EXPERIMENTAL

Normobaric Hyperoxygenation Enhances Initial

Survival, Regeneration, and Final Retention in
Fat Grafting
Harunosuke Kato, M.D.
Background: Fat grafting is a promising modality for soft-tissue augmentation/
Jun Araki, M.D.
reconstruction. However, grafted fat tissue is not initially perfused and relies
Kentaro Doi, M.D.
on plasmatic diffusion from the recipient bed until revascularization occurs.
Shinichiro Kuno, M.D. The authors evaluated the therapeutic effects of normobaric hyperoxygenation
Kahori Kinoshita, M.D. for enhancing fat graft retention.
Kazuhide Mineda, M.D. Methods: Aspirated human fat tissue was cultured under tissue hypoxia (1%
Koji Kanayama, M.D. oxygen), normoxia (6%), and hyperoxia (20%) levels, and evaluated for adipo-
Kotaro Yoshimura, M.D. cyte viability. Inguinal fat pads were autografted under mouse scalps (n = 36),
Tokyo, Japan and mice were housed in either 20% (control) or 60% (normobaric hyper-
oxygenation) atmospheric oxygen for the first 3 days, and then returned to
normoxia. Samples harvested at 0, 1, 2, 4, 8, and 12 weeks were analyzed im-
munohistochemically for adipocyte viability and regeneration.
Results: Organ culture adipocytes died more quickly under lower oxygen ten-
sions; thus, hyperoxygenation of recipient tissues may delay adipocyte death after
fat grafting. Autografted mouse adipose tissue underwent dynamic remodeling,
from ischemic degeneration to partial regeneration, over 12 weeks. Normobaric
hyperoxygenation grafted samples showed significantly larger survival zones and
engraftment scores (calculated using sample weight and adipocyte viability) at 1
and 12 weeks, respectively, than control samples. In addition, adipocyte regen-
eration (number of perilipin-positive preadipocytes), which peaked at 4 weeks,
was significantly increased in normobaric hyperoxygenation samples.
Conclusion: The normobaric hyperoxygenation protocol using 60% oxygen
can be safely applied to enhance adipocyte survival, regeneration, and final
engraftment after fat grafting.  (Plast. Reconstr. Surg. 134: 951, 2014.)

F
at grafting has been increasingly used for soft-
tissue augmentation and reconstruction.1
Clinical cases that used fat grafting to treat
radiation damage2,3 and scar contracture4 suggest
the clinical potential of fat grafting not only for
tissue enlargement, but also for tissue harmoniza-
tion and revitalization. Although there have been
many attempts to optimize and standardize fat
grafting surgical procedures, inconsistent engraft-
ment and complications such as oil cysts and calci-
fications remain problematic.
In nonvascularized tissue grafting, the con-
dition of the recipient bed is a critical factor in
graft success, because the recipient tissue alone is
responsible for providing the graft with plasmatic
From the Department of Plastic Surgery, University of Tokyo
School of Medicine.
Received for publication October 14, 2013; accepted March
26, 2014.
Copyright © 2014 by the American Society of Plastic Surgeons
DOI: 10.1097/PRS.0000000000000600
diffusion until the ingrowing neovasculature
develops. Ischemic tissues with low nutrient and
oxygen provision are not good recipients for fat
grafting. It is also difficult to obtain desirable graft
retention in stem cell–depleted recipient tissues
such as irradiated tissue and nonhealing ulcers.
Blood perfusion and tissue oxygen consumption
are key factors in determining tissue oxygen tension.5
Disclosure: The authors report no commercial asso-
ciation or conflicts of interest, financial or otherwise,
with regard to this article.
Supplemental digital content is available for
this article. A direct URL citation appears in
the text; simply type the URL address into any
Web browser to access this content. A clickable
link to the material is provided in the HTML
text of this article on the Journal’s Web site
(www.PRSJournal.com).

