Artificial Organs

36(2):161169, Wiley Periodicals, Inc.

2012, Copyright the Authors
Artificial Organs 2012, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Liposome-Encapsulated Hemoglobin Accelerates Skin

Wound Healing in Mice
*Tsuyoshi Fukui, Akira T. Kawaguchi, Susumu Takekoshi, *Muneo Miyasaka,
and *Rica Tanaka
Departments of *Plastic Surgery, Cell Transplantation and Regenerative Medicine, and Pathology, Tokai University
School of Medicine, Kanagawa, Japan

Abstract: Effects of liposome-encapsulated hemoglobin

with high O2 affinity (m-LEH, P50O2 = 17 mm Hg) on skin
wound healing in mice were examined. Two full-thickness
dorsal wounds 6 mm in diameter encompassed by silicone
stents were created in Balb/c mice. Two days later (day 2),
the animals randomly received intravenous m-LEH (2 mL/
kg, n = 12), homologous blood transfusion (red blood cell
[RBC], n = 11), or saline (n = 12). The same treatment was
repeated 4 days after wounding (day 4), and the sizes of the
skin defects and ulcers were monitored on days 0, 2, 4, and
7, when all animals were euthanized for morphological
studies. While the size of the skin defect in relation to the
stent ring remained the same in all groups, the size of the
ulcer compared with the skin defect (or silicone stent)
became significantly reduced on days 4 and 7 in mice

treated with m-LEH (46 10% of pretreatment size, P <

0.01) compared with mice treated with RBC transfusion
(73 6%) or saline (76 7%). m-LEH treatment significantly accelerated granulation, increased epithelial
thickness, suppressed early granulocyte infiltration, and
increased Ki67 expression in accordance with the ulcer size
reduction, while there was no difference in surface blood
flow or CD31 expression among the groups. The results
suggest that m-LEH (2 mL/kg) may accelerate skin wound
healing in Balb/c mice via mechanism(s) involving reduced
inflammation and increased metabolism, but not by
improved hemodynamics or endothelial regeneration.
Key Words: Artificial oxygen carrierSkin ulcer
IschemiaHypoxia-inducible
factor
1aHypoxia
Ki67CD31Liposome-encapsulated hemoglobin.

Skin wound healing is important not only for accelerating recovery but also for preventing propagation
of inflammation or likelihood of infection. As topical
malperfusion with resultant ischemia and/or hypoxia
(1) has been considered the leading cause of persistent ulcer rather than anemia (2) or malnutrition (3),
studies have sought effective treatments, including
topical decompression (4) and hyperbaric oxygen
therapy (5). However, topical decompression is not
always possible, and hyperbaric oxygen therapy is
sometimes difficult to apply, depending on the clinical
situation and anatomy behind the disease (5).

Therefore, most patients are left with indirect measures to support wound healing, such as transfusion
to correct anemia (2) and parenteral alimentation to
improve nutritional status (3).We have been studying
the action of liposome-encapsulated hemoglobin
(LEH) as an artificial O2 carrier (68), buffer, or antioxidant at reperfusion. Its characteristics include
nanometer size, comparable O2-carrying capacity to
red blood cells (RBCs), and adjustable O2 affinity
allowing targeted O2 delivery (68). Unlike the existing cell-free hemoglobin-based O2 carriers (9), hemoglobin is separated by the liposome capsule, similar in
structure to RBCs, protecting it against nitric oxide
scavenging and resultant adverse effects (9). Because
of its size (250 nm), which is much smaller than
RBCs, LEH was considered to freely perfuse collaterals and capillaries and protect the brain from
ischemic and/or reperfusion damage in a rodent (6,8)
as well as in a primate model (7). Moreover, LEH has
been modified to have a higher O2 affinity (h-LEH,

doi:10.1111/j.1525-1594.2011.01371.x
Received March 2011; revised June 2011.
Address correspondence and reprint requests to Dr. Akira T.
Kawaguchi, Tokai University School of Medicine, Shimokasuya
143, Isehara, Kanagawa 259-1193, Japan. E-mail: akira@is.icc.utokai.ac.jp

161

aor_1371

161..169

T. FUKUI ET AL.
A

RBC
n=4

RBC

D
Day 7

LEH

n=11
11

Sttudy

n=4
n
4

Saline

n=12

Histo
ol

LEH

D
Day 4

n=4

Sttudy

Saline

2nd IV

Histo
ol

1st IV

Random
mization

P50O2 = 10 mm Hg) to transport more O2 than RBCs

under hypoxic conditions (10). Based on these
characteristics, we evaluated the hypothesis that
LEH with moderately high O2 affinity (m-LEH,
P50O2 = 17 mm Hg) may improve microcirculation
and supply O2 to promote aerobic metabolism in skin
wounds and thereby accelerate healing in a murine
model of skin defect, a simulation of skin ulcer
healing.

