EXPERIMENTAL

The Effect of Hyperbaric Oxygen Therapy on

Human Adipose-Derived Stem Cells
Yuriko Yoshinoya, M.D.
Arne H. Böcker, M.D.
Tim Ruhl, Ph.D.
Ullrich Siekmann, M.D.
Norbert Pallua, M.D.
Justus P. Beier, M.D.
Bong-Sung Kim, M.D.
Aachen and Düsseldorf, Germany
Downloaded from http://journals.lww.com/plasreconsurg by BhDMf5ePHKbH4TTImqenVG1FEF7qxsIEfAX5oNU27jnyv5+yGFCTPD7mTYCO1P7g on 07/31/2020
Background: Adipose-derived stem cells are considered as candidate cells for
regenerative plastic surgery. Measures to influence cellular properties and
thereby direct their regenerative potential remain elusive. Hyperbaric oxygen
therapy—the exposure to 100% oxygen at an increased atmospheric pressure—
has been propagated as a noninvasive treatment for a multitude of indications
and presents a potential option to condition cells for tissue-engineering pur-
poses. The present study evaluates the effect of hyperbaric oxygen therapy on
human adipose-derived stem cells.
Methods: Human adipose-derived stem cells from healthy donors were treated

with hyperbaric oxygen therapy at 2 and 3 atm. Viability before and after each
hyperbaric oxygen therapy, proliferation, expression of surface markers and
protein contents of transforming growth factor (TGF)-β, tumor necrosis factor-
α, hepatocyte growth factor, and epithelial growth factor in the supernatants
of treated adipose-derived stem cells were measured. Lastly, adipogenic, osteo-
genic, and chondrogenic differentiation with and without use of differentia-
tion-inducing media (i.e., autodifferentiation) was examined.
Results: Hyperbaric oxygen therapy with 3 atm increased viability, prolifera-
tion, and CD34 expression and reduced the CD31−/CD34+/CD45− adipose-de-
rived stem cell subset and endothelial progenitor cell population. TGF-β levels
were significantly decreased after two hyperbaric oxygen therapy sessions in
the 2-atm group and decreased after three hyperbaric oxygen therapy sessions
in the 3-atm group. Hepatocyte growth factor secretion remained unaltered
in all groups. Although the osteogenic and chondrogenic differentiation were
not influenced, adipogenic differentiation and autodifferentiation were signifi-
cantly enhanced, with osteogenic autodifferentiation significantly alleviated by
hyperbaric oxygen therapy with 3 atm.
Conclusion: Hyperbaric oxygen therapy with 3 atm increases viability and pro-
liferation of adipose-derived stem cells, alters marker expression and subpopu-
lations, decreases TGF-β secretion, and skews adipose-derived stem cells toward
adipogenic differentiation.  (Plast. Reconstr. Surg. 146: 309, 2020.)
CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

M
esenchymal stem cells are probably the
most well-discussed cells in regenerative
medicine, and the discovery of fat tissue
as a rich depot for adipose-derived mesenchymal
stem cells has revolutionized regenerative plastic
surgery in recent years. Despite extensive research
efforts, understanding of their biological behavior
From the Department of Plastic Surgery and Hand Surgery–
Burn Center and the Department of Anesthesiology, Univer-
sity Hospital RWTH Aachen; HBO-Center Euregio Aachen;
and Aesthetic Elite International.
Received for publication August 6, 2019; accepted February
13, 2020.
The first two authors contributed equally to this article.
Copyright © 2020 by the American Society of Plastic Surgeons
DOI: 10.1097/PRS.0000000000007029
and potential appears to be far from exhausted.1
Experimental data and a rising number of clinical
studies have reported promising results of stromal
vascular fraction and adipose-derived stem cell
therapy for many plastic surgical applications.2
Furthermore, adipose-derived stem cells represent
a feasible cell source for tissue engineering, as they
are abundantly available and easily accessible.3
Disclosure: The authors have no financial interest
to declare in relation to the content of this article.
Related digital media are available in the full-text
version of the article on www.PRSJournal.com.

www.PRSJournal.com 309
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 Plastic and Reconstructive Surgery • August 2020

