Mesenchymal 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 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
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 pressure. 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 Hyperbaric 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 varying effects of oxygen tension and hyperbaric oxygen therapy on human and animal mesenchymal stem cells including adipose-derived stem cells,6 a detailed description of the influence of hyperbaric 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, proliferation, marker expression, differentiation, and secretion of key cytokines and growth factors of human adipose-derived stem cells was investigated in vitro.
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, undergoing abdominoplasties at the Department of Plastic and Reconstructive Surgery, Hand Surgery–Burn Center, University Hospital RWTH Aachen. The study was approved by the regional ethics committee (EK163/07), and all experiments were conducted in compliance with the principles of the Declaration of Helsinki. Adipose-derived stem cells were isolated as described earlier.7
Hyperbaric Oxygen Chamber and Hyperbaric Oxygen Therapy
For hyperbaric oxygen therapy, an experimental 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/temperature (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 pressure 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 as an indicator for cell viability was measured by the PrestoBlue assay (Invitrogen Corp., Carlsbad, Calif.) as described earlier.7 Before the first hyperbaric oxygen session, cell viability was measured by PrestoBlue (set as 100 percent; PBx) (Fig. 2). After the measurement, cells underwent hyperbaric oxygen therapy 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.
Adipose-derived stem cells were treated for 5 consecutive days. After the time point PA5, adipose-derived stem cell proliferation was analyzed by Crystal Violet solution (Carl Roth GmBH, Karlsruhe, Germany) according to the manufacturer’s instructions. Absorbance was measured on a FLUOstar OPTIMA Microplate reader (BD, Heidelberg, Germany).
CD31, CD34, CD45, CD73, CD90, and CD105 Expression
Fluorescence-activated cell sorting analysis for CD31, CD34, CD45, CD73, CD90, and CD105 was performed according to earlier protocols 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 sorting 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 chondrogenic was measured by Oil red O, Alizarin red, and Alcian blue staining, respectively. Representative slides for Oil red O (above), Alizarin red (center), and Alcian blue (below) with scale bars are presented. For quantification of the difference in differentiation, absorption was measured for Oil red O and Alizarin red staining, whereas the circumference of spheres was measured by software, http://links.lww.com/PRS/E120.] Three hyperbaric oxygen therapy treatments were performed; after that, cells were collected at PB4, as this time point showed the highest metabolic differences 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 factor (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 guidelines. Supernatants were collected at time points PB3 and PB4, as these time points showed the highest metabolic differences between the groups and over time.
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 differentiation medium. Hyperbaric oxygen therapy was performed for 5 consecutive days. All differentiation 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.]
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 differentiation 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 experiment, no differentiation medium was added. Alizarin 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% Alizarin 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.
For chondrogenic differentiation, adipose-derived stem cells were prepared in a pellet culture. 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 content of chondrogenic differentiation medium. The medium was used to induce chondrogenic differentiation 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. Chondrogenic 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.), incubated for 15 minutes, and dehydrated in ascending 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.).
Each experiment was repeated once, and each experiment was performed in triplicate. The statistical analysis is based on the mean of the independent 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 Friedman 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.
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 control 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 similar 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).
The proliferation of adipose-derived stem cells treated with 3 atm was significantly higher when compared to the control groups and markedly higher than that of adipose-derived stem cells undergoing 2-atm treatment, although no statistical difference was reached (Fig. 4); 2 atm therapy was merely altered when compared to the control group.
The stem cell marker CD34 was significantly increased between the control group and the 2-atm and 3-atm groups (Fig. 5, above). The endothelial 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 hematopoietic 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 significantly 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 latter 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 control, 2-atm, and 3-atm groups. Also, in the subpopulation of CD31−/CD34+/CD45− adipose-derived stem cells, hyperbaric oxygen therapy had no 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 significantly 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 significantly 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 secretion 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, chondrogenic, 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.
When treated with adipogenic differentiation medium, adipose-derived stem cells from the 3-atm group showed a significantly increased adipogenic differentiation capacity when compared to the control group (Fig. 8, left). Without adipogenic differentiation medium (i.e., autodifferentiation), 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 (autodifferentiation), however, the 3-atm group showed a significantly lower osteogenic autodifferentiation (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.
The effect of hyperbaric oxygen therapy at the cellular level has been established for many cell types. Studies have revealed that hyperbaric oxygen 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 oxygen tension are mesenchymal stem cells, as they are physiologically adapted to low oxygen levels. 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 stemness.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 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 noninvasive 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 molecules 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 evaluate 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, regulator of cell proliferation, motility, morphogenesis, 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 healing.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-activated cell sorting analysis showed altered expression 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, differentiation, 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 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 adipose-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 tissue revascularization, significantly dropped after 3-atm hyperbaric oxygen therapy.
There is no consensus on the exact level of atmospheric pressure that should be applied during 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 positive 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 adipogenic differentiation of mesenchymal stem cells. Hyperbaric oxygen therapy–induced reactive oxygen species were shown to increase mesenchymal stem cell proliferation, support adipogenic differentiation, 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 oxygen therapy with 2.5 atm supported osteogenic differentiation.24 The most obvious explanation for up-regulation of adipogenic but down-regulation 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 adipose-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 possible that the high adipogenic differentiation capacity under hyperbaric oxygen therapy inhibits the osteogenic differentiation.26
According to earlier studies, hypoxia appears to be a stimulator for chondrogenic differentiation 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 osteogenic differentiation, the adipogenic commitment of the adipose-derived stem cells may be an explanation for the missing effect on chondrogenic 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 regeneration 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 beneficial role of hyperbaric oxygen therapy on tissue regeneration (e.g., wound healing) may be explained by increased adipose-derived stem cell viability and proliferation, and alteration of adipose-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 adipose tissue-engineering purposes. Several groups in fact have reported positive effects of hyperbaric oxygen therapy through in vivo tissue-engineering 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 adipogenic differentiation of adipose-derived stem cells was measured by Oil red O staining. To confirm these results and dissect the mechanisms of altered adipogenic differentiation by hyperbaric oxygen therapy, additional analysis on gene and protein level should be performed.
Our study is the first to investigate the influence of hyperbaric oxygen therapy on human adipose-derived stem cells in detail. Hyperbaric oxygen with 3 atm increases viability and proliferation 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 experiments are solely descriptive, the functional consequence of our results remains subject to future studies.
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 German Research Foundation (Deutsche Forschungsgemeinschaft, KI 1973/2-1 to B.S.K.).
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