modified as fol-
lows: 0 = normal; 1 = mild increase; 2 =
mild to moderate; 3 = moderate; 4 = mod-
erate to marked; 5 = marked increase (28).
For the degree of inflammatory cell infil-
tration, sections were assigned a score of 0,
1, 2, 3, 4 or 5 when less than 3, 3-10, 11–20,
21–30, 31-40, or greater than 41 inflamma-
tory cells (neutrophils, lymphocytes, plas-
ma cells, macrophages, eosinophils, and
mast cells) were detected per high power
field (HPF) (x400), respectively. Moreover,
the number of blood vessels and capillary
buds was measured in 6 HPF per section.
Statistical analysis
Data were presented as mean ± standard
deviation (SD). The normality of data dis-
tribution was tested with the aid of Shapi-
ro-Wilk test. Regarding tissue perfusion
and histological measurements, in order to
assess any statistically significant differ-
ences between the two groups (PRP-treated
and control flaps) at each measurement
time, as well as between measurement
times within each group, a general linear
model for repeated measures was used. Re-
garding the percentage survival, the Wilc-
oxon signed ranks test was utilized. Differ-
ences were considered significant at p
Vet Comp Orthop Traumatol 5/2014
382 M. Karayannopoulou et al.: Platelet-rich plasma and skin flap survival in dogs

compared to only two of six PRP-treated

25 flaps in which the necrotic area was smaller.
proximal control mid control distal control On day 10 (▶Figure 2), when the viable and
proximal PRP mid PRP distal PRP necrotic regions on each flap could be more
20 easily distinguished, calculations of the per-
centage flap survival were performed. The
mean percentage survival of the PRP-treated
15 flaps (96.3%) was significantly higher than
that of controls (79.7%), (p = 0.046).

10 Laser-Doppler flowmetry (LDF)

The mean tissue perfusion values
5 measured at the proximal, mid and distal
part of PRP-treated and control flaps are
shown in ▶ Figure 3 and ▶ Table 1. Tissue
0 perfusion values did not differ significantly
day 0 day 2 day 4 day 6 day 10 before and immediately after flap creation
(day 0), in either group. The postoperative
Days after surgery values increased gradually in both groups,
and on day 10 they remained significantly
increased compared to the postoperative
Figure 3 Tissue perfusion mean values (in perfusion units) at the proximal, mid and distal part of
platelet-rich plasma (PRP) -treated and control flaps, on days 0 (immediately after flap creation), 2, 4, 6 value on day 0 in both PRP-treated (p
and 10 post-surgery, measured by laser-Doppler flowmetry. <0.001) and control (p = 0.002) flaps. With
regard to the site of LDF measurements,
tissue perfusion was significantly lower at
≤0.05. All statistical analyses were per- solutions compared with the whole blood. the mid and distal compared to the proxi-
formed by using commercial softwareh. The mean platelet number in the PRP sol- mal part of the control flaps (p = 0.035 and
utions was 5795166 ± 3100553/μL (range: p = 0.006, respectively), whereas in the
3183000/μL to 11092000/μL) compared to PRP-treated flaps it was significantly lower
Results 348833 ± 90801/μL (range: 283000/μL to only at the distal compared to the proximal
521000/μL) in blood. part of the flap (p = 0.011). Between PRP-
Platelet concentration in PRP treated and control flaps, irrespective of
There was an 11 to 21-fold increase in pla- Skin flap survival measurement time, a significant difference
telet concentration in the prepared PRP in tissue perfusion was found in favour of
Macroscopic evaluation PRP at the proximal (p = 0.012), the mid (p
h SPSS version 19.0, IBM-SPSS Science, Chicago, IL, On day 4 post-surgery, five of six control <0.001), and the distal (p = 0.031) part of
USA flaps showed necrosis at their distal part the flap. Tissue perfusion values were sig-

Table 1 Tissue perfusion values (mean ± standard deviation) measured at the proximal, mid and distal part of platelet-rich plasma (PRP)-treated and
control flaps in dogs. Values are expressed in perfusion units.

Flap part Group Tissue perfusion units (mean ± standard deviation)

Day 0 Day 2 Day 4 Day 6 Day 10
Pre Post
Proximal Control 6.12 ± 1.6 5.52 ± 1.9 6.56 ± 2.3 12.64 ± 5.9 12.12 ± 7.7 10.58 ± 3.1
PRP-treated 7.14 ± 2.0 5.06 ± 2.1 8.59 ± 2.3 13.24 ± 5.1 19.10 ± 4.3 16.59 ± 4.5
Mid Control 5.70 ± 1.3 4.16 ± 1.2 4.72 ± 3.6 5.78 ± 2.4 7.74 ± 6.0 7.75 ±3.4
PRP-treated 6.84 ± 2.9 5.76 ± 1.6 7.38 ± 1.7 14.86 ± 5.5 16.95 ± 7.1 16.58 ± 6.5
Distal Control 4.99 ± 2.1 3.97 ± 2.8 2.16 ± 1.0 4.37 ± 2.6 3.47 ± 3.0 6.74 ± 0.6
PRP-treated 6.05 ± 2.9 5.28 ± 3.3 4.17 ± 2.1 6.41 ± 2.5 8.28 ± 4.9 11.66 ± 4.8

PRP: platelet-rich plasma. Day 0: Pre = before flap creation; Post = after flap creation.

