Tissue and Cell 78 (2022) 101886

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Tissue and Cell

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The effect of hypergravity, hyperbaric pressure, and hypoxia on osteogenic

differentiation of adipose stem cells
L.F. Lingens *, T. Ruhl , J.P. Beier , W. Mende , G. Freund , R. Götzl
Department of Plastic Surgery, Hand Surgery–Burn Center, University Hospital RWTH Aachen, Pauwelsstraße 30, Aachen 52074, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: Human adipose stem cells (ASCs) hold great potential for regenerative medicine approaches, including osteo­
Adult stem cells genic regeneration of bone defects, that fail to heal autonomously. Osteogenic differentiation of stem cells is
Differentiation dependent on the stimulation of biophysical factors. In the present study, the effects of hypergravity, hypoxia,
Proliferation
and hyperbaric treatment were investigated on adipose stem cell (ASC) metabolic activity, quantified by Pres­
Osteogenesis
Physico-chemical stimulation
toBlue conversion, and cell numbers, evaluated by crystal violet staining. Osteogenic differentiation was assessed
by alkaline phosphatase (ALP) activity and cresolphthalein staining of calcium deposition. Differentiation was
performed for 12 days, which was accompanied by periodical stimulation. Increasing gravity forces up to 50x g
did not affect ASC viability, but it enhanced osteogenic markers with a strongest effect between 20 and 30x g.
Hyperbaric stimulation at 3 bar decreased ASC cell numbers but increased ALP activity and calcium deposition.
Hypoxia at 8 % atmospheric oxygen did not affect ASC proliferation, while cell numbers were reduced at 3 %
oxygen. Furthermore, hypoxic conditions produced opposing results on osteogenic markers, as ALP activity
increased whereas cresolphthalein staining decreased upon stimulation. These data demonstrated that inter­
mittent short duration of basal physical or chemical impulses interfere with the osteogenic differentiation of
ASCs. Our findings could be of specific relevance in ASC based therapies for regenerative medicine and bone
tissue engineering approaches.

1. Introduction including the bone morphogenetic protein (BMP)− 2, growth/differ­

entiation factor (GDF)− 5, retinoid acid and tumor necrosis factor
Bone defects fail to heal physiologically due to critical size or because (TNF)-α, among others (Cho et al., 2010; Min et al., 2014; Zeng et al.,
of systemic medical conditions. In these cases, bone tissue engineering 2007). GF supplementation is associated with certain disadvantages
using stem/stromal or progenitor cells on scaffolds made of a variety of when used in tissue engineering, i. e. regulatory issues or interference
materials is a promising therapeutic option for repair or replacement. with other cell types in vivo. An alternative approach is based on
The usage of adipose stem cells (ASCs) for bone tissue engineering is well manipulation of gene activity in ASCs, to upregulate the genes and their
established, but its application bears obstacles such as sufficient cell products that are crucially involved in ASC osteogenesis (Lee et al.,
numbers and differentiation towards the osteogenic lineage in a short 2011; Lin et al., 2008). Genetic transfection is a laborious process and
time period (Mende et al., 2021). In order to overcome these obstacles when mediated by viral transduction, it also puts the recipient at risk of
and to generate artificial bone tissue, it is essential to precondition the permanent genetic changes with currently unforeseeable consequences.
stem cells into the osteogenic lineage in the most effective way (Seo Bone tissue is constantly reorganized, therein mechanical, physical and
et al., 2019). Currently, there are different approaches to induce ASC chemical stimuli are important for the bone remodelling processes
osteogenesis. The stimulation with pharmacologic or biochemical (Chen et al., 2010; Morseth et al., 2011).
compounds is widely used, e. g. via supplementing the medium with Different physical stimuli were already examined for their effect on
ascorbic acid, β-glycerolphosphate, dexamethasone and 1,25-dihydrox­ osteogenesis of adult stem or progenitor cells. Tensile strain increases
yvitamin D3 (Jing et al., 2010; Zuk et al., 2001). Further approaches osteogenic differentiation in human ASCs, as it stimulates the Wnt
stimulate osteogenesis by adding cytokines and/or growth factors (GFs) pathway (Shafaei and Kalarestaghi, 2020). This stimulation increases

* Corresponding author.
E-mail addresses: [email protected] (L.F. Lingens), [email protected] (T. Ruhl), [email protected] (J.P. Beier), [email protected] (W. Mende),
[email protected] (G. Freund), [email protected] (R. Götzl).

https://doi.org/10.1016/j.tice.2022.101886
Received 5 January 2022; Received in revised form 1 August 2022; Accepted 2 August 2022
Available online 3 August 2022
0040-8166/© 2022 Elsevier Ltd. All rights reserved.