Woundin
ng

162

n=12

MATERIALS AND METHODS

1st Photo

LEH
The characteristics of LEH (Terumo Co. Ltd.,
Tokyo, Japan) have been reported (68,10). Briefly, it
is a liposome capsule measuring 250 nm in mean
diameter, containing hemoglobin eluted from
donated human RBCs outdated for transfusion. The
liposome capsule is coated with polyethylene glycol
to reduce aggregation and capture by the reticuloendothelial system, allowing its prolongation of circulation half-life to 13 h in the rodent (68) and to 70 h in
nonhuman primates (7,11). Inositol hexaphosphate
was included as an allosteric effecter for 2,3diphosphoglycerate so as to increase O2 affinity to a
higher level (P50O2 = 17 mm Hg, m-LEH) than that of
rodent RBCs (P50O2 = 30 mm Hg) in the current
study. LEH is suspended in saline to a hemoglobin
concentration of 6 g/dL or 20% of volume (LEHcrit).
LEH is precipitated between plasma and RBC by a
centrifuge at 50 000 g for 2 h. A sibling mouse
donated homologous blood, which was collected
in a syringe containing citrate-phosphate-dextrose
solution. The blood was then centrifuged to separate
RBCs, which were diluted with saline and washed at
least three times to 20% hematocrit to serve as a
control solution containing a comparable amount of
hemoglobin to LEH.
Animals and skin wounding
All experiments were approved by the institutional review board of Tokai University School of
Medicine. Animals received humane care as
requested in National Institutes of Health publications (1985) amended in 2005. Male Balb/c mice
were purchased from CLEA Co. Ltd. (Yokohama,
Japan) and used at 8 weeks of age (20 1 g). Mice
were anesthetized with 2% sevoflurane, and trunk
hair was removed by skin hair remover on the day
before skin defect creation (Fig. 1A). On day 0, skin
wounds were created using a circular skin tome
(6 mm diameter) in the bilateral back of the anesthetized mice (Fig. 1B). A donut-shaped silicone
skin holder (Fuji Systems, Yokohama, Japan) was
Artif Organs, Vol. 36, No. 2, 2012

2nd Photo

4th Photo

3rd Photo

Days After Wounding

c
1 2

2 1

FIG. 1. Experimental protocol. Experimental protocol (A) is

illustrated. A mouse received two dorsal skin lesions (B) surrounded by a silicone skin holder (a), which was fixed in place
firmly by an adhesive and eight 6-0 monofilament sutures. The
wound (b) was evaluated by photograph to measure the area
inside the ring (area 1), skin defect (area 2), and ulcer (area 3)
(c).

fixed around each skin defect, first by adhesives

and then by eight 6-0 monofilament sutures
(Fig. 1B) to prevent skin contraction. These skin
defects were photographed and covered with an
occlusive skin protector to prevent contamination.
Finally, a plastic corset was placed around the trunk
of the body to keep animals from reaching the
wounds themselves.
Drug administration
Two days later (day 2), animals were randomly
assigned to three groups so that each group with the