Factors that influence adipose-derived stem
cell functions, however, are not well investigated
to date. A potential way to influence adipose-
derived stem cell biology in a noninvasive way
is hyperbaric oxygen therapy—the exposure to
100% oxygen at an increased atmospheric pres-
sure. In specialized hyperbaric oxygen therapy
chambers, patients inhale 100% oxygen at an
ambient pressure that is higher than two-fold
atmospheric pressure (2 atm), which increases
tissue oxygenation. The first hyperbaric oxygen
therapy chamber for medical purposes was built
in 1622. In the nineteenth century, hyperbaric
oxygen therapy was increasingly applied to treat
systemic diseases such as tuberculosis, anemia,
and cholera. Today, hyperbaric oxygen therapy is
applied for the treatment of various indications,
including gas embolism, decompression sickness,
gas gangrene, carbon monoxide, critical wounds
such as diabetic ulcers, and osteomyelitis.4 Hyper-
baric oxygen therapy also may be relevant for
tissue engineering, as oxygen not only is crucial
for the survival of engineered constructs but also
serves as a signaling molecule for differentiation
of stem cells within the scaffold.5
Although some studies have reported vary-
ing effects of oxygen tension and hyperbaric oxy-
gen therapy on human and animal mesenchymal
stem cells including adipose-derived stem cells,6
a detailed description of the influence of hyper-
baric oxygen therapy is still lacking, including
the effect of various pressures on adipose-derived
stem cells. In the present work, the impact of
hyperbaric oxygen therapy on the viability, pro-
liferation, marker expression, differentiation,
and secretion of key cytokines and growth factors
of human adipose-derived stem cells was investi-
gated in vitro.
Hyperbaric Oxygen Chamber and Hyperbaric
Oxygen Therapy
For hyperbaric oxygen therapy, an experimen-
tal portable hyperbaric oxygen chamber provided
by the HBO-Center Euregio Aachen GmbH & Co.
KG (Aachen, Germany) (Fig.  1) was used. The
chamber consists of a cylindrical main chamber
filled with water and a Plexiglas lid connected to an
oxygen tank. Continuous pressure and temperature
measurement guaranteed consistent pressure/tem-
perature (21°C) during hyperbaric oxygen therapy.
Cells were placed on a rack with no contact with
the water. Adipose-derived stem cells were treated
for 5 consecutive days. A single hyperbaric oxygen
therapy session lasted 90 minutes at 2 or 3 atm pres-
sure at room temperature. The control was placed
under 1 atm at room temperature for 90 minutes.
Ninety minutes before hyperbaric oxygen therapy,
adipose-derived stem cells were taken out of the
incubator (37°C with 5% carbon dioxide) and were
held at room temperature for 90 minutes.
Metabolic Activity/Viability
Metabolic activity as an indicator for cell
viability was measured by the PrestoBlue assay

MATERIALS AND METHODS

Human Samples and Adipose-Derived Stem Cell
Isolation
Adipose tissue was harvested from seven
healthy patients (three men and four women),
with a mean age of 44.29 ± 13.08 years and a mean
body mass index of 27.04 ± 4.38 kg/m2, undergo-
ing abdominoplasties at the Department of Plastic
and Reconstructive Surgery, Hand Surgery–Burn
Center, University Hospital RWTH Aachen. The Fig. 1. Hyperbaric oxygen chamber. Cells were installed within
study was approved by the regional ethics com- the chamber and were exposed to 2 or 3 atm of hyperbaric oxy-
mittee (EK163/07), and all experiments were gen therapy. Each session consisted of 90 minutes of hyperbaric
conducted in compliance with the principles of oxygen therapy treatment at room temperature. For the control
the Declaration of Helsinki. Adipose-derived stem group, the cells were exposed to normal atmospheric pressure
cells were isolated as described earlier.7 (1 atm) at room temperature.

