Ghép Da: Chỉ Định, Ứng Dụng Và Các Nghiên Cứu Hiện Tại (2011)

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15
The Effect of Human Recombinant
Erythropoietin (rHuEPO) and Tacrolimus
(FK506) in Autologous and Homologous
Full Thickness Skin Graft (FTSG)
Take and Viability in a Rat Model
Dimitrios Karypidis, Despina Perrea,
Othon Papadopoulos and Alkiviadis Kostakis
2nd Propedeutic Surgical Department and Department
of Experimental Surgery Athens
University School of Medicine
Greece
1. Introduction
The use of autologous skin grafts comprises a well established method of reconstruction in
large areas involving partial thickness defects. Additionally, when skin availability is
limited due to associated trauma or injury (e.g. burns) the use of homologous skin grafts
from a compatible donor appears to be a valid alternative. The effort to maximize
effectiveness of skin grafting in terms of increasing graft take, protecting viability, reducing
full healing time and achieving improved postoperative outcomes without impairing
function has also been constant especially in cases of impaired general condition.
Human recombinant erythropoietin (rHuEPO) is a glycoprotein with primarily a
hemopoietic role due to the inhibition of precursor erythrocytes and the promotion of their
proliferation. Its genetic expression is being regulated by hypoxia. It is primarily
synthesized by the fetal kidneys and liver and by the adult kidneys (Paschos et al. 2008).
The biological activity of rHuEPO is promoted by its interaction with the cell surface
erythropoietin receptor (EPOR) (Brines and Ceramic 2008) which is a type I cytokine
expressed by precursor erythrocytes as well as in other non hemopoietic systems such as the
central and the peripheral nervous system in response to trauma (Galeano et al. 2004), in
cardiac muscle cells, vascular endothelial smooth muscle cells and in subcutaneous mast
cells with potent anti-ischaemic action (Brines and Ceramic, 2008; Galeano et al. , 2004).
Current research has focused on its non-hemopoietic role. Many studies report the local
production of erythropoietin (EPO) in response to trauma prior to the local concentration of
pre-inflammatory cytokines and EPO acting antagonistically to them by reducing edema,
inhibiting cellular apoptosis, promoting neovascularization by enhancing endothelial cell
mitosis and promoting wound healing (Paschos et al. 2008).
Since endogenous local EPO production is inadequate, it has been shown that the exogenous
administration of rHuEPO enhanced wound healing and revascularization in experimental

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198
studies on healthy rats, rats with genetically induced diabetes mellitus and rats with burn
injuries (Galeano 2006). In clinical studies including human subjects, the wound healing
promoting properties of rHuEPO have been reported following γ-knife for brain surgery
and recently in patients who underwent destruction osteogenesis (Mihmanli et al. 2009)
Vascular endothelial growth factor (VEGF) comprises a heparin-binding, dimeric
glycoprotein which initiates the proliferation and migration of endothelial cells to
participate in the development of new vascular lumens, and increases the penetration and
extravasation of plasma macromolecules (Hom et al., 2005; Paschos et al., 2008 ). VEGF
receptors are found in endothelial cells and are expressed under conditions of hypoxia and
following endothelial damage. Two high-affinity endothelial cell receptors, KDR/Flk-1 and
flt-1 mediate VEGF effects on tissue physiology (Lantieri et al. 1998)
VEGF has been shown to participate in wound healing enhancement, embryo development,
growth of certain solid tumors, and ascites formation (Isogai et al., 2006; Schultze-Mosgau et
al., 2003). In this study, the immunohistochemical detection of VEGF expression is used as a
method of skin graft take evaluation.
Tacrolimus (FK-506), has recently been established as a valid immunosuppressant and
comprises a well-known calcineurin (a serine/threonine phosphatise) inhibitor. It has been
shown that FK-506 inhibits TGF-β induced VEGF production by antagonizing calcineurin
which leads to the attenuation of the activation and translocation of the nuclear factor that
activates T-cells (nft) (Mori et al 1997). FK-506 also inhibits: a) interleukin-2 induced
interleukin-5 production by CD4+ T cells and b) T-cells proliferation stimulated by
interleukin-2 and interleukin-7 (Mori et al 1997). Confirmation was provided by similar
studies which showed that rapamycin, another immunosuppressant which does not inhibit
calcineurin, did not have any effect on TGF-β induced VEGF production while cyclosporine
A, which is also a calcineurin inhibitor, resulted in reduction of VEGF production (Marumo
et al. 1995). The action of FK-506 is mediated by immunophilins, a class of proteins which
bind to immunosuppressive drugs, giving to FK-506 the ability to interact with calcineurin
and to interfere with its access and dephosphorylation of various substrates. The primary
immunophilin was shown to be FKBP 13 which is localized to the endoplasmic reticulum
lumen, where processing and presentation of antigen in the immune system are thought to
take place (Nigam et al. 1993). The liver and the intestinal mucosa are the main sites of FK-
506 metabolism by the cytochrome P4503A4 enzyme. Subsequently, FK-506 is eliminated
through biliary excretion. (Lampen et al., 1995; Nakazawa et al., 1998; Plosker & Foster,
2000). FK-506 has been widely used systemically, maintaining a profile of safety during the
long-term experience with the drug. Most side effects appear to be related to whole-blood
concentrations and refer to nephrotoxicity, cardiovascular toxicity, metabolic and
neurotoxicity. However, almost all of the reports of FK-506 toxicity are derived from its use
in solid-organ transplantation, in which much higher doses are used compared to those for
skin transplantation (Mayer et al., 1997; Pirsch et al. 1997). The aim of this animal study is to
investigate the effect of human recombinant erythropoietin (rHuEPO) and tacrolimus (FK-
506) on autologous and homologous full thickness skin grafts in an experimental research
design including Wistar rats.
2. Methods and material
Thirty adult inbred female Wistar rats (n=30), with weights ranging from 220 to 300 gr, were
selected for this study. Two control and 3 experimental groups were formed, each consisting

The Effect of Human Recombinant Erythropoietin (rHuEPO) and Tacrolimus (FK506) in
Autologous and Homologous Full Thickness Skin Graft (FTSG) Take and Viability in a Rat Model
199
of 6 subjects. This experimental study followed the National Research Council’s Guide for
the Care and Use of Laboratory Animals and was approved by the National Bioethics
Committee. This study used the minimum number of animals needed in order to establish a
valid conclusion and statement regarding the specific subject. All necessary refinements and
design methods have been performed and planned according to accredited established
protocols. All surgical procedures were performed under general anesthesia with the use of
ketamine
hydrochloride
(80
mg/kg
intramuscularly)
and
xylazine
(2
mg/kg
intramuscularly).
After induction of general anesthesia the surgical site was shaved and prepped with
povidone iodine and chlorhexidine solution. The animals were covered with sterile surgical
drapes with only the operative site exposed.