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 M. Karayannopoulou et al.: Platelet-rich plasma and skin flap survival in dogs 383

Table 2 Histological variables in platelet-rich plasma (PRP)-treated and untreated (control) flaps in dogs. Values are presented as mean ± standard
deviation (SD).

Histological Day 4 Day 10 Irrespective of time

variable Group Biopsy location Biopsy location (p-value)
Proximal Distal Proximal Distal Proximal Distal
(mean ± SD) (mean ± SD) (mean ± SD) (mean ± SD)
Oedema score Control 1.6 ± 0.73 4.76 ± 0.39 p = 0.036§ 3.25 ± 0.5 4.0 ± 0.55 p = 0.002¶
p = 0.01* p = 0.012¤ p = 0.008¤
PRP 1.22 ± 0.86 4.33 ± 0.75 1.92 ± 1.11 2.42 ± 0.97 p = 0.001¶
Collagen production Control 1.84 ± 0.43 1.54 ± 0.67 p = 0.001§ 2.52 ± 0.91 4.32 ± 0.26 P = 0.019¶
score PRP 1.32 ± 0.83 2.08 ± 0.80 p= 0.002§ 3.03 ± 0.74 3.92 ± 1.08
Mesenchymal cell Control 2.0 ± 0.89 1.9 ± 0.49 p= 0.035§ 2.62 ± 1.07 3.9 ± 0.95
proliferation score PRP 1.83 ± 0.75 2.08 ± 0.92 p= 0.016§ 2.45 ± 0.6 3.42 ± 1.19
Number of new Control 10.83 ± 3.5 12.17 ± 9.83 p = 0.021§ 17.33 ± 5.09 32.5 ± 13.1 p = 0.023¶
vessels PRP 10.50 ± 5.17 17.33 ± 8.94 16.67 ± 4.8 22.83 ± 10.4 p = 0.002¶

PRP: platelet-rich plasma. p§: Significant difference between measurement times (days 4 and 10) in PRP-treated or control flaps, irrespective of biopsy
location (proximal or distal). p*: Significant difference between PRP-treated and control flaps, irrespective of biopsy location (proximal or distal), on
day 10. p¤: Significant difference between PRP-treated and control flaps, at the proximal or distal part of the flap, irrespective of measurement time.
p¶: Significant difference between the proximal and distal part of the flap, irrespective of measurement time. Proximal: biopsy located near base of the
flap; Distal: Biopsy of viable tissue in the distal part of the flap.

nificantly higher in PRP-treated compared
to control flaps on day 4 (p = 0.036), day 6
(p = 0.013), and day 10 (p = 0.003) post-
surgery, irrespective of measurement site.
Histological evaluation
Changes in histological variables are shown
in ▶ Table 2 and ▶ Figure 4 A and B. His-
tological and tissue perfusion results spe-
cifically concerning the distal (and most
vulnerable to necrosis) part of the flaps are
also presented separately in ▶ Table 3. In-
flammatory cell infiltration was normal or
mild in most cases. Oedema, irrespective of
measurement time, was higher at the distal
compared to the proximal part in both
PRP-treated (p = 0.001) and control (p =
0.002) flaps, whereas between the two
groups it was lower in the PRP-treated
flaps, at both the proximal (p =0.012) and
distal (p = 0.008) part. Irrespective of the
flap’s part, comparisons between measure-
ment times revealed that the mean oedema
score on day 10 was significantly higher
compared to day 4 only in control flaps (p
= 0.036). The mean oedema score was

A B

Figure 4 Histological pattern of the distal part of a control (A) and a platelet-rich plasma (PRP)-treated (B) flap on day 4 showing the improvement in
tissue healing after PRP treatment. A) Marked oedema, poor collagen production, and B) less oedema, increased collagen production and angiogenesis.
Haematoxylin and eosin stain, Bar = 190 μm.