310
Copyright © 2020 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
 Volume 146, Number 2 • Hyperbaric Oxygen Therapy
Fig. 2. Hyperbaric oxygen therapy sessions and time points for assay measurements. Hyperbaric oxygen therapy with 2 and 3 atm
was conducted on days 3 or 5 depending on the assay. X1 through X5 stand for the hyperbaric oxygen therapy or control treatment
each consisting of 90 minutes of therapy. Time points PB1 to PB5 stand for measurements 90 minutes before hyperbaric oxygen
therapy, the time points PB1 to PB5 directly after hyperbaric oxygen therapy or control treatment.
(Invitrogen Corp., Carlsbad, Calif.) as described
earlier.7 Before the first hyperbaric oxygen ses-
sion, cell viability was measured by PrestoBlue (set
as 100 percent; PBx) (Fig. 2). After the measure-
ment, cells underwent hyperbaric oxygen ther-
apy or control treatment. Cell viability was again
measured after hyperbaric oxygen therapy before
the adipose-derived stem cells (PAx) (Fig.  2)
were stored in the incubator. This procedure was
repeated for 5 consecutive days.
Proliferation
Adipose-derived stem cells were treated for
5 consecutive days. After the time point PA5, adi-
pose-derived stem cell proliferation was analyzed
by Crystal Violet solution (Carl Roth GmBH,
Karlsruhe, Germany) according to the manufac-
turer’s instructions. Absorbance was measured on
a FLUOstar OPTIMA Microplate reader (BD, Hei-
delberg, Germany).
CD31, CD34, CD45, CD73, CD90, and CD105
Expression
Fluorescence-activated cell sorting analy-
sis for CD31, CD34, CD45, CD73, CD90, and
CD105 was performed according to earlier pro-
tocols on a LSR II cytometer (BD Bioscience,
San Jose, Calif.).8 The following antibodies were
purchased from eBioscience (San Jose, Calif.)
and used for all fluorescence-activated cell sort-
ing measurements: CD31-eFluor450, CD34-FITC,
CD45-PerCP-Cy5.5, CD73-PE-Cy7, CD90-PE, and
CD105-APC. [See Figure, Supplemental Digital
Content 1, which shows representative staining
controls, Oil red O, Alizarin red, and Alcian blue
staining. The differentiation of adipose-derived
stem cells into adipogenic, osteogenic, and chon-
drogenic was measured by Oil red O, Alizarin red,
and Alcian blue staining, respectively. Represen-
tative slides for Oil red O (above), Alizarin red
(center), and Alcian blue (below) with scale bars
are presented. For quantification of the differ-
ence in differentiation, absorption was measured
for Oil red O and Alizarin red staining, whereas
Copyright © 2020 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
the circumference of spheres was measured by
software, http://links.lww.com/PRS/E120.] Three
hyperbaric oxygen therapy treatments were per-
formed; after that, cells were collected at PB4, as
this time point showed the highest metabolic dif-
ferences between the groups.
Transforming Growth Factor-β, Tumor Necrosis
Factor-α, Hepatocyte Growth Factor, and
Epithelial Growth Factor Measurement
Transforming growth factor (TGF)-β, tumor
necrosis factor (TNF)-α, hepatocyte growth fac-
tor (HGF), and epithelial growth factor (EGF)
contents were measured in the supernatants
of adipose-derived stem cells by enzyme-linked
immunosorbent assay Duo-Sets (R&D Systems,
Minneapolis, Minn.) on a FLUOstar OPTIMA
reader according to the manufacturer’s guide-
lines. Supernatants were collected at time points
PB3 and PB4, as these time points showed the high-
est metabolic differences between the groups and
over time.
Adipogenic Differentiation
Adipogenic differentiation was determined
by Oil red O staining as reported earlier.9 We
treated adipose-derived stem cells with or without
(i.e., autodifferentiation) adipogenic differen-
tiation medium. Hyperbaric oxygen therapy was
performed for 5 consecutive days. All differentia-
tion experiments included only 3-atm hyperbaric
oxygen therapy and control groups. Absorption
was quantified on a FLUOstar OPTIMA reader.
[See Figure, Supplemental Digital Content 2,
where representative staining of adipogenic,
osteogenic, and chondrogenic differentiation is
depicted. The expression levels for the adipose-
derived stem cell surface markers CD31, CD34,
CD45, CD73, CD90, and CD105 were measured
by flow cytometry. Histograms for the respective
antibodies (red) versus their isotype controls
(blue) are depicted. Images were created by
FlowJo (FlowJo, LLC, Ashland, Ore.), http://links.
lww.com/PRS/E121.]
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 Plastic and Reconstructive Surgery • August 2020
Osteogenic Differentiation
Osteogenic differentiation was induced using
osteogenic differentiation medium on the first
day of hyperbaric oxygen therapy. Differentiation
medium was replaced every 2 days. (See Table,
Supplemental Digital Content 3, which lists the
content of osteogenic differentiation medium.
The medium was used to induce osteogenic dif-
ferentiation of human adipose-derived stem cells
in vitro. Alizarin red staining was used to quantify
osteogenic differentiation, http://links.lww.com/
PRS/E122.) In the autodifferentiation experi-
ment, no differentiation medium was added. Aliz-
arin red staining (Sigma-Aldrich Corp., St. Louis,
Mo.) was used to detect osteogenic differentiation.
After 5 hyperbaric oxygen therapy days, cells were
fixed in 4% paraformaldehyde. Next, 0.5% Aliza-
rin red solution was added and incubated for 20
minutes. After washing steps, 10% acetic acid was
applied for 30 minutes. The cell monolayer was
then dissolved and heated to 85°C for 10 minutes.
The cells were cooled on ice and centrifuged for
15 minutes at 18,000 g. After addition of sodium
hydroxide, absorption was measured at 405  nm
using a FLUOstar OPTIMA reader.
Chondrogenic Differentiation
For chondrogenic differentiation, adipose-
derived stem cells were prepared in a pellet cul-
ture. Chondrogenic differentiation was induced
using chondrogenic differentiation medium on the
first day of hyperbaric oxygen therapy. [See Table,
Supplemental Digital Content 4, which lists the con-
tent of chondrogenic differentiation medium. The
medium was used to induce chondrogenic differen-
tiation of human adipose-derived stem cells in vitro.
Alcian (periodic acid–Schiff) blue staining was used
to quantify chondrogenic differentiation, http://
links.lww.com/PRS/E123.] In the autodifferentiation
experiment, no differentiation medium was added.
After 5 consecutive hyperbaric oxygen therapy days,
differentiation medium was replaced every 4 days
over a 4-week differentiation period. Chondro-
genic spheres were fixed with paraformaldehyde
and then deep-frozen at −80°C. Spheres were cut
on a cryotome (Leica, Wetzlar, Germany) in 35-μm-
thick slices and dried for 24 hours at 37°C. Slides
were stained in Alcian (periodic acid–Schiff) blue
solution (Merck Millipore, Burlington, Vt.), incu-
bated for 15 minutes, and dehydrated in ascend-
ing alcohol series. After two washes with xylene,
the cuts were finally covered with Entellan (Merck
Millipore). The circumference of the spheres was
measured by ImageJ (National Institutes of Health,
Bethesda, Md.).
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Copyright © 2020 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
Statistical Analysis
Each experiment was repeated once, and each
experiment was performed in triplicate. The sta-
tistical analysis is based on the mean of the inde-
pendent repeated experiments. The data from all
experiments were grouped and the mean value ±
standard error was determined using GraphPad
Prism Version 5.03 (GraphPad Software, Inc., La
Jolla, Calif.). Statistical testing was performed with
IBM SPSS Version 22 (IBM Corp., Armonk, N.Y.).