Fig. 1. Preoperative design of the FTSG on the back of the subject

Skin Grafts – Indications, Applications and Current Research
200













Fig. 2. Dissection and harvesting of the FTSG and then rotation of the FTSG on the wound
bed by 180o so as the previously caudal edge becomes rostral and the donor site can be used
as the recipient site

The Effect of Human Recombinant Erythropoietin (rHuEPO) and Tacrolimus (FK506) in
Autologous and Homologous Full Thickness Skin Graft (FTSG) Take and Viability in a Rat Model
201

Fig. 3. Cleaned wound area and dressing application on the FTSG
A full thickness skin graft was harvested from the area between the two scapula bones on
the dorsum of each rat. The skin graft was elliptical in shape, with a long diameter of 2 cm
and a small diameter of 1 cm (Figure 1). During harvesting, the panniculus layer was
carefully removed from the bed of the skin graft and recipient site. The skin graft was kept
in phosphate-buffered saline before being transferred to the recipient site. In this
experimental model, each skin graft was repositioned in the same area from which it was
harvested after being rotated by 180 degrees in the vertical axis, so as the severed vascular
axons in the skin graft remain misaligned to the vascular axons of the recipient bed. In this
way each donor site also serves as a recipient site since revascularization and new vascular
connections between skin graft and recipient bed are still required due to the preserved
vascular misalignment in the new graft position. When a homologous FTSG was required,
animals underwent surgery in pairs and a FTSG was harvested in the aforementioned way
from each animal and it was then transplanted into the donor site of the other after being
similarly rotated by 180 degrees (see subject groups below).
The location of the recipient bed is conveniently situated between the scapular bones,
unreachable by the rats and well fixated on a minimally mobile area causing negligible
irritation to the rats. No animal involved in this project was subjected to discomfort, pain, or

Skin Grafts – Indications, Applications and Current Research
202
distress. Any such discomfort, pain, or distress was alleviated with the appropriate
approved medication (Figure 1-3). Nylon 5-0 non-absorbable sutures were invariably used.
The animals were placed in a warm environment with the aid of heating lamps and were
continuously monitored. Pain management was carried out with the use of Buprenorphine
at 0.05-0.10 mg/Kg, SC, and then twice daily. The animals were observed for their pattern of
breathing, alertness, ability for food and drink uptake. After surgery rats were carefully
observed for dehydration and diarrhea. Rats were kept separately in their housing cages
which were standard filter top secured cages.
The animals were randomized into the following groups according to the treatment that was
used:
Group A (First control group, n=6): An autologous FTSG was harvested and repositioned.
Local infiltration with water for injection was also performed as a control.
Group B (Second control group, n=6): Animals underwent surgery in pairs. Consequently
there were 3 pairs in this group. A homologous FTSG was harvested from each animal of
each pair and transplanted into the donor site of the other. FK506 (tacrolimus) was
administered systematically to prevent rejection. Local infiltration with water for injection
was also performed as a control.
Group a (experimental, n=6): An autologous FTSG was harvested and repositioned. Local
infiltration with rHuEPO was performed.
Group b (experimental, n=6): Animals underwent surgery in pairs as in group B, forming a
total of 3 pairs. A homologous FTSG was harvested from each animal and it was
transplanted into the donor site of the other while FK506 (tacrolimus) was administered
systematically (local infiltration) to prevent immunologic response. Local infiltration with
rHuEPO was performed.
Group c (experimental, n=6): An autologous FTSG was harvested and repositioned and local
infiltration with FK506 was performed. Table 1 summarizes the details regarding each
animal group.

Group
Type
Number of
subjects
FTSG
FK506
rHuEPO
wfi
A
control
6
autologous
no
no
yes
B
control
6
homologous
for
immunosuppression
no
yes
a
experimental
6
autologous
no
factor
no
b
experimental
6
homologous
for
immunosuppression
factor
no
c
experimental
6
autologous
factor
no
no
Table 1. The animal groups in the study. FTSG: full thickness skin graft, for
immunosuppression: FK506 is used in order to prevent rejection, not as tested factor, factor:
FK506 or rHuEPO are used as local infusion around the recipient bed and their effect on
graft viability and healing is tested, wfi: water for injection used for local infiltration as a
placebo, when one of the previously mentioned factors were not used
FK-506 (Fujisawa USA, Inc.) was dissolved in 80% ethanol and 20% cremaphor to create a
stock solution with a concentration of 10 mg/ml. This stock was then diluted with 75%

The Effect of Human Recombinant Erythropoietin (rHuEPO) and Tacrolimus (FK506) in
Autologous and Homologous Full Thickness Skin Graft (FTSG) Take and Viability in a Rat Model
203
propylene glycol and 25% water to a final concentration of 0.5 mg/ml. The diluted solution
was replaced every 3 days.
In groups B and b, FK-506 was subcutaneously administered daily at a dose of 2mg/Kg. The
use of FK-506 either as a systematic immunosuppressant (groups B and b) or as a local agent
(group c) always involved local, subcutaneous infiltration. In groups a and b, 400UI/kg in a
100μL solution of rHuEPO were subcutaneously administered, daily. Local infiltration of
agents was invariably performed around and under the recipient bed.
The effect of FK-506 and rHuEPO administration in the aforementioned experimental
groups was investigated. The investigation consisted of the clinical, histological and
immunohistochemical evaluation of the grafts and their viability-take on the 10th post
grafting day. All subjects were euthanized on the 10th post grafting day by prolonged
inhalation in a closed diethyl-ether inhalation chamber.
Skin graft take area and viability were assessed using digital photographs and manual
measurements. The photographs were processed by digital image surface area analysis
software (Pixcavator Image Analysis Software 2.3). Specimens of autologous and
homologous skin grafts were then harvested together with a marginal recipient bed skin,
along the perimeter of the graft for histological examination and immunohistochemistry.
The formalin fixed, paraffin embedded tissue sections were treated with hematoxylin-eosin
stain as well as by two antibodies. Standard procedures as recommended by the
manufacturer were performed.
The antibodies used in our study were:
Monoclonal Mouse Anti-Human Vascular Endothelial Growth Factor, Clone VG1, isotype:
IgG1, kappa antibody was used to label the VEGF-121, VEGF-165, and VEGF-189 isoforms
of vascular endothelial growth factor (VEGF) Clone VG1.
Monoclonal Mouse Anti-Human CD31, Endothelial Cell, Clone JC70A, isotype: IgG1, kappa,
(Dako A/S, Glostrup
Denmark Autostainer/Autostainer Plus) antibody was used to label endothelial cells of
newly formed vessels to determine angiogenesis.
Histology findings were evaluated for vascular presence and endothelial condition,
lymphocytic infiltration, dermal/epidermal interphase reaction (spongiosis, incomplete,
complete epidermal separation) and necrosis. More specifically, due to the relatively
variable macroscopic presentation of homologous graft rejection, histology findings and
criteria were solely used to describe and categorize potential rejection according to the
established method of Tatsuya et al (1997) which includes the following three grades:
Grade 1: intraepidermal blister formation,
Grade 2: incomplete epidermal separation from the dermis;
Grade 3: complete epidermal separation from the dermis.
Immunochemistry analysis aimed at demonstrating the degree of angiogenesis in the skin
grafts as a direct sign of vascular network rearrangement and development, viability and
effective graft take.
Manual measurement and clinical assessment of graft take was performed using light
microscopy and corresponded adequately with the digital image analysis.
Spleens were harvested and subjected to the mixed lymphocyte reaction, to assess the
degree of immunosuppression. Liver and kidneys were also harvested and examined for
signs of potential hepato- and nephrotoxicity.

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3. Statistical analysis
Areas of graft take comprise numeric data which are presented with approximation of
two decimal digits. Means are presented ±SD where applicable, and the t test for
independent samples has been used for statistical analysis of the results. SPSS 17.0 (IBM
Corporation Somers, NY 10589) was used. Statistical significance was considered for
p<0.05.
4. Results
Clinical results were classified according to the mean area of inadequate graft take – graft
necrosis (N). In control group A N=40.98 mm2, in control group B N=71.2 ± 4.67mm2, in
experimental group a N= 15.22 ± 4.76 mm2 in experimental group b N= 29.2± 4.70 mm2, in
experimental group c N=46.52 ± 4.60 mm2. Manual measurements corresponded well with
the aforementioned digital image analysis measurements. The corresponding percentages
(over the total area of each graft which was 20 x 10 mm2= 200 mm2) of mean graft necrotic
areas were: group A: 20.49%, group B: 35.6%, group a: 7.6%, group b: 14.6%, group c: 23.26%
(p<0.001 in all group comparisons: A-a, B-b and A-c) (Figures 4-9).
Histological findings using hematoxylin-eosin stain showed the differences in graft
revascularization and take among groups (10th post-grafting day) (Figures 10-19)
Immunohistochemistry
resulted
in
significant
observations
regarding
graft
revascularization in the each group (Figures 20-27).
Clinical, histological and immunohistochemical findings consistently show the resulting
quality of graft take and degree of revascularization among the various groups: graft take in
group a>group b> group A> group c> group B.
No signs of hepato- and nephro-toxicity were found.