ARTIFICIAL O2 CARRIER ACCELERATES WOUND HEALING IN MICE

same size of ulcer received the initial infusion;
2 mL/kg of body weight of m-LEH (n = 12), transfusion of homologous washed RBCs at the same
amount of hemoglobin (n = 11), or saline (n = 12) was
intravenously infused from the tail vein under 2%
sevoflurane anesthesia (Fig. 1A). Each solution was
diluted five times with saline (approximately 0.2 mL
per animal) for volume accuracy at administration.
After administration, animals were placed back in
cages in room air, with access to food and water ad
libitum until 2 days later for the second medication
(day 4) and 5 days later for the last observation and
sacrifice (day 7, Fig. 1A).
Laser Doppler flow measurement
Four additional mice (n = 4) in each group, treated
in the same way, were followed for surface blood flow
and euthanized for histological observation on day 4,
2 days after initial treatment (Fig. 1A). To evaluate
changes in surface blood flow in response to the
administration of solutions, surface perfusion was
monitored in normal skin (area 1, Fig. 1B), edge of
the ulcer (area 2), and center of the ulcer (area 3)
before and after solution administration on day 2 by
Laser Perfusion Imager (moorFLPI, Moor Instruments, Devon, UK). This was repeated 2 days later,
on day 4, when the animals were then sacrificed for
histological studies (Fig. 1A).
Skin lesions
The area of each skin lesion was measured from
photos taken on day 2 as ring (inside the ring, area 1,
Fig. 1B), skin defect (faintly dotted area 2), and ulcer
(darkly dotted area 3). Then, the area ratios, skin
defect/ring (area 2/area 1), ulcer/ring (area 3/area 1),
and ulcer/skin defect (area 3/area 2) were determined
for the right and left wounds, which served as control
(100%) for the ratios of the later observations on
days 4 and 7.
Morphological studies
After all the animals were photographed on days 4
(n = 12) and 7 (n = 35), they were sacrificed under
deep anesthesia, and the back skin lesions (two in
each animal, Fig. 1B) were removed in full depth for
morphological studies. Each lesion was divided down
the middle; one half was placed in 4% paraformaldehyde for Ki67 staining and methanol for CD31 staining, and the other half was frozen in liquid nitrogen
for later studies. The ulcer specimen was prepared so
that the wound could be observed in full length along
the ulcer diameter. The specimens were fixed and
stained with hematoxylineosin (H&E) for histopathological observations, to determine the degree of

163

neutrophil infiltration, titrated as none (scored 0),

mild (scored 1), moderate (scored 2), and severe
(scored 3) by two independent observers blinded to
the study protocol. Granulation development and
epithelial thickness were also measured in the same
way for each edge of the ulcer (Figs. 1B and 4A).
Immunohistochemical studies
Immunohistochemical staining for hypoxiainducible factor 1a (HIF-1a), CD31 (12), and Ki67
(13) was carried out. CD31- and Ki67-positive cell
numbers and whole cell numbers as background were
counted by two independent observers. Five fields
were randomly selected and counted using digital
image analyzing software (Image J1.37v) developed
at the National Institutes of Health, Bethesda, MD.
Statistics
The two skin lesions in each animal were considered independently. Data were presented as
mean standard deviation. The variables from each
lesion were averaged for each group, and compared
among groups by KruskalWallis analysis. A P value
of less than 0.05 was considered significant.
RESULTS
Laser Doppler flow measurement
The surface blood flow determined by Laser
Doppler flow meter (Fig. 2) was greater in the order
of the center, edge of the ulcer, and skin surrounding
the defect. There were no significant or consistent
changes between before and after administration of
solution (day 2) and 2 days later (day 4). Among the
animal groups, the m-LEH-treated mice had a tendency toward reduced flow on day 4 compared with
the animals treated with RBC or saline.
Ulcer size
The process of skin wound healing was followed
(Fig. 3A); only lesions properly created according to
the study design were included (day 0). Two days
later, animals were randomly assigned to three
groups so that each group had the same ulcer size,
and they received the initial infusion (day 2). Two
days later (day 4), some differences in macroscopic
appearance of the ulcers were noted, and the
animals received the second infusion of the same
solution. Three days thereafter (day 7), the ulcers
were dry and largely covered by epithelium in the
m-LEH-treated mice, whereas ulcers were still wet
in the other treatment groups (Fig. 3A). There was a
highly significant correlation between the left and
right lesions in ulcer/skin defect (area 3/area 2) on
Artif Organs, Vol. 36, No. 2, 2012

164

T. FUKUI ET AL.

FIG. 2. Changes in surface blood flow. In

four additional mice (n = 4) in each group
treated in the same way, surface blood
perfusion was monitored in normal skin
(area 1), edge of the ulcer (area 2), and
center of the ulcer (area 3) before and
after the administration of the solution on
day 2 and again on day 4 by Laser Perfusion Imager (moorFLPI, Moor Instruments,
Devon, UK).