All data were checked for normal distribution
with the Kolmogorov-Smirnov test. Nonnormally
distributed data were evaluated with the Fried-
man test with Dunn-Bonferroni post hoc test or
the U test. For normal distribution, the data were
analyzed using either the single-factor analysis of
variance or the t test for nonconnected samples. A
value of p < 0.05 was considered significant.
RESULTS
Viability
In the control group, a decrease of adipose-
derived stem cell viability was observed at days 1
and 2 (Fig. 3, above). On days 3, 4, and 5, the con-
trol treatment resulted in an increase of adipose-
derived stem cell viability. Adipose-derived stem
cell viability recovered in the interval between
adipose-derived stem cells and PrestoBlue when
cells had time to rest. In the 2-atm group, the early
stage of adipose-derived stem cell viability was sim-
ilar to that of the control group, with dropping
adipose-derived stem cell viability after the first
and second hyperbaric oxygen therapy sessions
and an increase after the third, fourth, and fifth
sessions (Fig. 3, center). After the fifth session, the
metabolic activity significantly dropped for the
2-atm group. At 3 atm, a more pronounced zigzag-
shaped viability pattern was observed where the
hyperbaric oxygen therapy resulted in a significant
decrease of viability (interval between PrestoBlue
and adipose-derived stem cells) (Fig.  3, below).
However, overnight adipose-derived stem cell
viability recovered significantly (interval between
adipose-derived stem cells and PrestoBlue). The
highest viability was measured at PB5 with 225.2
percent. When compared to the control and
2-atm groups, viability after day 5 increased (PB6 =
177.1 percent).
Proliferation
The proliferation of adipose-derived stem cells
treated with 3 atm was significantly higher when
compared to the control groups and markedly
 Volume 146, Number 2 • Hyperbaric Oxygen Therapy
Fig. 3. Metabolic activity of adipose-derived stem cells. Adi-
pose-derived stem cells were treated for 5 days with 2-atm or
3-atm hyperbaric oxygen therapy and control therapy. Viabil-
ity was measured by PrestoBlue assay at the time points found
in Figure  2. In all of the groups, metabolic activity decreased
directly after hyperbaric oxygen therapy/control therapy. Com-
pared to the control and 2-atm groups, the 3-atm group showed
a zigzag-shaped metabolic activity during the course of 5 days.
higher than that of adipose-derived stem cells
undergoing 2-atm treatment, although no statistical
difference was reached (Fig.  4); 2 atm therapy
Copyright © 2020 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
Fig. 4. Proliferation of adipose-derived stem cells. Adipose-
derived stem cells were treated for 5 days with 2- or 3-atm
hyperbaric oxygen therapy and control therapy. Adipose-
derived stem cell proliferation was measured by Crystal Violet
at the time point PA5. Adipose-derived stem cells treated with 3
atm showed significantly higher proliferation when compared
to the control group. No difference between the control and
2-atm groups was observed.
was merely altered when compared to the control
group.
Marker Expression
The stem cell marker CD34 was significantly
increased between the control group and the
2-atm and 3-atm groups (Fig. 5, above). The endo-
thelial marker CD31 was significantly reduced
in the 3-atm group when compared to the 2-atm
group, whereas no statistical difference was seen
compared to the control group, and the hemato-
poietic marker CD45 was significantly increased
in the 2- and 3-atm groups when compared to the
control group (data not shown).
We measured the CD31−/CD34+/CD45−
adipose-derived stem cell subset, which was sig-
nificantly reduced in the 3-atm group when
compared to the control group. In addition,
2-atm hyperbaric oxygen therapy also tended to
reduce CD31−/CD34+/CD45− populations; this
effect, however, was not significant (Fig. 5, center).
Endothelial progenitor cells were defined as
CD31+/CD34+/CD45− populations. Both 2 atm
and 3 atm led to a reduction of the endothelial
progenitor cell population, although only the lat-
ter reached statistical significance (Fig. 5, below).
The expression of the mesenchymal stem cell
markers CD73, CD90, and CD105 on all vital cells
showed no significant differences between con-
trol, 2-atm, and 3-atm groups. Also, in the subpop-
ulation of CD31−/CD34+/CD45− adipose-derived
stem cells, hyperbaric oxygen therapy had no
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 Plastic and Reconstructive Surgery • August 2020
Fig. 5. Marker expression of adipose-derived stem cells. Adipose-
derived stem cells (ASCs) were treated for 3 days with 2-atm
hyperbaric oxygen therapy, 3-atm hyperbaric oxygen therapy,
or control therapy. Surface marker expression was measured by
flow cytometry at the time point PB4. A significantly increased
expression of CD34+ was seen in the 2- and 3-atm groups (above).
However, beneficial effects of hyperbaric oxygen therapy were
not observed for endothelial progenitor cells (EPCs), which were
defined as CD31+/CD34+/CD45− cell populations (center) and the
CD31−/CD34+/CD45− adipose-derived stem cell subset (below).
314
Copyright © 2020 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
significant influence on the expression of CD73,
CD90, CD105, and CD73/CD90/CD105 (results
not shown).
TGF-β, TNF-α, HGF, and EGF Secretion
At time point PB3, the 2-atm group showed sig-
nificantly lower TGF-β levels when compared to
the control and 3-atm groups (Fig. 6, above, left). At
PB4, by contrast, the 3-atm group secreted signifi-
cantly less TGF-β when compared to the control
and 2-atm groups (Fig. 6, above, right). By taking a
closer look at the time course of TGF-β secretion
in each group, a significant increase of TGF-β in
the 2-atm group between the time points PB3 and
PB4, and an inverse decrease of TGF-β between
the same time points, was observed (Fig. 6, below).
In the control group, no change of TGF-β secre-
tion was seen over time.
For HGF, no statistical differences between
the control, 2-atm, and 3-atm groups were seen
(Fig. 7). When compared to the control and 3-atm
groups, adipose-derived stem cells treated by 2
atm tended to decrease HGF secretion between
the time points PB3 and PB4, although this effect
was not significant. Despite several repetitions
and optimization efforts of the respective enzyme-
linked immunosorbent assays, TNF-α and EGF
levels were below the detection limits in all of our
measurements of control, 2-atm, and 3-atm groups.
Differentiation of Adipose-Derived Stem Cells
As previous experiments showed little effect of
the 2-atm group, we compared adipogenic, chon-
drogenic, and osteogenic differentiation capacity
only between the control group and the 3-atm
group. For all differentiation experiments, 5 days
of hyperbaric oxygen therapy was applied.
Adipogenic Differentiation
When treated with adipogenic differentia-
tion medium, adipose-derived stem cells from
the 3-atm group showed a significantly increased
adipogenic differentiation capacity when com-
pared to the control group (Fig. 8, left). Without
adipogenic differentiation medium (i.e., autodif-
ferentiation), the effect was even more evident
(Fig. 8, right).
Osteogenic and Chondrogenic Differentiation
No difference was seen between the control
and 3-atm groups in the presence of osteogenic
differentiation media (Fig. 9, above, left). Without
osteogenic differentiation media (autodifferen-
tiation), however, the 3-atm group showed a sig-
nificantly lower osteogenic autodifferentiation
 Volume 146, Number 2 • Hyperbaric Oxygen Therapy