www.PRSJournal.com 951
 Plastic and Reconstructive Surgery • November 2014

Subcutaneous tissue has a low oxygen consumption
rate and has a higher tissue oxygen tension than other
organs such as the brain and muscle.5,6 Grafted fat tis-
sue is not initially perfused and has to rely on plas-
matic diffusion from the recipient bed. The driving
force of this diffusion is the gradient of oxygen partial
pressure6; therefore, a high oxygen partial pressure is
needed to force oxygen into the grafted fat.
In this study, we tested normobaric hyper-
oxygenation to therapeutically manipulate the
recipient microenvironments. Normobaric
hyperoxygenation is easily performed and com-
monly used during or after surgery and anesthe-
sia. Experimental normobaric hyperoxygenation
therapy was applied after fat grafting in mice, and
the therapeutic value was evaluated carefully.
MATERIALS AND METHODS
Human Adipose Tissue Sampling
We obtained lipoaspirates from three healthy
female donors (mean age, 42.0 ± 8.5 years; mean
body mass index, 21.9 ± 1.0 kg/m2; n = 3) who
underwent abdominal liposuction, for use in an in
vitro study (organ culture). Participants provided
written informed consent, and our study proto-
col had been approved by our institutional review
board. Subcutaneous fat tissue was suctioned using
a 2.5-mm (inner diameter) cannula at −500 to −700
mmHg with a conventional liposuction machine,
under general anesthesia, after infiltration of
saline solution containing epinephrine (0.001%).
Organ Culture under Different Oxygen
Concentrations
Human aspirated adipose tissue was cultured in
a petri dish at 37°Cunder 5% carbon dioxide and
different oxygen concentrations (1%, 6%, or 20%
oxygen), in humidified air. Culture medium was Dul-
becco’s Modified Eagle Media containing 10% fetal
bovine serum, 100 IU of penicillin, and 100 mg/ml
streptomycin. A cover glass was placed over the fat to
prevent exposure to the air. Organ culture fat sam-
ples were fixed (Zinc Fixative; BD Biosciences, San
Jose, Calif.) and paraffin-embedded for histologic
analysis after 1, 2, and 3 days. Freshly harvested tissues
samples were immediately fixed to serve as controls.
Mouse Autologous Fat Grafting Model
Animals were cared for in accordance with our
institutional guidelines. Eight-week-old BL6/Jjcl
mice (n = 36) were purchased from Japan CLEA, Inc.
(Tokyo, Japan) and were anesthetized with intraper-
itoneal pentobarbital (50 mg/kg). The subcutane-
ous inguinal fat pad (150 to 200 mg) was aseptically
excised for autologous grafting. The fat pad was
small enough to mimic the human aspirated fat tis-
sue used for clinical fat grafting. Autologous mouse
fat pads were transplanted by insertion through an
incision into a small subdermal pocket underneath
the scalp of the donor mouse. Grafted mice were
randomly divided into two groups, control and nor-
mobaric hyperoxygenation. Control animals were
housed under room air (20% oxygen), whereas nor-
mobaric hyperoxygenation animals were kept under
normobaric 60% oxygen/40% air for the first 3 days
after fat grafting and under room air for the remain-
ing period (Fig. 1). Animals were given free access to
food and water, and were killed after 0 (before graft-
ing), 1, 2, 4, 8, or 12 weeks (n = 3 at each time point).
The grafted fat samples were weighed and the nor-
malized sample weight ratio, which is the ratio of the
harvested sample weight to total body weight, was
calculated to evaluate the change in sample weight.
Each harvested sample was fixed (Zinc Fixative) and
paraffin-embedded for histology.

Fig. 1. Postoperative normobaric hyperoxygenation therapy (NBO)

protocol. After autologous fat grafting, mice were housed under
room air (control) or 60% oxygen (normobaric hyperoxygenation)
for the first 3 days. From day 4, both models were kept under room
air. Grafted fat samples were evaluated before transplantation and
at 1, 2, 4, 8, and 12 weeks after grafting.