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384 M. Karayannopoulou et al.: Platelet-rich plasma and skin flap survival in dogs
Table 3 Histological variables and tissue perfusion (mean ± standard deviation) evaluated at the distal part of platelet-rich plasma (PRP)-treated and
control flaps in dogs.
Variable Group
Oedema score Control
PRP-treated
Collagen production score Control
PRP-treated
Mesenchymal cell Control
proliferation score PRP-treated
Number of new vessels Control
PRP-treated
Tissue perfusion Control
PRP-treated
PRP: platelet-rich plasma.
lower in the PRP-treated flaps than in con-
trols at both measurement times, but the
difference was significant (p = 0.01) only
on day 10. Mesenchymal cell proliferation
and collagen production were significantly
increased on day 10 compared to day 4 in
both PRP-treated flaps (p = 0.016 and
0.002, respectively) and controls (p = 0.035
and 0.001, respectively), but no significant
differences between the two groups were
found at any measurement time. Irrespec-
tive of measurement time, collagen pro-
duction was greater (p = 0.019) at the distal
compared to the proximal part only in con-
trol flaps. Angiogenesis was significantly
higher at the distal compared to the proxi-
mal part of the flaps in both PRP-treated (p
= 0.002) and controls (p = 0.023). Between
measurement times, a significant increase
in the number of blood vessels was reveal-
ed on day 10 compared to day 4 only in
control flaps (p = 0.021). No significant dif-
ferences in angiogenesis were observed be-
tween the two groups, although more new
vessels were measured at the distal part of
PRP-treated flaps on day 4 and of controls
on day 10.
Discussion
Distal flap necrosis, particularly in subder-
mal plexus skin flaps with a high length-to-
width ratio as in our study, constitutes a
serious problem in both human and vet-
erinary plastic and reconstructive surgery.
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Day 0 Day 2
3.97 ± 2.77 2.16 ± 1.0
5.28 ± 3.26 4.17 ± 2.07
The major factors implicated in flap necro-
sis are inadequate blood perfusion result-
ing from damage to the subdermal plexus
during surgery, thrombosis or tension on
the flap edges, and ischemia-reperfusion
injury (1, 2, 4, 24, 29).
Platelets are aggregated in response to
tissue injury and vascular disruption,
whereas their activation results in blood
clot formation and in the release of numer-
ous bioactive proteins essential for tissue
repair including growth factors, cytokines
and chemokines (16, 17, 23). The concen-
tration of growth factors at the wound site
increases with increasing platelets; there-
fore, increased platelet numbers in a
wound bed or adherent to a flap after PRP
treatment may lead to a better healing out-
come (14, 16, 22). Most investigators sug-
gest that therapeutic PRP should contain a
platelet concentration at least five-fold
higher than blood values (16, 22, 30). In
our study, the number of platelets in PRP
solutions was found increased 11 to
21-fold, which is as reported in other simi-
lar studies (14, 31).
The wound healing properties of PRP
depend on the activation of platelets, which
can be accomplished by the addition of
thrombin or calcium chloride before appli-
cation or alternatively at the wound site by
contact of PRP with elements of injured en-
dothelium such as collagen and addition-
ally by the formation of thrombin during
coagulation (15, 23, 30-32). We opted to
allow the endogenous activation of PRP,
Day 4 Day 6 Day 10
4.76 ± 0.39 4.0 ± 0.55
4.33 ± 0.75 2.42 ± 0.97
1.54 ± 0.67 4.32 ± 0.26
2.08 ± 0.80 3.92 ± 1.08
1.9 ± 0.49 3.9 ± 0.95
2.08 ± 0.92 3.42 ± 1.19
12.17 ± 9.83 32.5 ± 13.1
17.33 ± 8.94 22.83 ± 10.4
4.37 ± 2.63 3.47 ± 3.03 6.74 ± 0.59
6.41 ± 2.49 8.28 ± 4.94 11.66 ± 4.81
because it may result in a slower release of
bioactive proteins, which in turn better
benefits wound healing (23).
In the present study, we found that flap
survival was significantly improved in
PRP-treated flaps with a high length-to-
width ratio (5:1) compared to control flaps.
In two experimental studies performed in
rats, the survival rate of PRP-treated ran-
dom skin flaps with a length-to-width ratio
of 4:1 was lower than in our study (64.9%
and 61.2% compared to 61.2% and 28% of
controls, respectively), and only in the sec-
ond study the difference between PRP-
treated and controls was significant (20,
21). In those studies, however, the flaps
were made ischemic before PRP appli-
cation. In another experimental study in
rats evaluating the effect of various routes
of VEGF administration on the survival of
random skin flaps with a length-to-width
ratio of 5:1 (as in our study), the best sur-
vival rate (80.4%) was achieved when
VEGF was injected into the distal third of
the flap before its transposition to the re-
cipient area; in our study, however, PRP
was injected between the flap and the re-
cipient bed after suturing (6).
The process of wound healing is roughly
divided into three overlapping phases, the
inflammatory (including haemostasis), the
proliferative, and the remodelling phase.
During the phase of inflammation, re-
leased vascoactive cytokines induce local
vasodilatation and capillary permeability
resulting in the attraction of neutrophils
© Schattauer 2014