Fig. 6. TGF-β concentration in the supernatants of adipose-derived stem cells. Adipose-derived stem cells were
treated for 3 days with 2-atm hyperbaric oxygen therapy, 3-atm hyperbaric oxygen therapy, and control therapy.
TGF-β concentrations in the supernatants of adipose-derived stem cells were measured by enzyme-linked immu-
nosorbent assay at time points PB3 and PB4. TGF-β concentrations seem to be influenced not only by the atmo-
spheric pressure applied, but also by the count of hyperbaric oxygen therapy therapies. Before the third hyperbaric
oxygen therapy intervention (PB3), 2 atm presented significantly decreased TGF-β concentrations compared with
the control group and the 3-atm group (above, left). In contrast, before the fourth intervention (PB4), TGF-β signifi-
cantly decreased (above, right). The time course between PB3 and PB4 shows distinct changes (below).

(Fig. 9, above, right). In the case of chondrogenic
differentiation, no statistical difference was seen
between the control and 3 atm group (Fig.  9,
below). Autodifferentiation was not measurable in
the 2-atm, the 3-atm, or the control group.
DISCUSSION
The effect of hyperbaric oxygen therapy at the
cellular level has been established for many cell
types. Studies have revealed that hyperbaric oxy-
gen therapy induces fibroblast proliferation and
keratinocyte differentiation.10,11 Osteoblasts were
shown to accelerate differentiation and increase
alkaline phosphatase activity and bone nodule
formation on hyperbaric oxygen therapy, whereas
increased oxygen or pressure alone proved less
effective.12
Cells that may be sensitive to increased oxy-
gen tension are mesenchymal stem cells, as they
are physiologically adapted to low oxygen lev-
els. Adipose-derived stem cells are known to fill
a niche with oxygen concentrations less than 4%
in human adipose tissue,13 which maintains or
even increases adipose-derived stem cell stem-
ness.14 Studies to date have mainly investigated the
influence of hyperbaric oxygen therapy on bone
marrow–derived mesenchymal stem cells in tissue
engineering or other stem cells such as umbilical
cord–derived stem cells. This being said, adipose-
derived stem cells are at the forefront of clinical
application and tissue engineering, as the harvest