Fig. 4. Mean inadequate graft take-necrosis percentage per group. A, B, a, b and c
correspond to animal groups

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Fig. 5. Group A macroscopic presentation. Mild peripheral necrosis, good overall take


Fig. 6. Group a macroscopic presentation. Full graft take, no signs of necrosis, excellent graft
viability

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206

Fig. 7. Group B macroscopic presentation. Considerable peripheral graft rejection. Central
graft take and viability


Fig. 8. Group b macroscopic presentation. Minimal peripheral rejection and nearly complete
graft take.

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Fig. 9. Group c macroscopic presentation. Partial graft necrosis, not uniform graft take
compared to control group A.
Groups A-a

Fig. 10. Formalin fixed, paraffin embedded FTSG x50 in group A. Normal graft take with
intermediate inflammatory inflammation demonstrating FTSG take without the use of
exogenous active factors.

Skin Grafts – Indications, Applications and Current Research
208
v
p l
Fig. 11. Formalin fixed, paraffin embedded autologous FTSG x200 in group A. Vascular
lumens with erythrocytes (v), polymorphonuclear cells(p), lymphocytes (l) and other
inflammatory cell intermediate concentrations are also shown. The number of vessels is
indicative of the quality of graft take and viability


Fig. 12. Formalin fixed paraffin embedded autologous FTSG x50 in group a. Higher vessel
concentration, more uniform and higher epidermal layer (double arrow line), less
inflammatory infiltration than in control group A

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v
p l
Fig. 13. Formalin fixed, paraffin embedded autologous FTSG x200 in group a. Vascular
lumens with erythrocytes (v), polymorphonuclear cells(p), lymphocytes (l) and other
inflammatory cell concentrations are also shown. Note the increased number of vessels and
the milder inflammatory response which are indicative of better quality of graft take and
viability than in the control group

s
b
Fig. 14. Homologous FTSG x50 in group B. Increased intraepidermal blisters (b),
dermoepidermal lack of cohesion (s) and increased inflammatory infiltration compatible
with grade 2-3 of rejection according to Tatsuya et al (1997).

Skin Grafts – Indications, Applications and Current Research
210
Groups B-b
v
e
Fig. 15. Formalin fixed, paraffin embedded homologous FTSG x200 in group B.
Inflammatory response and mild revascularization compatible with the initial stages of
rejection, impairing the ongoing course of graft revascularization (v) and take. Inflammatory
cells include large eosinophils concentrations (e) characteristic of the aforementioned
response type.

b
Fig. 16. Formalin fixed, paraffin embedded homologous FTSG x50 in group b.
Intraepidermal blisters (b) but lack of partial dermoepidermal separation. Better quality of
graft take and minimal rejection response than in control group B.

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v
p l e
Fig. 17. Formalin fixed, paraffin embedded homologous FTSG x200 in group b. Inflammatory
response with concentrations of eosinophils (e), lymphocytes (l) and polymorphonuclear cells
(p) but also nearly unimpaired revascularization (v) demonstrating a considerably better
quality of graft take and milder rejection response than in control group B.
Group c

Fig. 18. Formalin fixed, paraffin embedded autologous FTSG x50 in group c. Nearly normal
graft take with minimal inflammatory response, firm dermoepidermal cohesion but
marginally decreased new vessels development than in control group A.

Skin Grafts – Indications, Applications and Current Research
212


Fig. 19. Formalin fixed, paraffin embedded autologous FTSG x200 in group c. Nearly normal
graft take with minimal inflammatory response and decreased revascularization compared
to group A.
Groups A-a

Fig. 20. VEGF expression in group A, as detected by treating tissues with human VEGF
monoclonal antibody. Low or absent stain in the normal autologous grafting group.

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Fig. 21. VEGF expression in group a, as detected by treating tissues with human VEGF
monoclonal antibody. Stained-activated macrophages (brown) are noted indicating an
increase in revascularization process compared to the control group A






Fig. 22. CD31 stained endothelial cells of newly formed vessels (copper color) in control
group A (left) and group a (right). Notably increased angiogenesis is shown in group a

Skin Grafts – Indications, Applications and Current Research
214
Groups B-b



Fig. 23. VEGF expression in group B, following tissue treatment with human VEGF
monoclonal antibody. Low or absent stain in the homologous grafting group. Blisters are
also noted, indicative of early rejection response signs.






Fig. 24. VEGF expression in group b after the use of VEGF monoclonal antibody. Stained-
activated macrophages (brown) are noted compared to their absence in group B tissues,
indicative of increased angiogenesis.

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Fig. 25. CD31 stained endothelial cells of newly formed vessels (copper color) in control
group B (left) and group b (right). Note the absence of endothelial concentrations in group B
and the increased angiogenesis in group b
Groups c



Fig. 26. VEGF expression in group c, after treating tissues with human VEGF monoclonal
antibody. Minimal stain in the autologous grafts included in group c.

Skin Grafts – Indications, Applications and Current Research
216

Fig. 27. CD31 stained endothelial cells of newly formed vessels in group c. Considerable
decrease in angiogenesis compared to control group A
5. Discussion
Between days 3 and 8 after grafting, the transient formation of spherical protrusions in the
graft capillaries resembling angiogenic buds is taking place in what appears to be an
angiogenic response of the autochthonous graft capillaries (Lindenblatt et al 2010).
Proangiogenic molecules in the recipient bed and the graft tissue were revealed by
immunohistochemical analysis which showed that the wall of the aforementioned buds
expressed CD31 and desmin, indicating the presence of both endothelial cells and pericytes
(Gerhardt & Betsholtz 2003). A docking of sprouting vascular formation takes place from the
recipient bed which connects to the aforementioned spherical protrusions of the pre-existing
graft vessels unlike the previously suggested neovascularization theory which requires
weeks to complete. Additionally, there is rather no vessel inosculation from the recipient
bed to the graft as it was previously accepted but there is what appears to be a gradual
angiogenic response leading to newly formed vascular buds (Goretsky et al., 1995; Okada
,1986; Young et al., 1996).
The traditional vessel inosculation theory implies vessel re-formation and lumen restoration
of already existing vessels via direct approximation of vessels of the recipient bed and the
ones in the graft. Since it is nearly impossible to assume that simple graft positioning leads
to an effective number of successful graft-bed vessel couplings, the aforementioned
angiogenetic process has been recently described. Conversely, it has also been suggested
that pre-existing channels in the graft allow vessels from the recipient bed to invade the
graft from the periphery replacing a considerable number of graft vessels (Capla et al. 2006)
In order to provide the physiologic mechanism of graft take process it could be suggested
that during the initial stages (hours) following transplantation, blood, nutrients and
angiogenic factors cover the graft area and bed. Hypoxia along with the substrate of
available factors creates the signal for the angiogenic response which commences at 2-3
postgrafting days. According to our findings it was noted that the center of the graft