and monocytes and in the leakage of serous
fluid into the wound bed creating oedema
(32, 33). In this study, the degree of inflam-
matory cell infiltration into the wound site
was scored as normal or mild (<10 inflam-
matory cells per HPF) in both PRP-treated
and control flaps, as it was also reported by
Kryger and colleagues in a study in rats on
random skin flaps treated with VEGF (9).
On the contrary, Li and colleagues ob-
served significantly fewer polymorphonu-
clear cells in PRP-treated compared to un-
treated ischemic random skin flaps in rats
(21). In our study, oedema was less pro-
nounced in the PRP-treated flaps com-
pared to controls at both their proximal
and distal parts, a change more evident on
day 10. As the inflammatory response sub-
sides and the repair of injured tissues be-
gins, many growth factors (PDGF, TGF-β,
VEGF, bFGF) contribute to angiogenesis,
fibroblast proliferation, collagen produc-
tion and epithelialisation (32–34). Follow-
ing angiogenesis and the restoration of tis-
sue perfusion, microcirculation is re-estab-
lished at the wound site and thus oedema is
diminished. Vascular endothelial growth
factor plays a major role in angiogenesis
and lymphangiogenesis (35). Indeed, some
authors have reported increased angiogen-
esis and increased levels of VEGF or PDGF
in PRP-treated skin flaps in rats (20, 21). In
addition, decreased oedema was noticed
after infusion of VEGF into the distal por-
tion of random skin flaps (4x11 cm) in a
porcine model (36). Consistent with the
above, in the present study, the number of
new vessels on day 4 was increased at the
distal part of PRP-treated flaps (which were
also less oedematous) compared to con-
trols, albeit not significantly. The resolution
of oedema, therefore, in our PRP-treated
flaps could be attributed partially to angio-
genesis and the angiogenic effect of VEGF
or other angiogenic growth factors. We
found no significant differences in mesen-
chymal cell proliferation and collagen pro-
duction in favour of PRP. Contrary to our
findings, in a study in rats, the thickness of
granulation tissue and the amount of
myofibroblasts at demarcation sites be-
tween surviving and necrotic areas was
higher in PRP-treated than in untreated
flaps (20). Increased granulation tissue
formation was also reported when single
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M. Karayannopoulou et al.: Platelet-rich plasma and skin flap survival in dogs
growth factors (VEGF or bFGF) were
evaluated for enhancing random skin flap
survival in rats (8, 9). In our study, how-
ever, the absence of any significant differ-
ences in angiogenesis and collagen produc-
tion in favour of PRP may be due to find-
ings such as the increased angiogenesis on
day 10 compared to day 4 in control flaps
and the increased collagen production at
their distal compared to proximal part,
which might reflect the need for restora-
tion of the necrotic areas present in most of
these flaps.
Laser-Doppler flowmetry is a non-
invasive way to evaluate skin flap survival.
The measurement of tissue perfusion by
LDF has been widely established as an ob-
jective method for detecting cutaneous
microcirculatory changes and, thus, evalu-
ating skin flap viability, even before physi-
cal signs of ischemia or necrosis become
evident (27, 37-39). In our study, the sig-
nificantly increased tissue perfusion found
in PRP-treated flaps compared to controls
is in accordance with the increased percen-
tage survival of these flaps. Since angiogen-
esis did not differ significantly between the
two groups, the higher tissue perfusion
found in the PRP-treated flaps compared to
controls could be due to increased dila-
tation of pre-existing blood vessels within
the flaps. This dilatation of blood vessels
occurs in response to an increased produc-
tion of the endothelium-derived vasorelax-
ing factor nitric oxide induced by VEGF (4,
10, 39, 40). Increased blood flow in
ischemic tissues due to the vasodilator ef-
fect of VEGF has also been reported in
other studies (10, 24, 40). The diminished
oedema, therefore, found in our PRP-
treated flaps compared to controls could be
perhaps mainly attributed to this vasodi-
lator effect of VEGF, presumably due to its
increased release from the injected PRP.
In conclusion, the use of locally injected
autologous PRP enhanced the microcircu-
lation (tissue perfusion) and decreased
oedema in canine long subdermal plexus
skin flaps, thus significantly improving
their survival.
Acknowledgments
The authors would like to acknowledge
Professor D. Papadimitriou (Medical
385
School, Aristotle University of Thessaloni-
ki, Greece) for provision of the laser-
Doppler velocimeter. We are also grateful
to Hospital Line SA for providing the
Magellan-Medtronic (Minneapolis, MN,
USA) centrifuge without any financial
claim.
Conflicts of interest
The authors declare that they have no
conflict of interest.
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