Skin (Area 1)

Edge (Area 2)

Center (Area 3)

day 4 (r = 0.827, P < 0.05) as well as on day 7

(Fig. 3B, P < 0.01). When bilateral lesions were independently evaluated in the time sequence (Fig. 3C),
skin defect/ring (area 2/area 1) was reduced without
difference among the treatment groups, and ulcer/
ring (area 3/area 1) and ulcer/skin defect (area
3/area 2) were significantly more reduced in
m-LEH-treated mice than in mice receiving RBC or
saline, with no difference between the latter two
groups on day 4 or on day 7.

the other treatment groups by day 7, and no significant difference was observed among the groups.
While CD31-positive cells showed no significant difference among the m-LEH (114 29), RBC
(104 33), and saline (95 9) groups, Ki67-positive
cells were significantly more numerous in the
m-LEH-treated mice (115 17, P < 0.05) than in
mice treated with RBC (96 14) or saline (95 9)
on day 4, and also on day 7 (m-LEH-treated mice,
104 14, P < 0.05; RBC, 86 10; saline, 84 8).

Granulation and epithelium

Granulation formation and epithelial thickness
were determined in each lesion (Fig. 4A) and averaged for each treatment group. Granulation was
better developed in mice treated with m-LEH
(617 142 mm, P < 0.05) compared with mice treated
with RBC (486 125 mm) or saline (446 116 mm).
Also, the epithelium was thicker in mice treated
with m-LEH (114 27 mm, P < 0.05) than in mice
receiving RBC transfusion (70 24 mm) or saline
(74 17 mm) on day 7.

DISCUSSION

Neutrophils, Ki67-positive cells, and

CD31-positive cells
HIF-1a was not clearly stained in any animal on
day 4 or day 7. Granulocyte infiltration and Ki67- and
CD31-positive cells were counted in each lesion (Fig.
5A), and averaged for the treatment groups at 2 days
(day 4) after the initial treatment and 3 days (day 7)
after the second administration (Fig. 5B). Although
granulocyte infiltration was significantly suppressed
in the m-LEH-treated mice (1.4 0.5, P < 0.05) compared with mice treated with RBC (2.3 0.7) or
saline (2.8 0.5) on day 4, the infiltration subsided in
Artif Organs, Vol. 36, No. 2, 2012

LEH has been examined on an experimental

basis and was found to be protective in animal
models of cerebral ischemia (68,10), cochlear
ischemia/reperfusion (14), skeletal muscle ischemia/
reperfusion (15), gastrointestinal wound healing (in
preparation), and enhancing tumor radiotherapy
(16). Improved microcirculation, increased oxygen
delivery, and persistent aerobic energy metabolism
form the rationale for these applications of LEH
(17). In particular, LEH with high O2 affinity is considered to be able to, and has actually been
observed to, efficiently deliver O2 to tissues under
ischemia (10) or deranged perfusion after gastric
surgery (in preparation). Based on these experimental results, the current study was performed to
examine our hypothesis that LEH may accelerate
wound healing of the skin, where O2 metabolism
may be different from other organs or tissues
(68,1416).
Since surface blood flow as determined by Laser
Doppler flow meter (Fig. 2) showed no differences
among the treatment groups on days 2 and 4, hemodynamic effects were likely not responsible for the

ARTIFICIAL O2 CARRIER ACCELERATES WOUND HEALING IN MICE

165

Day 2

Day 4

Day 7

Ulcer / Skin Defect (%)

(Day 7)

LEH
RBC
Saline

20

RBC

60

LEH

40

Right Lesion (%)

80

Day 0

100

r=0.867

Right = 0.95 Left + 2.3

Saline

20

40
60
Left Lesion (%)

80

100

100 (%)

2nd dose

1stt

2ndd dose

1stt

2ndd dose

60

80

1st

Saline
RBC
LEH

Saline
RBC
LEH

20

40

Saline
RBC
LEH

Ulcer / Ring

Ulcer / Skin Defect

Skin Defect / Ring

2

Days After Wounding

FIG. 3. Macroscopic changes in ulcers. Macroscopic sequential observation was carried out in all animals (A). There was a highly
significant correlation between the left and right lesions in ulcer/skin defect (area 3/area 2) on day 4 (r = 0.827, P < 0.05), as well as on
day 7 (B). When bilateral lesions were independently evaluated in the time sequence (C), skin defect/ring (area 2/area 1) decreased
without difference among the treatment groups, ulcer/ring (area 3/area 1), and ulcer/skin defect (area 3/area 2) were significantly more
reduced in m-LEH-treated mice than in mice receiving RBC or saline (*P < 0.05), with no difference between the latter two groups.

significant differences among the treatment groups.