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Volume 134, Number 5 • Hyperoxygenation for Fat Engraftment
Immunohistochemistry
After preparing 5-μm-thick sections of the
harvested tissue samples, we performed immu-
nostaining with the following primary antibodies:
guinea pig anti-mouse perilipin, a cytoplasmic
marker of viable adipocytes (dilution, 1:200; Pro-
gen Biotechnik, GmbH, Heidelberg, Germany)
and rat anti-mouse MAC-2 (also known as galec-
tin-3; dilution, 1:200; Cedarlane, Burlington,
Ontario, Canada). MAC-2 is a cell surface mol-
ecule expressed by macrophage subpopulations.
For double-fluorescence staining, the following
secondary antibodies were used: Alexa Fluor
488–conjugated goat anti–guinea pig immuno-
globulin G (dilution, 1:200; Invitrogen, Carlsbad,
Calif.) and Alexa Fluor 568–conjugated donkey
anti-rat immunoglobulin G (dilution, 1:200; Invit-
rogen). Appropriate species-specific fluorescently
labeled isotype-matched immunoglobulin Gs
were used as negative controls for immunostain-
ing. Nuclei were stained with Hoechst 33342 (dilu-
tion, 1:200; Dojindo, Tokyo, Japan). The number
of matured small (diameter <20 μm) adipocytes
(perilipin-positive cells) was counted in at least
four field images for each sample.
Viable adipocyte area was measured by evalu-
ating the perilipin-stained area in sections. Dead
adipocytes do not express perilipin, whereas viable
adipocytes are strongly positive for perilipin. An
engraftment score was calculated to evaluate the
magnitude of graft retention, as follows: Engraft-
ment score = Normalized fat sample weight × Per-
centage of viable adipocyte area.
Statistical Analysis
Results are expressed as mean ± standard error
of the mean. The statistical significance between
control and normobaric hyperoxygenation in
animal experiments was determined using an
unpaired t test in. The Tukey-Kramer method was
used for the organ culture experiment. Values of p
< 0.05 were considered indicative of statistically sig-
nificant differences between comparator groups.
RESULTS
Adipocyte Viability in Adipose Tissue Cultured
under Different Oxygen Tensions
Organ cultures of aspirated adipose tissue
were generated to mimic the nonvascularized
condition that exists initially after fat grafting.
By using the organ culture system, we examined
sequential changes in adipocyte viability in non-
vascularized aspirated adipose tissues subjected
to hypoxia (1%), normoxia (6%), and hyperoxia
(20%). The oxygen tension of the culture medium
was almost the same as that of the culture vessel
gas (1%, 8 mmHg; 6%, 47 mmHg; and 20%, 152
mmHg). Immunohistology for perilipin showed
that adipose tissue organ cultures progressively
degenerated during the first 3 days of culture,
and this degeneration was significantly influ-
enced by oxygen tension (Fig. 2, above). [See Fig-
ure Supplemental Digital Content 1, which shows
perilipin expression in human aspirated adipose
tissue cultured for 1 to 3 days after isolation (com-
plete version of Fig. 2, above). Stained adipose
tissue sections (green, perilipin for viable adipo-
cytes; blue, Hoechst 33342 for nuclei) revealed
that adipocytes degenerated more quickly under
lower oxygen tensions, in both time- and oxygen
concentration–dependent fashions. Scale bars = 50
μm, http://links.lww.com/PRS/B108.) The number
of viable adipocytes (perilipin-positive round cells)
in fat tissue cultures was significantly greater under
20% oxygen than 1% oxygen at 3 days (p = 0.038)
(Fig. 2, below). The finding suggests that mature
adipocytes die more quickly under lower versus
higher oxygen tensions; thus, generating higher
oxygen tension of the recipient tissue may help to
delay adipocyte death after fat grafting.
Sequential Changes in Autologous Grafted
Mouse Adipose Tissue with and without
Normobaric Hyperoxygenation Treatment
The normobaric hyperoxygenation tissue
samples were exposed to normobaric hyperoxy-
genation conditions for 3 days before return to
normoxia. These normobaric hyperoxygenation
tissues appeared to have less fibrous scarring
compared with control samples (Fig. 3, left). Nor-
mobaric hyperoxygenation samples also showed
greater normalized weight throughout the 12
weeks examined than the control samples (Fig. 3,
right). At 12 weeks, the normalized normobaric
hyperoxygenation sample weight (0.55 ± 0.08 per-
cent of total body weight, n = 3) was significantly
larger that the control samples (0.19 ± 0.05 per-
cent, n = 3) (p = 0.006) (Fig. 3, right). Weight reten-
tion rate was calculated by dividing the normalized
sample weight by the normalized initial sample
weight; normobaric hyperoxygenation samples
showed a significantly larger rate (81.4 ± 1.6 per-
cent) than control (31.2 ± 3.9 percent) (p = 0.008).
Fat Graft Remodeling after Grafting
Before grafting, adipose tissue was filled with
viable mature adipocytes that were stained strongly
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 Plastic and Reconstructive Surgery • November 2014