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contains a higher density of vascular buds and vessels suggesting that angiogenesis begins
there and continuous outwardly towards the periphery of the graft. It is controversial
whether angiogenesis takes place before the 2-3td postgrafting day since there are authors
who claim that the graft on its own has the physiologic potential to perform angiogenesis
much earlier (Laschke et al., 2008; Shepherd et al., 2004). However, it is generally accepted
that revascularization is a process that involves the presence of both the recipient bed and
the graft itself since it is their interaction which leads to effective angiogenesis. Additionally,
after the initial angiogenic response, vessels from the recipient bed gradually take over and
invade-merge with the existing vascular infrastructure of the graft and with the newly
formed vascular buds. Following that, endothelial cells initially migrate into the
perivascular space creating gradually enlarging vascular lumens which in turn are covered
by a migrating pericytes from the inner side of the vascular wall. Pericytes control and
further organize the formation of effective vascular structure by producing VEGF and by
signalling the end of aberrant angiogenesis where it takes place.
Wang et al (1996) in their study including 22 Sprague-Dawley rats, used human platelet
derived wound healing factor (HPDWF) from burn patients and porcine pituitary extract on
the recipient bed of 6 pieces of FTSG placed 1 cm apart on the back of each rat and
investigated their effect on wound healing. The recipient site was actually the bed of a
previously raised dermocutaneous local flap which was subsequently re-positioned on top
of the grafts after interposing a completely occlusive sheet between the flap and the grafts.
They found approximately 14.4% for the HPDWF and 13.16% for the PPE group
improvement in terms of the time needed to bridge the gaps between the FTSGs. However,
limitations in their study are the fact that there was no account of the role of the overlying
flap in graft wound healing despite its suggested occlusive nature by adding for example a
control group without the flap covering technique as well as the lack of evidence regarding
the histochemical compatibility of the used factors with the subjects and the potential
adverse effects of their use. Additionally the study refers to autologous skin grafts only.
Tatsuya et al (1997) found that the topical application of FK-506 on rat skin allografts may
prolong their viability and prevent the rejection cascade phenomena. Nevertheless there was
no attempt to further improve graft take results like in our study where the additional use of
rHuEPO provided additional graft healing improvement in terms of lowering the
percentage of post-grafting necrosis by 21% compared to the control group where only FK-
506 was used (35.6% vs 14.6). In our study, rHuEPO had a considerably favorable effect on
graft take and viability by achieving 12.89% less graft necrotic area (20.49%-7.6%).
According to Kaemmer et al. (2010) the formation of granulation tissue in soft tissue wounds
is promoted by rHuEPO primarily under tissue hypoxia. More specifically, rHuEPO
promotes cell migration, proliferation, myo-fibroblasts and VEGF production, solely under
hypoxia but not under normoxia. Consequently, hypoxia in the graft tissue comprises the
signal for the initiation of rHuEPO promoting effect on the graft healing process.
In group b, the additional administration of rHuEPO achieved 21% smaller area of graft
necrosis. The favorable effect of rHuEPO was shown in homologous skin grafting. New
drugs have been developed in order to achieve prevention of the rejection process without
causing toxicity is a goal in transplantation with calcineurin inhibitors (CNIs) comprising
one o them. Rejection was prevented by the use of FK-506, which is a CNI, while rHuEPO
promoted gradual healing of the graft area due to its effect on cell migration and new vessel
formation. It could be suggested that preventing excessive inflammatory response and
tissue rejection 'bought' the necessary time for rHuEPO to have its favorable effect in graft
and wound healing. Additionally, it has been demonstrated that rHuEPO inhibits the
production of pro-inflammatory cytokines by inflammatory cells, antagonizing the

Skin Grafts – Indications, Applications and Current Research
218
activation of local mechanisms which trigger potential innate injury response (Strunk et al.
2008). Thus it could be suggested that the tissue protective action of rHuEPO complemented
indirectly the rejection preventing role of FK-506.
Graft take and revascularization were lower in group c where FK-506 was administered in
subjects with autologous skin grafts than in the control group A. According to Mori et al
(1997) the explanation could be suggested to be the multiple inhibitory action of FK-506 to
many wound healing mediator molecules, primarily to TGF-β induced VEGF, leading to
angiogenesis impairment and consequently to graft take interference. However, it was
shown that graft take obstruction by FK-506 is neither irreversible nor detrimental since the
process of graft take appears to progress achieving optimum or nearly optimum levels. It
could therefore be suggested that FK-506 creates a new equilibrium, unfavorable to graft
revascularization, prolonging the required time for complete graft take and increasing the
risk of impaired viability for a part of the graft tissue. Nevertheless, more studies are
required to clarify the exact FK-506 mechanism of action in graft take and healing.
The potential clinical investigations that the finding of this study could include are the use of
rHuEPO in immunologically challenged patients (such as patients with extensive trauma, burn
injuries, transplanted organs, cancer, immunocompromising infectious disease, special
medication treatments, radioactivity injuries, hematological diseases etc) in order to promote
or/and sustain good quality skin graft take during the course of their hospitalization or even
permanently, as a definitive treatment. In our knowledge this is the first time rHuEPO has
been used in conjunction with FK-506 in homologous skin grafting with favorable results.
When it comes to the potential use of skin graft promoting factors to patients, considerations
regarding adverse effects also concern the use of rHuEPO as its systemic administration has
been associated with strong pro-coagulant and hemodynamic effects which are likely to lead
to thrombotic complications especially in patients with cancer, infection, or trauma.
Exacerbating the abnormal retinal angiogenesis in patients presenting with retinopathy and
the tumor growth supporting effect of rHuEPO have also been reported (Bennett et al., 2008;
Corwin et al., 2007). Nevertheless, there is not enough evidence to support any of the
aforementioned adverse effects. Cautious use in patients with history of diabetes, heart
disease, cancer and extensive trauma is always required.
6. Conclusion
Human recombinant erythropoietin promotes autologous FTSG take and viability in rats. It
also appeared to have a favorable effect on homologous FTSG in rats, with the use of FK-506
as an immunosuppressant. When used alone in rats with autologous skin grafts, FK-506 was
shown to have a moderately unfavorable effect on graft take. Overall, rHuEPO was shown
to promote graft take and viability in rats. Clinical considerations regarding both the
favorable and the adverse effects of special factors when used in patients, require future
studies which could provide additional insight and feedback.
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16
Recent Innovation in Pretreatment
for Skin Grafts Using Regenerative
Medicine in the East
Hitomi Sano1 and Shigeru Ichioka2
1Department of Surgical Science, Graduate School
of Medicine, the University of Tokyo
2Department of Plastic and Reconstructive Surgery
Saitama medical university hospital
Japan
1. Introduction
Chronic ulcers remain a significant health care concern today, especially in the elderly and
immobile population. Pedicled or free flap transfers are usually applied to deep wounds in
which bone and/or tendon are exposed. Despite improvements in reconstructive surgery
techniques, operative invasion and complications, including flap necrosis and infection, are
serious problems in patients whose general condition is unfavorable. For these patients, skin
graft provides less invasive coverage of wounds as compared to the flap procedures.
However, skin graft cannot survive on deep and poorly vascularized defects. Conversion of
such defects to applicable beds to simple skin graft requires technologies to induce high-
quality granulation tissue on compromised wounds. Stimulating the repair process after
surgical debridement may achieve granulation tissue formation and shorten the period for
skin grafts. For this purpose, regenerative medicine has attracted our attention. The
characteristics of the methodology include the use of the three main tools of regenerative
medicine; biomaterials, cytokines, and cells. This section introduces the technologies of
regenerative medicine for wound bed preparation with particular focus on their progress in
Asia.
All the patients illustrated in this chapter consented to the treatment. Clinical trials were
approved by the Institutional Review Board (IRB) of Saitama Medical University.
2. Regenerative medicine for wound bed preparation
As already mentioned, the methodology includes the use of biomaterials, cytokines and
cells, regenerative medicine’s three main tools.
2.1 Biomaterials
Collagen is a natural substratum for various types of animal cells and is contained in tissues
in large amounts as compared to other proteins (Linsenmayer, 1985). Advanced purification
techniques can feasibly extract biocompatible and biodegenerative collagen matrix from