315
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 Plastic and Reconstructive Surgery • August 2020

Fig. 7. HGF concentration in the supernatants of adipose-derived stem cells. Adipose-derived stem cells were
treated for 2 days with 2-atm hyperbaric oxygen therapy, 3-atm hyperbaric oxygen therapy, and control ther-
apy. HGF concentrations in the supernatants of adipose-derived stem cells were measured by enzyme-linked
immunosorbent assay at the time points PB3 and PB4. HGF concentrations were not influenced by hyperbaric
oxygen therapy before the third (PB3) intervention (left) and the fourth (PB4) intervention (right).

and isolation process of adipose-derived stem cells
is considerably less complicated when compared
to bone marrow–derived mesenchymal stem cells
in tissue engineering or other stem cells such as
umbilical cord–derived stem cells.
To our knowledge, the present study is the
first in-depth analysis on the impact of hyperbaric
oxygen therapy on adipose-derived stem cells.
The rationale to study the impact of hyperbaric
oxygen therapy on adipose-derived stem cells
was twofold: (1) to add to the understanding of
adipose-derived stem cell behavior in patients
undergoing hyperbaric oxygen therapy; and (2)
to evaluate hyperbaric oxygen therapy as a non-
invasive way of preconditioning adipose-derived
stem cells in the context of tissue engineering.
Our results show that hyperbaric oxygen
therapy with 3 atm altered metabolic activity and
increased the proliferation of adipose-derived stem
cells, which is in line with experiments on other
cell types.15,16 Adipose-derived stem cells exert their
regenerative and regulatory function through—
among others—soluble factors. We focused on
the upstream cytokines TNF-α and TGF-β and the
growth factors EGF and HGF as candidate mole-
cules of inflammation and regeneration. EGF and
TNF-α levels are rather consumed than secreted
by mesenchymal stem cells.17 Nevertheless, our
rationale to still measure TNF-α and EGF in the
adipose-derived stem cell supernatants was to eval-
uate a possible stimulation by means of hyperbaric
oxygen therapy. TNF-α and EGF levels, however,
remained below the detection limit independent
of hyperbaric oxygen therapy.
HGF is considered a key regenerative
growth factor that contributes to organogenesis,

Fig. 8. Adipogenic differentiation of adipose-derived stem cells. Adipose-derived stem cells were
treated for 5 days with 3-atm hyperbaric oxygen therapy and control therapy. Adipogenic dif-
ferentiation was measured with (left) or without adipogenic (right) induction media (autodiffer-
entiation) by Oil red O staining. Adipogenic differentiation was significantly increased by 3-atm
hyperbaric oxygen therapy in the presence of induction media (left). The effect was even more
pronounced in the autodifferentiation setting (right). OD, optical density.