Fig. 2. Organ culture of human aspirated fat tissue. Aspirated fat samples were cultured under 1% (hypoxia), 6% (normoxia),
or 20% (hyperoxia) oxygen for 3 days. (Above) Stained sections (green, perilipin for viable adipocytes; blue, Hoechst 33342
for nuclei) revealed that adipocytes degenerated differently, depending on the oxygen tension. See also Supplemental
Digital Content 1, http://links.lww.com/PRS/B108, for the complete version. Scale bars = 50 μm. (Below) By day 3, viable
adipocyte (round perilipin-positive cells) numbers were significantly reduced in fat tissue cultured under 1% oxygen versus
20% oxygen (*p < 0.05).

for perilipin (Fig. 2, above). The grafted adipose
tissue underwent dynamic remodeling from isch-
emic degeneration to partial regeneration and
showed three distinctly demarcated zones: the
most superficial surviving zone, an intermediate
regenerating zone, and a central necrotic zone
(Fig. 4). The demarcation between the surviving
and regenerating zones was apparent at 1 week
after grafting, whereas that between the regenerat-
ing and necrotic zones was obvious between 2 and
4 weeks after fat transplantation. In the surviving
zone, adipocytes maintained their original size,
shape, and strong expression of perilipin, suggest-
ing that they remained viable after fat grafting. In
the regenerating and necrotic zones, all adipocytes
lost their expression of perilipin within 1 week
after grafting, indicating that they died because of
ischemia. New small preadipocytes (monolocular
or multilocular cells that were strongly positive for
perilipin) appeared as early as 2 weeks after trans-
plantation in the regenerating zone, whereas no
adipogenesis was observed in the necrotic zone.
These results confirmed adipocyte turnover after
fat grafting that we previously reported.7
Surviving Zone in Autografted Mouse
Adipose Tissue with or without Normobaric
Hyperoxygenation Treatment
Although adipose remodeling in the three
different graft zones was similarly seen in both

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Volume 134, Number 5 • Hyperoxygenation for Fat Engraftment
Fig. 3. Autologous fat grafts in mice with or without normobaric
hyperoxygenation. (Above) Macroscopic cross-sectional view of
grafted fat samples at 12 weeks after transplantation. Mice were
kept under either room air (left, control) or 60% oxygen (right,
normobaric hyperoxygenation) for the first 3 days. Dashed lines
outline cross-section edge of samples. (Below) Weight changes
of harvested fat graft samples. Normalized sample weights were
calculated (sample weight divided by body weight). Normobaric
hyperoxygenation fat graft sample weight was significantly
larger than control samples at 12 weeks after transplantation
(*p < 0.01). NBO, normobaric hyperoxygenation.
control and normobaric hyperoxygenation mice,
the surviving zone (perilipin-positive area mea-
sured 1 week after fat grafting) was significantly
larger in normobaric hyperoxygenation mice
(27.1 ± 5.6 percent) than control mice (14.4 ± 4.9
percent) (Fig. 5, above). The engraftment score
(Normalized sample weight/Normalized initial
sample weight × Percentage of viable adipocyte
area) of normobaric hyperoxygenation samples
(29.1 ± 4.1) was significantly larger than in con-
trols (14.4 ± 4.1) (p = 0.011) (Fig. 5, below). The
average thickness of the fat graft surviving zone
was much greater in normobaric hyperoxygen-
ation mice (505 ± 197 μm) than in controls (259
± 88 μm). This suggests that normobaric hyper-
oxygenation for the first 3 days after fat trans-
plantation promotes adipocyte survival in the
superficial graft region. Beginning at 2 weeks
after fat transplantation, perilipin-positive small
adipocytes (newly born preadipocytes) indicating
adipogenesis appeared and increased in number
in the regenerating zone in both control and nor-
mobaric hyperoxygenation grafts.
Fig. 4. Three zones with different adipocyte fates in grafted
mouse autologous adipose tissue. A control sample at 4 weeks
after transplantation was stained for perilipin (a marker of viable
adipocytes) and Hoechst 33342 (general nuclear stain). (Above) A
cross-section view indicated three histologically distinct regions
from the periphery to the center. S, surviving; R, regenerating; N,
necrotizing zones. White scale bar = 1 mm. (Below) A higher-mag-
nification image clearly shows that the regenerating zone has
many new preadipocytes (small round cells strongly positive for
perilipin) around dead adipocytes (dark round areas without peril-
ipin staining surrounded by MAC-2+ macrophages). The surviving
zone was filled with mature adipocytes, whereas adipogenesis
was not seen in the necrotizing zone. Yellow scale bar = 200 μm.
Adipocyte Regeneration in Autografted Mouse
Adipose Tissue with or without Normobaric
Hyperoxygenation Treatment
Adipogenesis was observed in control fat
grafts from 2 weeks to 8 weeks, based on per-
ilipin expression profiles (Fig. 6). The number
of small new adipocytes with strong perilipin
expression was comparable to baseline at 12
weeks after grafting, suggesting that adipose
regeneration was complete by 12 weeks. How-
ever, the necrotic area showed many inflamma-
tory cells and unresolved oil drops, indicating
that stabilization (phagocytosis and cicatriza-
tion) was still ongoing. The number of small
round monolocular or multilocular cells strongly
positive for perilipin in the regenerating zone,
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 Plastic and Reconstructive Surgery • November 2014