Skin Grafts – Indications, Applications and Current Research
222
animal tissues. These properties support the collagen matrix as one of the most suitable
components of artificial tissue substitutes for reconstruction of damaged tissues and organs
(Chvapil et al., 1973; Doillon ＆ Silver, 1986). Several types of collagen-based artificial skin
have been reported since the initial description by Yannas and Burke (1980) (Leipziger et al.,
1985; Doillon et al., 1986; Boyce et al., 1988; Bel et al, 1981).
In the West, Integra ® (Siad Healthcare, Milano, Italy) has been generally used, and recently
approved in Japan, but only for the treatment of burns. On the other hand, clinicians in
Japan have widely used two types of artificial collagen matrix substitute dermis composed
of an atelocollagen matrix (collagen matrix) with a silicone layer, namely TERUDERMIS
(Terumo Corp, Tokyo, Japan) and Pelnac (Smith and Nephew Co., Ltd, Tokyo, Japan). These
have been officially approved for the treatment of various skin defects caused by acute
wounds as well as chronic wounds (Dantzer et al., 2001; Ichioka et al., 2003).
When these matrix substitutes are applied to a tissue defect, sprouting capillaries and
fibroblasts migrate into the collagen, which acts as a scaffold for regeneration, resulting in
induction of angiogenesis and fibroplasia. The autogenous regenerating tissue then
gradually replaces the atelocollagen, and poorly vascularized deep defects are resurfaced by
high-quality granulation tissue that can be easily covered with a simple skin graft (Ichioka et
al., 2003). Collagen matrices have been reported to achieve less invasive reconstruction of
severe defects with bone and/or tendon exposure that formerly required invasive tissue
transfer (Braden ＆ Bergstrom, 1989; Dantzer ＆ Braye, 2001).
2.2 Cytokines
Cohen (1962) noticed that the purification of submaxillary gland extracts led to earlier eyelid
separation and eruption of the incisor in mice, which eventually led to the isolation of the
first growth factor as epidermal growth factor (EGF) as part of his Nobel Prize winning
work. Since Cohen’s discovery, it has been revealed that many growth factors are implicated
in the processes of angiogenesis and wound healing (Greenhalgh, 1996; Stadelmann et al.,
1998). In the chronic wound, the balance between stimulation and inhibition is destroyed
(Stadelmann et al., 1998; Doughty & Sparks-Defriese, 2007) and fibroblasts have been
reported to be less responsive to growth factors (Harding et al., 2002). The analysis of the
supernatant from chronic pressure ulcers has shown decreased levels of growth factors,
compared with the values in acute wound supernatant (Cooper et al., 1994). Deficiencies of
growth factors in chronic ulcers suggest that the supplement of these factors might
accelerate tissue repair processes. Therefore, growth factors have been proposed as
therapeutic agents.
2.2.1 Cytokines for critical limb ischemia
Peripheral artery disease (PAD) is a major cause of the most difficult chronic limb ulcers.
The occlusion of large limb arteries leads to ischemia, and can progress to critical limb
ischemia (CLI). The treatment for ischemia is given priority in wound care. The main
treatment strategy for severe, limb-threatening ischemia is either surgical or endovascular
revascularization. If revascularization has failed or is not possible, major amputation is often
inevitable. This relates to about 30% of all cases of severe limb ischemia (Adam et al., 2005).
Regenerative medicine has been spotted as a new therapeutic option to induce angiogenesis.
Recently gene therapies using cytokines have been developed as novel treatment strategies.
The first in-human studies of gene therapy for the treatment of PAD and coronary artery

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disease(CAD) were reported in the late 1990s (Isner et al., 1996; Schumacher et al., 1998;
Laitinen et al., 1998). Since these initial reports, several gene therapies have been reported
using growth factors, including vascular endothelial growth factor (VEGF) (Isner et al.,
1996), fibroblast growth factor (FGF) (Nikol et al., 2008), hypoxia-inducible factor (HIF)
(Rajagopalan et al., 2007), developmentally regulated endothelial locus (Del-1) (Grossman et
al., 2007), and hepatocyte growth factor (HGF) (Powell et al., 2008). Among these studies,
VEGF has been the most commonly employed approach. The beneficial effects of
therapeutic angiogenesis using VEGF gene transfer have been reported in human patients
with CLI and CAD (Isner et al., 1998; Baumgartner et al., 1998; Baumgartner et al., 2000;
Losordo et al., 1998; Vale et al., 1999; Vale et al., 2000; Rosengart et al., 1999).
On the other hand, in Japan, Morishita et al (1999, 2002) have focused on HGF. HGF is also a
potent angiogenic growth factor (Belle et al. 1998; Hayashi et al., 1999; Taniyama et al., 2001;
Aoki et al., 2000), is a potent mitogen for a wide variety of cells and has angiogenic,
antiapoptotic, and antifibrotic properties (Azuma et al., 2006; Matsumoto et al., 1996;
Bussolino et al., 1992; Morishita et al., 2004). It is suggested that even in high-risk conditions
for atherosclerosis, over expression of HGF is enough to stimulate collateral formation to
treat ischemic symptoms (Taniyama e al., 2001). Furthermore, the mitogenic activity of HGF
has been reported to be more potent than that of VEGF (Belle et al., 1998; Nakamura et al.,
1996). Serum levels of HGF, but not VEGF, are elevated in CAD patients with collaterals,
and elevated HGF levels are associated with better prognoses in patients with acute
coronary syndromes (Lenihan et al., 2003; Heeschen et al., 2003). Morishita et al. (1999; 2004)
conducted preclinical investigations and initial human safety studies of HGF gene therapy,
which subsequently led to the phase II HGF-STAT trial. Patients with CLI were randomized
to treatment with placebo or 1 of 3 doses of HGF plasmid (Powell et al., 2008). TcPO2 was
significantly more improved in patients who received the highest dose of HGF plasmid than
in patients in whom a placebo or the lower HGF plasmid doses were administered. The
authors concluded that intramuscular injection of HGF plasmid has an acceptable safety
profile for the treatment of patients with CLI. Intramuscular injection of HGF plasmid may
have a favorable effect on limb perfusion as measured by TcPO2 in patients with CLI. Based
on these findings, the phase III trial is now underway to determine whether HGF has a
clinically meaningful effect on wound healing, limb salvage, and survival.
2.2.2 Cytokine products for topical application
Topical application of cytokines might be effective for wound healing. In the West, the
growth factor product generally used is recombinant human platelet-derived growth factor
(PDGF) (Regranex®). In South Korea, products based on PDGF, epidermal growth factor
(EGF) (Easyef®) and basic fibroblast growth factor (bFGF) (Fibrast®) are available. In Japan,
a bFGF product has been licensed since 2001 for use on various skin defects such as diabetic
foot wounds, pressure ulcers, venous ulcers and trauma. The following subsections
introduce EGF and bFGF for wound bed preparation.
2.2.2.1 Epidermal growth factor (EGF)
Epidermal growth factor (EGF) is a polypeptide of 53 amino acids that was first isolated
from the mouse submaxillary gland by Cohen (1962) as previously mentioned. EGF acts by
binding with the high affinity to epidermal growth factor receptor (EGFR) which is
expressed in almost all types of tissue, triggering an increase in the expression of certain