316
Copyright © 2020 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
 Volume 146, Number 2 • Hyperbaric Oxygen Therapy

Fig. 9. Osteogenic and chondrogenic differentiation of adipose-derived stem cells. Adipose-

derived stem cells were treated for 5 days with 3-atm hyperbaric oxygen therapy and control
therapy. Optical density (OD) was measured with (above, left) or without osteogenic (above, right)
induction media (autodifferentiation) by Alizarin red staining. Chondrogenic differentiation was
measured by evaluation of spheres only with induction medium (as autodifferentiation was tech-
nically not measurable) (below). Although the addition of differentiation medium did not induce
osteogenic differentiation under 3-atm hyperbaric oxygen therapy, the osteogenic autodifferen-
tiation was significantly decreased. Chondrogenic differentiation was not influenced by hyper-
baric oxygen therapy.

regulator of cell proliferation, motility, morpho-
genesis, angiogenesis, and mesenchymal stem cell
mobilization. Our observations, however, indicate
that adipose-derived stem cells are not a cellular
source for HGF secretion on hyperbaric oxygen
therapy, at least during the time/settings used in
our experiments.
TGF-β is a pleiotropic regulatory cytokine that
is abundantly synthesized in mesenchymal stem
cells, including adipose-derived stem cells with
pronounced action on immune cells. A level of 3
atm led to significantly reduced TGF-β secretion
over time. TGF-β is known to foster extracellular
matrix formation, to induce mesenchymal stem
cell mobilization, and is discussed as a regulator/
target gene of fibrosis and scarless wound heal-
ing.18 It is not clear whether decreased TGF-β
under 3 atm in our experiments is attributable
to lower secretion or probably higher resorption,
and additional analysis may offer the functional
result of hyperbaric oxygen therapy–induced
TGF-β down-regulation.
The phenotypic change of mesenchymal
stem cells in response to altering oxygen levels is
a matter of debate. In the peripheral blood from
patients undergoing hyperbaric oxygen therapy,
a change of circulating CD34+/CD45− cells was
observed.19 Interestingly, our fluorescence-acti-
vated cell sorting analysis showed altered expres-
sion of certain markers, and an increase of CD34+
cells under hyperbaric oxygen therapy was seen.
CD34 is one of the most prominent stem cell
markers involved in cell replication, differentia-
tion, stemness, and angiogenesis,20 and in fact was
specified as a negative marker by the International
Society for Cell and Gene Therapy.21 Additional
studies, nevertheless, showed that CD34 present
in the majority of in situ adipose-derived stem cells