As normal skin structure would not be able to
develop within the observed 7-day period, there was
no difference in the size of the skin defect in terms of
ring area among the treatment groups. Each mouse
had two dorsal skin lesions; the degree of healing in
the right and left lesions was similar (Fig. 3B), suggesting that wound healing was largely dependent on
the individual animal than the construction or maintenance of each lesion.
Hypoxia immediately after skin wounding is
known to increase the production of angiogenic

factors such as vascular endothelial growth factor,

platelet-derived growth factor receptor, and transforming growth factor-b and their dose-dependent
effect on angiogenesis (18). In the current study,
m-LEH was administered 2 days after wounding,
when these immediate hypoxia and/or angioproliferative signals had already been generated and
angiogenesis triggered in each animal, accounting for
little or no expression of HIF-1a in each animal and
no significant difference in CD31 expression among
treatment groups. While the first 2-day observation
without treatment allowed us to equally subject the
Artif Organs, Vol. 36, No. 2, 2012

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T. FUKUI ET AL.

Skin

Ulcer

100 m

Epithelium

Granulation

Ulcer

Skin
100 m

Epithelium

Granulation

Saline

LEH

Skin
100 m

Ulcer
Epithelium

Granulation

RBC

Epithelium (m)

Granulation (m)

800
600
400
200
0

Saline

mLEH

RBC

Saline

mLEH

RBC

150

100

50

FIG. 4. Microscopic observations. Granulation formation and epithelial thickness were determined in each lesion (A) and averaged for the
treatment group (B). Both granulation and epithelial thickness were significantly more accelerated in the m-LEH-treated mice than in mice
treated with RBC or saline (*P < 0.05), with no difference between the latter two groups.

wounds to the initial ischemic phase and to

evenly randomize the animals regarding the wound
in terms of size, coverage, and absence of infection,
later oxygenation by m-LEH on days 2 and 4 might
have caused the significant reduction in ulcerous
areas. In fact, these macroscopic and functional
observations were in accordance with the histological
findings; both granulation and epithelium were significantly better developed in the m-LEH-treated
mice compared with the other two groups, which
showed no difference between them.
In order to examine the mechanism(s) involved in
the benefits of m-LEH treatment, neutrophil infiltration to the lesions was counted. Consistent with
Artif Organs, Vol. 36, No. 2, 2012

the macroscopic observations, the infiltration of

neutrophils was suppressed in m-LEH-treated mice
2 days (day 4) after the initial treatment as compared with the other treatment groups. The difference was no longer significant 3 days following the
second treatment (day 7), highlighting the possibility that m-LEH treatment suppresses inflammatory
reaction early after skin wounding. Such early suppression of inflammatory response appears to be a
common phenomenon after LEH administration,
as it was also observed following gastric surgery
(in preparation). While HIF-1a was not clearly
stained in any of the animals on day 4 or day 7,
Ki67 expression was increased only in the

ARTIFICIAL O2 CARRIER ACCELERATES WOUND HEALING IN MICE

167

LEH

RBC

Ki67

CD31

Neutrophil

Saline

Neutrophil

3
2
1

CD31

0
100
50
0

Ki 67

120
80
40
0

Day 4

Day 7

FIG. 5. Immunohistochemical observations. Granulocyte infiltration (H&E, 300), CD31-positive cells (200), and Ki67-positive cells
(200, arrows) were counted in each lesion (A), and averaged for each treatment group (B) at 2 days (day 4) after the initial treatment
and at 3 days (day 7) after the second administration. Although granulocyte infiltration was significantly suppressed only in the
m-LEH-treated mice on day 4 (*P < 0.05), the extent became less in the other treatment groups on day 7, when there was no difference
among the groups. While the number of CD31-positive cells showed no significant difference among the groups on day 7, Ki67-positive
cells (arrows) were significantly more numerous in the m-LEH-treated mice than in the other mice both on days 4 and 7 (*P < 0.05).