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Fig. A1. Experimental design: Experiments were carried out with after the schema shown above, differing in the time and form of stimulation. OM = osteogenic
medium (low glucose medium, 10 % FBS, 1 % P/S, containing 2 % L-ascorbic acid, 1 % beta-glycerophosphate and 0.1 % dexamethasone), d=day.

Fig. B1. Representative photos of 1 measuring the metabolic activity with PrestoBlue, 2 assessing cell number by crystal violet stain, 3 the cresolphtaleine assay
targeting calcification, and 4 ALP activity. Examples of samples with high activity/ stain are presented in the top row and examples of samples with low activity/stain
are given in the lower row. Blanks are colorless and not shown.

the cellular calcium content and it elevates the expression of osteogenic upregulates the hypoxia induced factor (Hif-1α), causes cell prolifera­
markers, e. g. ALP, collagen type I (Col1a1), osteopotin (OPN) and osteo­ tion (Kakudo et al., 2015) and cell migration (Xu et al., 2020). In vivo
calcin (OCN). Vibration loading produces similar effects. This type of Hif-1α is an important factor for bone formation (Shomento et al., 2010).
stimulation increases the Ca2+-level of the extracellular matrix and in­ Hypoxic conditions affect ASCs’ osteogenic differentiation potential, if
creases osteogenic gene expression and protein level of osteopotin and the cells are continuously stimulated with oxygen levels of 1–2 % in
collagen type I (Prè et al., 2011). vitro. This down-regulates the gene-activity of osteogenic markers (Choi
Based on the observation that in space, hypogravity reduces bone et al., 2014; Fotia et al., 2015). In order to find an optimal oxygen range
density of astronauts (Stavnichuk et al., 2020), the inhibiting effect of for osteogenic differentiation, atmospheric level (21 %), mild hypoxia
microgravity on progenitor cell osteogenesis has been substantiated (8 %) and strong hypoxia (3 %) were applied for their effects on ASC
(Zayzafoon et al., 2004). While increasing gravitational (g-)force en­ osteogenesis.
hances differentiation of murine bone marrow (bm) mesenchymal stem The aim of our study is to establish a cost efficient and safe approach
cells (MSCs) (Huang et al., 2009), human MSCs (Zayzafoon et al., 2004) for in vitro enhancement of osteogenesis of ASC for bone tissue
and osteoblast like cells (Kacena et al., 2004), little is known about the regeneration.
impact of hypergravity on human ASC osteogenic differentiation in vitro.
Therefore, ASCs were stimulated in 2D cultures with increasing g-forces 2. Material and methods
during regular osteogenic differentiation.
Hyperbaric oxygen therapy is applied to increase oxygen levels, 2.1. Materials
especially when a local body region suffers from oxygen deficiency.
Thus, it is beneficial as an adjunct treatment for osteomyelitis (Chen PrestoBlue, fetal bovine serum (FBS), low glucose medium (1 g/L)
et al., 2004). It increases the expression of the osteogenic marker RUNX2 and Dulbecco’s Modified Eagle’s medium (DMEM/F-12) were from Life
in human MSCs in vitro. This shift is mediated through Wnt signalling Technologies, Darmstadt, Germany. Collagenase was purchased from
(Lin et al., 2014). Comparable investigations on ASCs have not been Worthington Biochemical Corp., Lakewood, USA. Ascorbate
performed. Thus, in a second experimental series cells were repeatedly 2–phosphate, β-glycerophosphate, paraformaldehyde (PFA), tryp­
exposed to 3 bar during osteogenic differentiation. sin–EDTA, o-cresolphthalein complexon, 8–hydroxyquinolinol, pen­
ASCs reside in a hypoxic niche, as the oxygen levels in fat tissue icillin–streptomycin (P/S), 2–amino-2–methyl-1–propanol (AMP),
range beneath the standard culture conditions of 21 %. Oxygen levels of 2–amino-2–methyl-1.3–propanediol (AMPED), Bovine serum albumin
healthy human bone range between 5.4 % and 7 % (Mas-Bargues et al., (BSA) and Tween®20 were obtained from Sigma-Aldrich, St. Louis, USA.
2019) and in adipose at 5.7–7 % (Lempesis et al., 2020). In vitro hypoxia HCl, sodium-pyruvate, acetic acid, crystal violet, para-