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Copyright © 2020 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
 Plastic and Reconstructive Surgery • August 2020
gradually disappears in culture.22 The hyperbaric
oxygen therapy–dependent increase of CD34 cells
may be an indicator for the increased stemness
of 3-atm hyperbaric oxygen therapy–treated adi-
pose-derived stem cells. On the contrary, the stem
cell markers CD73, CD90, and CD105 remained
unaffected by hyperbaric oxygen therapy and, in
fact, the CD31−/CD34+/CD45− adipose-derived
stem cell population and CD31+/CD34+/CD45−
endothelial progenitor cells, cells involved in tis-
sue revascularization, significantly dropped after
3-atm hyperbaric oxygen therapy.
There is no consensus on the exact level of
atmospheric pressure that should be applied dur-
ing hyperbaric oxygen therapy. Applied oxygen
pressures range from 1.2 to 4 atm. Also, cells of
different origins appear to react differently to
atmospheric pressure gradients. In keratinocytes
and fibroblasts, for instance, 2.5 atm had a posi-
tive effect on cell proliferation, whereas 3 atm
inhibited proliferation.11 For adipose-derived
stem cells, most of our experiments indicate an
effect of 3 atm, whereas 2 atm did not result in
significant changes; therefore, 3 atm was chosen
for our differentiation experiments.
Differentiation is another hallmark of adipose-
derived stem cells.21 Our experiments showed a
significant up-regulation of adipogenic but down-
regulation of osteogenic autodifferentiation,
whereas chondrogenic differentiation remained
unaltered. Several studies, mostly investigating
mesenchymal stem cells under reduced oxygen
tension, indicate an oxygen-dependent adipo-
genic differentiation of mesenchymal stem cells.
Hyperbaric oxygen therapy–induced reactive oxy-
gen species were shown to increase mesenchymal
stem cell proliferation, support adipogenic differ-
entiation, and inhibit osteogenic differentiation.23
In bone marrow–derived mesenchymal stem cells
in tissue engineering or other stem cells such as
umbilical cord–derived stem cells, hyperbaric oxy-
gen therapy with 2.5 atm supported osteogenic
differentiation.24 The most obvious explanation
for up-regulation of adipogenic but down-regu-
lation of osteogenic autodifferentiation is that
bone marrow–derived mesenchymal stem cells
in tissue engineering or other stem cells such as
umbilical cord–derived stem cells and adipose-
derived stem cells follow their commitment to
their respective lines. The overwhelming bulk of
data support a lower osteogenic potential of adi-
pose-derived stem cells when compared to bone
marrow–derived mesenchymal stem cells in tissue
engineering or other stem cells such as umbilical
cord–derived stem cells.25 Furthermore, it may be
318
Copyright © 2020 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.
possible that the high adipogenic differentiation
capacity under hyperbaric oxygen therapy inhib-
its the osteogenic differentiation.26
According to earlier studies, hypoxia appears
to be a stimulator for chondrogenic differentia-
tion of bone marrow–derived mesenchymal stem
cells in tissue engineering or other stem cells
such as umbilical cord–derived stem cells27 and
embryonic stem cells.28 In line with the osteo-
genic differentiation, the adipogenic commit-
ment of the adipose-derived stem cells may be
an explanation for the missing effect on chon-
drogenic differentiation in our experiments. It
may require additional stimuli (e.g., mechanical
stress, scaffold) for hyperbaric oxygen therapy
to increase chondrogenic differentiation, as Dai
et al. observed increased chondrogenic regen-
eration in an in vivo model where rabbits were
treated with human adipose-derived stem cells
seeded onto a gelatin/polycaprolactone scaffold
and submitted to hyperbaric oxygen therapy
at 2.5 atm.29
Translated into the clinical situation, the ben-
eficial role of hyperbaric oxygen therapy on tis-
sue regeneration (e.g., wound healing) may be
explained by increased adipose-derived stem cell
viability and proliferation, and alteration of adi-
pose-derived stem cell subsets. The results of the
viability and differentiation assays also indicate
that hyperbaric oxygen therapy may be a feasible
option to treat adipose-derived stem cells for adi-
pose tissue-engineering purposes. Several groups
in fact have reported positive effects of hyperbaric
oxygen therapy through in vivo tissue-engineer-
ing experiments (e.g., the healing of ligaments30
or bone defects31). A more detailed evaluation of
adipose-derived stem cells in vivo is demanded to
underpin our preliminary results. Also, the adip-
ogenic differentiation of adipose-derived stem
cells was measured by Oil red O staining. To con-
firm these results and dissect the mechanisms of
altered adipogenic differentiation by hyperbaric
oxygen therapy, additional analysis on gene and
protein level should be performed.
CONCLUSIONS
Our study is the first to investigate the influ-
ence of hyperbaric oxygen therapy on human
adipose-derived stem cells in detail. Hyperbaric
oxygen with 3 atm increases viability and prolifer-
ation of adipose-derived stem cells, alters marker
expression and subpopulations, decreases TGF-β
secretion, and skews adipose-derived stem cells
toward the adipogenic differentiation. As our
 Volume 146, Number 2 • Hyperbaric Oxygen Therapy
experiments are solely descriptive, the functional
consequence of our results remains subject to
future studies.
Arne Hendrik Böcker, M.D.
Department of Hand, Plastic and Reconstructive
Surgery–Burn Center
BG Trauma Center Ludwigshafen
Ludwig-Guttmann-Straße 13
67071 Ludwigshafen am Rhein, Germany
arne.boecker@icloud.com
Bong-Sung Kim, M.D.
Department of Plastic Surgery and Hand Surgery
University Hospital Zurich
Rämistrasse 100
8091 Zürich, Switzerland
bong-sung.kim@usz.ch
ACKNOWLEDGMENTS
This work was sponsored by the START program of
the Medical Faculty of the RWTH Aachen University
(START-17/17 to A.H.B. and B.S.K.) and by the Ger-
man Research Foundation (Deutsche Forschungsgemein-
schaft, KI 1973/2-1 to B.S.K.).
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