Artif Organs, Vol. 36, No. 2, 2012

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T. FUKUI ET AL.

m-LEH-treated animals on these days, suggesting

that protein synthesis was accelerated compared
with the other treatments. Such elevated synthesis
was apparently not related to the regeneration of
blood vessels, as CD31 expression did not differ
among the groups. Thus, LEH administration promotes skin wound healing not by angiogenesis, but
by increasing the fibroblast proliferation and collagen synthesis. Although the 5-day follow-up with
two m-LEH 2 mL/kg administrations in the current
study may not be long enough for vascular regeneration, LEH might have promoted wound healing
by increasing fibroblast proliferation, as confirmed
by the increased Ki67 expression and collagen formation observed in the accelerated granulation and
epithelial development.
Transfusion is a common therapeutic option for
reversing anemia, replacing blood loss, improving O2
delivery, and accelerating postoperative recovery
(2). Although most artificial O2 carriers have been
developed for this purpose, so far, no single product
has been accepted as an RBC substitute for clinical
use (9). LEH as blood substitute has been reported
by Ikegawa and colleagues (19), who demonstrated
that LEH with low O2 affinity (P50 = 40 mm Hg)
may be inferior to RBCs in maintaining systemic
metabolism and peripheral perfusion in rabbits
undergoing extended exchange hemodilution up to
86%. As the current LEH has high O2 affinity
(P50 = 17 mm Hg) as compared with RBC (P50 =
30 mm Hg in mice), it is not likely to unload oxygen
prematurely to induce vasoconstriction or tissue
hypoperfusion, but rather deliver O2 to hypoxic
tissue efficiently (17). In contrast, RBC transfusion failed to improve skin wound healing in the
current study, similar to mice receiving saline. As
hematocrit in all animals was considered to be
normal, it is conceivable that the additional RBC
transfusion might not add any benefit as also
suggested by Mandai et al. (2), who revealed that
hemoglobin over 10 g/dL was enough in wound
healing. Similar protective effects of LEH have
been demonstrated in cerebral ischemia and
reperfusion models in rats (6,8) as well as in nonhuman primates (7), another model of local perfusion derangement. As RBC transfusion failed to be
beneficial in either of these cases, the nanometer
size of LEH may in fact provide advantages over
RBC transfusion in animal models of cerebral
ischemia and reperfusion as well as wound healing.
Thus, the presence of hemoglobin-containing nanoparticles in plasma, but not the presence of hemoglobin in the form of RBCs, may be important for
wound healing.
Artif Organs, Vol. 36, No. 2, 2012

CONCLUSION
The current results support our hypothesis that
m-LEH may improve aerobic metabolism and
accelerate wound healing in skin ulcer, which is not
offered by homologous transfusion, nor by an
equivalent amount of hemoglobin in the form of
RBCs. The mechanism is not clear from this study,
but it is probably related to a suppressed immediate
inflammatory response and later proper oxygenation for fibroblast proliferation and collagen synthesis in the skin defect, and likely not via direct
hemodynamic effect or accelerated angiogenesis, a
possible
mechanism
in
other
therapeutic
approaches. In fact, such treatments might provide
synergistic effects. Optimal oxygen affinity, dose, and
timing of LEH administration will need to be clarified for their application.
Acknowledgments: This study was supported in
part by: (i) Grant-in-Aid for Scientific Research
14370365, 16209037, and 20249072 from the Ministry
of Education, Culture, Science and Technology,
Tokyo, Japan; (ii) New Energy Development Organization (NEDO), Tokyo, Japan; and (iii) JST, Tokyo,
Japan. We would like to thank Chihiro Fujinuma,
Naokatsu Ando, Yo Kawaguchi, and Hideyuki
Hanano (Tokai University School of Medicine) for
their help with the experiments and animal care. We
also thank the Tokai University Teaching and
Research Support Center, Research Development
Division for assistance with the experimental setup,
preparation, and administration.
Conflict of Interest: All authors, Tsuyoshi Fukui,
Akira T. Kawaguchi, Susumu Takekoshi, Muneo
Miyasaka, and Rica Tanaka, are clinicians/scientists
who organized and performed this study. At the time
this work was conducted and completed, they all
worked at the Tokai University School of Medicine,
where all animal experiments took place. Terumo Co.
Ltd. developed and supplied the LEH tested in this
study. ATK received the research grants specified
above, which provided materials and personnel to
carry out the experiments and summarize the results
in this manuscript. There were no other monetary
dependencies or conflicts of interest among the
authors.
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Artif Organs, Vol. 36, No. 2, 2012