Fig. 5. Surviving zones in autologous fat grafts in control and normobaric hyperoxygenation mice. Adipocytes
located in the most superficial layers (surviving zone) survived probably because of direct plasmatic diffusion of
required nutrients and oxygen from the surrounding intact recipient tissues. All adipocytes died within 1 week
in the deeper zones (regenerating and necrotizing zones). (Above) Immunostained fat graft sections (green,
perilipin for viable adipocytes; red, MAC-2 for macrophages; and blue, Hoechst 33342 for nuclei) at 1 week after
fat grafting. The surviving zone (perilipin strongly positive area) was clearly demarcated by 1 week. Dashed lines
indicate the border of the perilipin strongly and weakly positive areas. The surviving zone was much thicker
in normobaric hyperoxygenation samples than in control samples. Scale bars = 300 μm. S, surviving zone; R,
regenerating zone. (Below) Mature adipocytes survived significantly more in normobaric hyperoxygenation
grafts than in controls. Engraftment scores (Normalized sample weight/Normalized initial sample weight ×
Percentage of viable adipocyte area) were calculated for comparison.

indicating adipogenesis, increased after fat
grafting and peaked at 4 weeks in both control
and normobaric hyperoxygenation grafts. The
new adipocyte number at 4 weeks was signifi-
cantly larger in the normobaric hyperoxygen-
ation group (73 ± 27.5) than in controls (45
± 16.2) (p = 0.016) (Fig. 6, above), suggesting
that normobaric hyperoxygenation for the first
3 days after fat grafting promotes survival of
resident adipose-derived stem/progenitor cells,
and facilitates adipogenesis at later stages.
Adipose Tissue Engraftment in Mice with
or without Normobaric Hyperoxygenation
Treatment
Adipogenesis appeared complete by 12
weeks, as suggested by measurement of small
new adipocyte numbers. Thus, we evaluated final
fat transplant engraftment at this time point
(Fig. 7). The weight of control fat grafts dropped
quickly between 8 and 12 weeks (Fig. 3), probably
because the control grafted fat contained many
oil drops (failed replacement in the necrotic
zone) and they were partly absorbed between 8
and 12 weeks. The percentage of viable adipocyte
area (strong positive perilipin staining) was larger
in normobaric hyperoxygenation samples (57.5 ±
8.2 percent) than in control samples (34.3 ± 2.0
percent) at 12 weeks after grafting. The calculated
engraftment score for normobaric hyperoxygen-
ation grafts (46.6 ± 5.2) was significantly increased
over four-fold compared with controls (10.7 ± 2.4)
(p = 0.002) (Fig. 7, below).
DISCUSSION
This study tested the therapeutic poten-
tial of systemic hyperoxygenation for enhanc-
ing engraftment of nonvascularized fat tissue.
Hyperbaric oxygenation therapy is already used