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genes that ultimately lead to DNA synthesis and cell proliferation (Herbst, 2004). EGF has
been observed to stimulate the differentiation of epithelial cells, endothelial chemotaxis and
angiogenesis, and fibroblast migration and proliferation, and to regulate extracellular matrix
turnover (Deasy et al., 2002). Enhanced wound healing has been noted in dermal wounds
treated with topical or subcutaneous EGF (Starkey et al., 1975). Franklin & Lynch (1979)
recorded quicker epithelial regeneration and less scar contracture in EGF-treated wounds in
rabbits.
With the introduction of recombinant human EGF in the 1980s, the range of studies
increased to include burn wounds and chronic ulcers (Brown et al., 1989; Falanga et al.,
1992). Studies with partial thickness human burn wounds and topical EGF confirmed a
decrease in wound healing time (Brown et al., 1989). Although the use of EGF in human
impaired wound healing by Falanga et al. (1992) showed a modest improvement in wound
healing times and rate of epithelialization, the results were not statistically significant. Some
other reports also suggested that the efficacy of EGF in chronic wounds is limited (Franklin
& Lynch 1979; Brown et al., 1989). Therefore, no conclusions about the efficacy of EGF for
chronic wounds can be made at present.
2.2.2.2 Basic fibroblast growth factor (bFGF)
In 1974, Gospodarowicz isolated a protein that accelerated the proliferation of fibroblasts
from bovine pituitary glands and termed it the fibroblast growth factor (FGF)
(Gospodarowicz et al., 1987; Gospodarowicz et al., 1988; Greenhalgh et al., 1990). One of the
best-known FGF activities includes the stimulation of fibroblast proliferation leading to
granulation tissue formation (Floss et al., 1997). FGF also plays a key role in proliferation of
endothelial cells and keratinocytes and the mitogenesis of mesenchymal cells (Floss et al.,
1997; Menetrey et al., 2000). Experimental studies have demonstrated that bFGF accelerates
angiogenesis, granulation, and epithelialization (Gospodarowicz et al., 1988). Some clinical
studies have shown the efficacy and safety of bFGF for the treatment of various wounds
including diabetic ulcers, pressure ulcers and burns (Ishibashi et al., 1996; Ichioka et al.,
2005).
2.3 Cell therapies
Stimulation of the microcirculation and enhancement of angiogenesis may promote
granulation tissue formation on compromised wounds. For this purpose, several
technologies using autologous cells have been developed. Bone marrow firstly attracted our
attention as a possible beneficial material for wound healing, because it contains
multipotential progenitor cells that can differentiate into endothelial cells and secrete several
growth factors. Several reports have illustrated the potential of bone marrow to induce
angiogenesis and also that it might contain early stem cells that can differentiate into non-
hematopoietic tissues such as the skin (Ichioka et al., 2004; Yamaguchi et al., 2005). Asahara
and colleagues (1999; 1997). have shown that vascular endothelial progenitor cells (EPCs),
the population of mononuclear cells, from the bone marrow passed through the peripheral
circulation and migrated locally to tumors, injured or ischemic regions. Furthermore, it has
been suggested experimentally that bone marrow cells contribute to the formation of a
matrix (Fathke et al., 2004; Badiavas et al., 2003), vascular formation (Asahara t al., 1999),
secretion of angiogenic cytokines (Kamihata et al., 2001; Sanchez-Guijo et al., 2010), and
stimulation of muscle cells. (Tateno et al., 2006.)

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2.3.1 Cell therapies for critical limb ischemia
Several experiments have demonstrated therapeutic relevance of cell therapy for the
ischemic limb. Ex vivo-expanded human EPCs transplanted into limb ischemia mice models
showed a recovery of blood flow, an enhanced collateral density, and a 60% limb salvage
(7% in controls) (Kalka et al., 2000). Iba et al. (2002) demonstrated that implantation of
Peripheral blood mononuclear cells and platelets into ischemic limbs effectively induced
collateral vessel formation using rat models, suggesting that this cell therapy is useful for
therapeutic angiogenesis.
Some reports indicated the efficacy and feasibility of the clinical use of cell therapy (Tateishi-
Yuyama et al., 2002; Kajiguchi et al., 2007; Matoba et al., 2008; Miyamoto et al., 2004).
Therapeutic angiogenesis using cell transplantation (TACT) is a treatment strategy for no-
option patients with CLI in Japan. Tateishi-Yuyama et al. (2002) reported the first large
clinical study on the use of bone marrow derived mononuclear cells in the treatment of limb
ischemia. According to this report, injection of bone marrow derived mononuclear cells into
the muscle of the ischemic limb apparently improved the ankle-brachial index (ABI),
transcutaneous oxygen pressure (TcPO2), rest pain, and the pain free walking distance. The
Japanese Ministry of Health, Labour and Welfare has approved this technology, namely
“Therapeutic angiogenesis using bone marrow cell transplantation”, as advanced medicine
for critical limb ischemia. In addition, two other technologies using autologous cells,
“Therapeutic angiogenesis using peripheral mononuclear cells” and “Therapeutic
angiogenesis using peripheral blood stem cells”, have been also approved (Matoba et al.,
2008; Kajiguchi et al., 2007; Miyamoto et al., 2004).
2.3.2 Topical application of autologous cells
Topical application of bone marrow cells and platelet rich plasma (PRP) have been
experimentally and clinically well-reported for their efficacy in the promotion of
angiogenesis and wound healing (Wu et al., 2010; Lacci & Dardik, 2010).
2.3.2.1 Bone marrow
Bone marrow has long been known to participate in wound healing by providing
inflammatory cells which produce cytokines and orchestrate a cascade of events (Gillitzer &
Goebeler, 2001). Recent bodies of evidence suggest that bone marrow might also serve as a
resource to provide skin progenitor cells (Krause et al., 2001; Liang & Bickenbach, 2002).
Several reports have experimentally illustrated that topically applied bone marrow cells
might promote wound healing (Badiavas et al., 2003; Sivan-Loukianova et al., 2003;
McFarlin et al., 2006).
Badiavas et al. (2003) indicated that wounding stimulated the engraftment of bone marrow
cells to the skin and induced bone marrow-derived cells to be incorporated and differentiate
into non-hematopoietic skin structures using mice models. The authors concluded that bone
marrow might be a valuable source of stem cells for the skin and possibly other organs. The
application of peripheral blood mononuclear cells accelerated the neovascularization and
epidermal healing in a model of chronic full-thickness skin wounds in diabetic mice (Sivan-
Loukianova et al., 2003). McFarlin et al. (2006) found that local injection of mesenchymal
stem cells significantly improved wound healing in an animal wound model. Bone marrow-
derived cells have been reported as transit-amplifying cells in injured tissue where they
differentiate into keratinocytes, using a mouse wound model (Borue et al., 2004).
Based on the results of the experimental studies, bone marrow-derived stem cells have been
clinically used in the treatment of chronic wounds (Badiavas ＆ Falanga, 2003; Ramsey et al.,

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1999; Metcalfe ＆ Ferguson, 2007). Badiavas & Falanga (2003) showed that topically applied
autologous bone marrow-derived cells can bring about closure of long-standing and hard-
to-heal wounds. These authors reported that bone marrow-derived cultured mesenchymal
stem cells accelerated the healing of cutaneous wounds (Falanga et al., 2007). A similar
clinical approach has been performed by Rogers et al (2008) and reported that topical
application and injection into the wound periphery of bone marrow can be a useful and a
potentially safe adjunct to wound simplification and ultimate closure (Rogers et al., 2008). It
was reported that locally applied mononuclear bone marrow cells restored angiogenesis and
promoted wound healing of an ulcer of the lower leg in a type 2 diabetic patient (Humpert
et al., 2005).
In Japan, several methods of applying bone marrow derived cells for wound have also been
tried. Mizuno et al. (2010) employed the combination of mononuclear bone marrow cells
and allogeneic cultured dermal substitute for the treatment of intractable ulcers in critical
limb ischemia. The authors injected mononuclear cells intramuscularly into the lower leg
and around the wound area and applied allogeneic cultured dermal substitute on the
wound surface (Mizuno et al., 2010). In our strategy, we impregnated autologous bone
marrow cells into a collagen matrix (Terdermis): bone marrow-impregnated collagen matrix,
(Fig.1) that has been utilized for the treatment of chronic wounds.


Fig. 1. The macroscopic finding of a bone marrow-impregnated collagen matrix
We have experimentally and clinically suggested the efficacy of this procedure for chronic
ulcer treatment (Ichioka et al., 2005, 2009). One typical case is presented to illustrate the
outcomes.
[patient 1] A-48-year-old woman suffered from a venous leg ulcer that had not healed
despite seven years of standard conventional wound therapy (Fig. 2. A). Surgical
debridement followed by bone marrow-impregnated collagen matrix application was
undertaken under lumbar anesthesia (Fig. 2. B, C). Twenty-two days later, well-vascularized
healthy granulation tissue had developed (Fig. 2. D), split-thickness skin graft was
performed. The patient has remained free of complications for 29 months since treatment
(Fig. 2. E).