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Volume 134, Number 5 • Hyperoxygenation for Fat Engraftment

Fig. 6. Adipogenesis in the regenerating zone of autologous fat grafts in control and normobaric hyperoxygen-
ation mice. In the graft regeneration zone, new preadipocytes emerged adjacent to dead adipocytes beginning
2 weeks after transplantation. (Above) The number of new preadipocytes (small multilocular cells strongly posi-
tive for perilipin) increased after grafting, peaked at 4 weeks, and returned to baseline levels by 12 weeks. The
preadipocyte number was significantly larger in normobaric hyperoxygenation grafts than in controls at 4 weeks
(*p < 0.05). (Below) Immunostained fat graft sections (green, perilipin for viable adipocytes; red, MAC-2 for macro-
phages; and blue, Hoechst 33342 for nuclei) at 4 weeks. Dashed lines indicate the border between the surviving
and regenerating zones. In the regenerating zone, numerous preadipocytes (multilocular cells strongly positive
for perilipin; yellow arrowheads) were seen between dead adipocytes (dark round areas; asterisks) surrounded by
infiltrated macrophages (stained with MAC-2). Scale bars = 50 μm. S, surviving zone; R, regenerating zone; NBO,
normobaric oxygenation.

for various tissue repairs such as diabetic wounds
and radiation injury.8 However, oxygen toxicity
can be a serious complication, depending on the
concentration and pressure of oxygen used, and
short-term (60 to 90 minutes) treatment using
100% oxygen under high pressure remains con-
troversial in terms of balancing beneficial and
adverse effects.9–11 Thus, great care must be taken
for clinical hyperoxygenation use. Our prelimi-
nary studies revealed that normobaric hyperoxy-
genation using 60% oxygen was safe in mice even
when applied continuously for 30 days (data not
shown), whereas all mice died within 2 days under
normobaric 100% oxygen because of oxygen tox-
icity. We decided to use normobaric hyperoxy-
genation for 3 days postoperatively because the
first 3 days after fat engraftment are critical for
the survival of stem cells in ischemic environ-
ments.12,13 If revascularization is achieved within
the 3 days, the tissue can be partially repaired by
activated and recruited stem/progenitor cells;
however, in cases with more than 3 days, the tis-
sue will finally necrotize.
We previously proposed three zones in the
grafted fat tissue with different fates: survival
(superficial), regeneration (intermediate), and
necrosis (central).7,12 Grafted tissue is avascular
and is maintained only by plasmatic diffusion of
nutrients and gasses from the surrounding intact
tissue until revascularization occurs. This isch-
emic condition degenerates the grafted adipose
tissue (except for the most superficial surviving
zone), but also activates resident adipocyte stem
cells/progenitor cells that repair damaged graft

957
 Plastic and Reconstructive Surgery • November 2014

Fig. 7. Final adipose tissue engraftment at 12 weeks after fat transplantation in control and normobaric hyper-
oxygenation mice. Adipogenesis was complete by 12 weeks after grafting. (Above) Immunostained sections of
samples (green, perilipin for viable adipocytes; red, MAC-2 for macrophages; and blue, Hoechst 33342 for nuclei)
at 12 weeks. Scale bars  =  300 μm. (Below) Engraftment scores showed that grafted fat retention was signifi-
cantly increased in normobaric hyperoxygenation samples compared with control samples. NBO, normobaric
hyperoxygenation.