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A



B


C



D

E
Fig. 2. (A) Nonhealing ulcer on the left lower limb before wound debridement. (B)
Application of the bone marrow-impregnated collagen matrix after debridement. (C) Robust
granulation tissue 3 weeks after PRP collagen application. (D) Split-thickness mesh skin
grafting was performed. (E) Healed wound.
2.3.2.2 Platelet-rich plasma (PRP)
Besides the fundamental role in hemostatsis at the injured site, platelets initiate and enhance
wound healing by releasing numerous plasma proteins and various growth factors (Everts
et al., 2006; Knighton et al., 1986; Knighton et al., 1988). Platelets stimulate angiogenesis,
proliferation and migration, and collagen synthesis. The main growth factors produced by
platelets include platelet-derived growth factor (PDGF) (Bennett et al., 2003), transforming
growth factor beta (TGF-β) (Assoian ＆ Sporn., 1986), insulin-like growth factor (IGF-I)

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(Hock et al., 1988), endothelial growth factor (EGF), vascular endothelial growth factor
(VEGF) (Mohle et al., 1997), fibroblast growth factor (FGF) (Brunner et al., 1993). Platelet-
rich plasma (PRP) is defined as a portion of the plasma fraction of autologous blood having
a platelet concentration above baseline (Mehta ＆Watson, 2008; Marx, 2001). PRP can be
supplied with a simple centrifugation procedure using solely autologous blood, suggesting
that it is a minimally invasive method (Shashikiran et al., 2006; Bhanot ＆ Alex, 2002).
Roberts and Sporn (1993) and Marx (1998; 2001) have contributed a wealth of knowledge for
the topical application of PRP in dentistry. In wound treatments, several investigations have
reported the beneficial effects of PRP since its first report in 1985 (Driver et al., 2006).
Margolis et al. (2001) reported a retrospective cohort study devised to estimate the
effectiveness of platelet releasate (PR) in the treatment of diabetic neuropathic foot ulcers.
The wounds of the 26,599 patients enrolled in this study, 43.1% of patients healed within 32
weeks, including 50% of patients treated with PRP and 41% of patients not treated with PR
treatment. The investigators concluded that PRP was more effective than the standard care
(Margolis et al., 2001). Driver et al. (2006) carried out the first prospective, randomized,
controlled multicenter trial in the United States regarding the use of autologous PRP for the
treatment of diabetic foot ulcers. The authors found the PRP treatment group attained
significantly better outcomes as compared with the control group. (Driver et al., 2006). In
our strategy, PRP is adapted with a collagen matrix to prepare the wound bed for skin graft
or spontaneous closure (Fig. 3), and is applied to the debrided wound.


Fig. 3. Collagen matrix impregnated with PRP
Some recent studies have examined the clinical application of PRP using a drug delivery
system (DDS). O'Connell et al. (2008) reported that a newly developed material containing
PRP, which they named platelet-rich fibrin matrix membrane (PRFM), exhibited a gradual
steady-state release of platelet-derived growth factors for as long as 7 days and potentially
provided a fibrin scaffold to further facilitate the tissue repair process (O’Connell, et al.
2008). In Japan, Yazawa et al. (2003) reported that when a platelet concentrate was used in
conjunction with fibrin glue as a carrier, the contents were released over a period of about 1
week. We have also reported that platelet-protein film (PPF) as an autologous fibrin clot
which PRP and plasma proteins, might continuously release growth factors to the wound
bed (Fig. 4) (Tanaka et al., 2007).

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Fig. 4. The macroscopic finding of PPF
One successful case is presented to illustrate the possible outcome of treatment using PRP.
[Patient 1]
A 89-year-old female developed a pressure ulcer over the left lower limb; the ulcer had not
healed despite three months of standard conventional wound therapy (Fig. 5 A). Surgical
debridement followed by PRP collagen application was performed under general anesthesia
(Fig. 5 B). Three weeks later, well-vascularized, healthy granulation tissue had developed
(Fig. 5 C), and a split-thickness skin graft was performed to completely close the wound
(Fig. 5 D). The patient has remained free of complications for 4 months since treatment (Fig.
5 E).



A


B



C


D


E
Fig. 5. (A) Nonhealing ulcer on the left lower limb before wound debridement.(B)
Application of PRP collagen after debridement.(C) Good granulation tissue 3 weeks after
PRP collagen application.(D) Split-thickness mesh skin graft was performed.(E) Healed
wound.
3. Conclusion
Advanced regenerative medicine-based technologies currently provide successful and less
invasive wound closure with spontaneous healing or can be used in combination with skin

Skin Grafts – Indications, Applications and Current Research
230
grafting instead of invasive surgical tissue transfer. Recent developments in physical
therapies including negative pressure wound therapy (NPWT) reinforce the efficacy of
conservative wound treatment. However, we should always bear in mind that topical
therapeutic management strategies work effectively only on adequately perfused wound
beds under a moist environment without devitalized tissue or a critical bacterial burden.
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17
Application of the Nanocrystalline Silver in
Treatment of Burn Wounds in Children
Maya Argirova and Ognian Hadjiiski
N.I. Pirogov, Centre of Burns and Plastic Surgery, Sofia
Bulgaria
1. Introduction
Infection is one of the most frequent and major complications in patients with burn injuries
and is the main cause for prolonged in-hospital stay and death in cases of wide-spread
burns despite marked progress in the development of treatments methods for these patients.
Burn infections get complicated by the development of multi-resistant organisms. The
colonization and infection of these wounds are a dual clinical problem. On one hand, a
slowing of the healing process is possible based on a damaged immune system and
inadequate perfusion to the wound. On the other hand, the infected wound may be a
potential source of spreading of antibiotic-resistant microorganisms. The infected wound is
a cause for pain and discomfort for patients, as well as life-threatening septic conditions.
Thus, the treatment cost and the medical care increase, respectively.
The topical treatment of the wound is an integral part of the general treatment of the burns.
The topical treatment agents should posses some important characteristics such as a broad
spectrum of antibacterial activity, low resistance level, decreased vapour loss, dehydration
prevention, limited adverse effects, pain control, ease of use and limited toxic risk. Due to
the wide variety of suitable agents, the choice of the agent depends on the assessment of the
wound. The most important considerations for the choice of a burn wound dressing are the
level of its antiseptic effect, the influence of its bioactive abilities on the epithelization and its
abilities for management of the wound infection. The perfect dressing for children should
provide optimal protection, reparation and minimization of pain and burn sequelae. Over
the last 30 years, the knowledge of the mechanism of wound healing has made incredible
progress as a result of intensive technological and clinical research. The new technologies
have focused on the development of antiseptic products releasing the antiseptic agent
slowly and steadily, which allows a lower and more efficient concentration. The wide-
spread antiseptic agents containing silver have a long history of usage and a wide spectrum
of activity (Klasen, 2000; Landsdown, 2006; Fong & Wood, 2006). Silver dressings are the
most commonly and frequently used in burn wound treatment.
The perfect silver dressing should contain such silver concentration which creates a free (or
limited) side antibacterial effect. The silver nitrate solution is not applied any more due to its
toxic effect, change of ionic balance and low penetration in depth. The silver sulphadiazine
SSD (Flammazine, Dermazin, Silvadene, Silverdin®, etc.) is the most frequently used agent
for topical treatment of children with burns. SSD has a powerful antibacterial effect against a
broad spectrum of Gram-positive and Gram-negative bacteria, and especially MRSA and