zones. The present study clearly showed that
normobaric hyperoxygenation therapy applied
immediately after surgery is potentially ben-
eficial in promoting survival, regeneration, and
final engraftment of transplanted fat. Perilipin
immunostaining is currently the most reliable
tool with which to differentiate between viable
and dead adipocytes, which enabled us to evalu-
ate regenerating adipocytes and zone surviving/
regenerating/necrotizing areas and to calculate
engraftment scores. Previous studies showed that
oxygen therapies do not work well to improve
tissue oxygen tension if the blood supply is
insufficient, as in an ischemic flap.5,6 Although
the grafted adipose tissue had no blood supply,
the recipient graft bed in our model had intact
perfusion and thus its tissue oxygen tension was
markedly (two- to three-fold) elevated by 60%
oxygen normobaric hyperoxygenation. In clini-
cal situations when fat is grafted to a less well-
perfused recipient bed, the hyperoxygenation
benefits may be reduced.
Organ cultures of aspirated fat tissue in
our study seem to mimic the transplant recipi-
ent microenvironment during the first few days
(revascularization would normally begin) after
fat grafting. We previously cultured human aspi-
rated fat tissue, and found that the number of
viable adipocytes decreased significantly dur-
ing the first day, suggesting that mature adipo-
cytes die readily under ischemic conditions.12 In
this study, we regarded organ culture under 6%
oxygen (100% means 760 mmHg at 1 atm) as
physiologic normoxia, because the tissue oxygen
tension of subcutaneous fat tissue in humans is 40
to 60 mmHg. Our normobaric hyperoxygenation
treatment using 60% oxygen can elevate the tis-
sue oxygen tension of subcutaneous recipient
tissue approximately two-fold in vivo and three-
fold in vitro, as shown in organ cultures.14 This
effect was suggested to be the main reason for
the reduced tissue damage observed in the organ
cultures treated with hyperoxia (20% oxygen),
and supports the current finding that the surviv-
ing graft area 1 week after autologous adipose
transplantation is significantly larger in normo-
baric hyperoxygenation compared with control
animals. Thus, normobaric hyperoxygenation
treatment effectively promotes adipocyte survival
in the nonvascularized adipose graft tissue.
Our previous studies showed that adipocyte
stem cells/progenitor cells survive even in the

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Volume 134, Number 5 • Hyperoxygenation for Fat Engraftment
regenerating zones, although all mature adipo-
cytes die within a few days because of ischemic
damage.7,12 In the present study, normobaric
hyperoxygenation also enhanced adipogenesis
in the regenerating zone, which peaked approxi-
mately 4 weeks after fat grafting. In control mice,
adipocyte stem cells/progenitor cells in the
grafts were activated and functioning to repair
the degenerating graft tissue, but they would
die if the microenvironment did not change.
Thus, postoperative hyperoxygenation increases
the regenerating area and reduces the necrotiz-
ing area, likely because the higher oxygen ten-
sion of the diffusing plasma accelerates capillary
ingrowth from surrounding recipient tissues to
rescue the activated adipocyte stem cells/pro-
genitor cells under ischemic conditions. In the
necrotizing zone, adipocyte stem cells/progeni-
tor cells also died approximately 3 days after
fat grafting, resulting in failed adipogenesis, oil
droplet deposits, and cicatrization.7 In addition
to improving immediate survival and regenera-
tion of adipocytes, the final engraftment score
measured at 12 weeks was superior in normo-
baric hyperoxygenation samples compared with
control samples.
CONCLUSIONS
Our normobaric hyperoxygenation protocol
using 60% oxygen can be safely applied to
enhance adipocyte survival, regeneration, and
final engraftment after fat transplantation.
Although normobaric hyperoxygenation may
not work well for rescuing ischemic tissues, nor-
mobaric hyperoxygenation can enhance the tis-
sue oxygen tension of well-perfused recipient
graft beds, which seems to be the mechanism
of the therapeutic effects of normobaric hyper-
oxygenation after fat grafting. Normobaric
hyperoxygenation can be applied easily without
special facilities and at reasonable cost (by using
an oxygen mask and a portable oxygen bomb),
and even shorter term use (e.g., overnight) of
normobaric hyperoxygenation immediately after
surgery may be clinically valuable. Based on the
underlying mechanism, the beneficial effects of
normobaric hyperoxygenation may not be lim-
ited to fat grafting, and normobaric hyperoxy-
genation may work as an adjuvant therapy for
any nonvascularized tissue transplantation; skin
grafting and hair transplantation may also be
good candidates for clinical application of nor-
mobaric hyperoxygenation.
Kotaro Yoshimura, M.D.
Department of Plastic Surgery
University of Tokyo School of Medicine
7-3-1, Hongo, Bunkyo-Ku
Tokyo 113-8655, Japan
kotaro-yoshimura@umin.ac.jp
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