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Candida spp. The antibacterial effect can be achieved two hours after application and its
maximum activity lasts approximately six hours. The majority of the comparative clinical
studies, utilizing biological and non-biological dressings, involves silver sulphadiazine,
which shows that it is used as a standard agent for treatment in most burn centres (Huang
et al., 2007; Cuttle et al., 2007).
Creams and solutions containing silver have been used as main agents in burn wound
treatment for a long time. Attempts to utilize the excellent antiseptic properties of silver and
keep the principles of maintaining the moisture environment in the wound on the one hand,
and avoid the disadvantages of SSD and silver nitrate on the other, have led to the
development of a new generation of silver dressings. New technologies allowing
impregnating substances with “antimicrobial” effect in materials or medical equipment
have been exploited. Silver dressings have preventive and healing effect on the wound
surface (Burrell, 2003; Orvington, 2001).
The new silver-impregnated dressings, developed over the last 20 years, have overcome to a
great extent the disadvantages and limitations of the older silver dressings and especially
the fast silver inactivation. In these new dressings, when silver is consumed by interaction
with target cells or inactivated by protein and anion complexes in wound exudate,
additional silver is released in the wound. Thus, a sustained and steady supply of active
silver is achieved on the wound surface (Leaper, 2006). The innovation in these dressings is
not the properties of silver, which are well-known, but the amount of silver incorporated in
the dressings themselves. Since the properties of silver are beyond doubt, the choice of
dressing is determined by the characteristics of the silver-carrying material and by the way
of silver delivery to the wound. The dressing components vary as well including mesh,
nylon, hydrocolloid or methylcellulose. In order to be biologically active, the structure of
silver should have dissolution properties, e.g., Ag+ or Ag0. Ag+ is a known ionic form
present in silver nitrate, silver sulfadiazine and other ionic silver compounds. Ag0 is the
metallic or uncharged form of silver occurring in crystalline and nanocrystalline silver
structures (Demling & De Santi, 2001; Orvington, 2001).
In the treatment practice, various silver-impregnated dressings are used. Dressings of this
type differ mostly in their silver “reservoirs”. Some examples are Acticoat (Smith &
Nephew), Actisorb Silver 220 (Johnson & Johnson), Aquacel – Ag (Convatec), Arglaes
(Medline, Mundelein), Contreet – H (Coloplast, Marietta), SilvaSorb (Medline) and
Silverlon® (Argentum LLC). The comparison of the silver dressings is usually based on the
bacteriology (Cavanagh et al., 2010). Thus, differences are noticed in the field of inhibition,
with Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration
(MBC) as a result of the antimicrobial activity of the agent. The results of the in-vitro studies
of these products with respect to their antimicrobial effect are comparatively identical, and
most of them demonstrate that Acticoat has a more powerful and broader-spectrum
antibacterial effect (Yin, 2003; Yin, Langford & Burrell, 1999). For example, MIC is reached in
concentration of Ag at the range of 5-50 ppm for the most bacteria of clinical interest and
60.5 ppm for MRSA. Ag concentration for Acticoat is 70 ppm. Acticoat and Aquacel-Ag are
shown to be most efficient among the silver-impregnated dressings. It has to be pointed out
that only few prospective clinical studies on the use of these products in patients with burns
have been published. The nanocrystalline silver dressing was produced using
nanotechnology and was introduced by R.E. Burrell in 1998 (Wright et al., 1998; Burrell et
al., 1999).

Application of the Nanocrystalline Silver in Treatment of Burn Wounds in Children

239
Acticoat has biological, clinical and practical advantages. The biological advantages are: i)
excellent antimicrobial activity; ii) decrease in the inflammatory reaction of the wound by
diminishing exudation and control of wound contamination; and iii) creation of optimal
environment for the healing process of the wound by maintaining moisture, stimulating
epithelization and controlling wound contamination (Gravante et al., 2009).
The antimicrobial activity of silver ions has been studied in detail. It has been found that this
activity is due to: i) interruption of the cell respiratory chain; ii) intervention in the electronic
and ionic transport of the bacterial cell; iii) attaching to bacterial DNA and influence on cell
replication; and iv) interaction with the cell membrane and damaging of its structure and
receptor function.
The aim of our study was to assess the quality and efficacy of the nanocrystalline silver
dressing in treatment of burn wounds and to determine the potential advantages over the
older silver dressings.
2. Materials and methods
2.1 Prospective clinical study involving children treated with Acticoat
A prospective clinical study involving a clinical contingent of 112 children treated with
Acticoat has been carried out. The protocol was approved by the local ethics committee and
written informed consent was provided by the children’s parents prior the commencement
of the study. The approval of reporting the results of the study has been obtained. The
nanocrystalline silver dressing has been applied on children of both genders, stationary
treated for a period of one and a half year in 2009 and 2010. ActicoatTM and Acticoat Flex
with 3- and 7-day-wear time have been used. ActicoatTM consists of three layers – two outer
and one inner. The inner layer is an absorbent core of rayon and polyester, which controls
the silver release. The two outer layers are a silver-coated, high-density polyethylene mesh.
The silver dressing coating has a specific physical structure consisting of silver
nanocrystalline, organised in a coarse columnar structure. The thickness of the silver layer is
approximately one micrometer and contains 0.24±0.4 mg silver per mg polyethylene mesh.
Acticoat-7 dressing consists of three layers of polyethylene mesh coated with
nanocrystalline silver and two layers of rayon polyester.
Acticoat Flex is a new, modern formula of the unique silver nanotechnology. Acticoat Flex
has an open-weave structure. The open weave structure allows good contact with the
wound and easy exudate migration, thus reducing the risk of maceration and does not
require constant moistening. The dressing can be perfectly combined with vacuum negative
pressure therapy in cases of soft tissue defects.
The monitored children were in the group age of 9 months to 18 years (mean age – 5.26
years) and had burn area of 0.5% to 95% (mean – 7.89%). The burn wounds had various
depth and localization. Among them predominated the second-degree superficial burns
(IIAB. The studied contingent was grouped into five groups:
-
The first and largest group comprised 78 children with second-degree superficial burns
(IIAB) having mean burn area of 6.82%;
-
The second group involved 19 children with deep burn wounds – mean area of the
deep burn wounds of 15.76%.
-
The third group comprised 5 children on whom the Acticoat dressing was applied on
excised wounds with mean area of 6.75%.
-
The fourth group comprised 5 children with application of the dressing on autografts.

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-
The fifth group comprised 5 children with application of the dressing on fresh donor
sites.
The assessment methods used included clinical, microbiological, statistical and photo
documentation.
2.2 Comparative analysis
A comparative clinical and cost analysis between Acticoat™ and SSD (Dermazin) has been
carried out, since the latter agent has proven antiseptic properties and is widely used for the
treatment of burns in children in comparison with the other epithelotonic and less antiseptic
agents such as iodic and chlorine-hexidine (Tonkin & Wood, 2005). The clinical contingent
for this comparative analysis is different from the previous one and involved 111 children.
The patients were randomized into two groups. The comparative assessment has been
performed for 68 children with Acticoat™ and 43 children with Dermazin and the
following parameters have been monitored:
-
Clinical – wound type (initial and post dressing change), wound clearing time,
epithelization time and hospital stay;
-
Microbiological – dynamically monitoring the microbial flora, prior and post each
dressing change;
-
Pharmacological and cost parameters.
2.3 Method of use
The way of application depends on the type of Аcticoat. The application is easy and
identical regardless of the age, area, localization and the depth of the burn, excised area,
area covered with autograft or donor site. The wound areas are mechanically cleaned by
hydro-procedure using Hibiscrub. Prior to use, ActicoatTM is moistened with sterile water
and applied on the burn wound, excised area, autograft or donor site. The dressing is
trimmed according to the shape of the treated area. ActicoatTM is covered with a four-layer
moistened gauze followed by four layers of dry gauze whose purpose is to drain the surplus
amount of fluid. The dressing is held in place with a bandage or mesh. The moisture
maintenance is an important condition for the dressing efficacy and can be done by means
of the following two ways:
-
Moistening the dressing by irrigation or watering once or twice daily. The outer gauze
layer is changed.
-
Covering the dressing with polyethylene foil which helps maintain moisture.
An alternative but a more expensive way to maintain the moisture is to use Intrasite gel or
Allevyn foam.
In case of heavy exudate wounds, additional moistening of the wounds is not necessary. The
dressing is changed 3 or 7 days after application, but this period may differ depending on
the estimation. Acticoat Flex is used after an identical preparatory wound treatment and is
moistened with sterile water prior to application. It is cut to the desired shape and size, and
exploiting its elasticity applied to the wound area. The dressing is covered with a four-layer
moistened gauze followed by four layers of dry gauze. It is not additionally moistened until
the next dressing. In wide spread burns, the dressing change is performed by using a
general anaesthesia (Tancheva, 2008). In cases of limited and small burn areas, a general
anaesthesia is not necessary due to the painless dressing change.
Microbiological quantitative (tissue biopsy samples) and qualitative (wound surface swab)
analysis of the flora in the treated areas were carried out before initial application and at