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Fig. C1. Influence of hypergravity on metabolic activity of ASCs – 1 immedi­ Fig. D1. Impact of stimulation with hypergravity of 10x g, 20x g, 30x g and 40x
ately after exposure and – 2 24 h after exposure. Cells were stimulated with 10x g on osteogenic differentiation of ASC after 14 days of intermitting stimulation
g, 50x g, 100x g, 500x g, 1000x g, 2000x g, 3000x g, 4000x g and the control for 3 min every 48 h. Osteogenesis was analyzed by 1 cresolphthalein staining
with 1x g for 3 min in a centrifuge with a swing out rotor. Metabolic activity for quantification of the extracellular matrix calcification and 2 quantification
was measured by PrestoBlue, and quantified with optical density. For statistical of enzymatic conversion of ALP substrate para-nitrophenylphosphate. No
analyses were performed the Kolmogorov-Smirnov test for normality and the treatment (1x g) served as control. Statistical analyses were performed with the
one-way ANOVA with Bonferroni’s multiple comparison test for significances; Mann–Whitney U-test; *p < 0.05, * *p < 0.01 and * **p < 0.001 versus 1x g.
* *p < 0.01 and * **p < 0.001 versus 1x g, OD = optical density.
expanded at standard culture conditions and splitted after reaching 90 %
nitrophenylphosphate (pNpp), NaOH, MgCl2, were from Roth. Acetic confluence. Experiments were performed with cells from P1 to P5.
acid and isopropyl alcohol were obtained from Merck, Darmstadt, Ger­ Prior to all experiments the plates were coated with 0.01 % poly-L-
many. Dulbecco’s phosphate buffered saline (PBS) was bought from lysin to improve attachment of the cells. 3 × 104 cells per cm2 were
Biochrom, Berlin, Germany. seeded on 24-well plates or 48-well plates (VWR®, Leuven, Belgium).
Osteogenic differentiation was induced by exchanging proliferation
against differentiation medium (low glucose medium, 10 % FBS, 1 % P/
2.2. Cell culture
S) containing 2 % L-ascorbic acid, 1 % beta-glycerophosphate and 0.1 %
dexamethasone as described earlier (Yoshinoya et al., 2020). During
ASCs were isolated from human abdominal fat tissue from six
differentiation, medium was exchanged prior to each stimulation every
healthy human donors, with a mean age of 50 ± 9.59 (age 35, 45, 47, 51,
second day (Fig. A1).
56 and 66) years and a mean body mass index of 31.77 ± 7.08 kg/m2.
The fat tissue was harvested during abdominoplasty after informed
2.3. PrestoBlue assay
consent of all patients. The study was approved by the regional ethics
committee (EK163/07), and all experiments were conducted in
The PrestoBlue assay was performed following the manufacturer’s
compliance with the principles of the Declaration of Helsinki.
instructions (Life Technologies, Darmstadt, Germany). Briefly, 10 %
The fat tissue was minced and incubated for 45 min at 37 ◦ C in 0.2 %
PrestoBlue diluted in osteogenic differentiation medium was added to
collagenase solution (Worthington Biochemical Corp., Lakewood, NJ) at
the cells and incubated for 1 h. Afterwards 70 μl of the supernatant were
a volume ratio of 1:1. Afterwards, the solution was filtered through a
transferred to a plate reader (BMG Labtech, Ortenberg, Germany) in
250 µm mesh and centrifuged at 300x g for 10 min. The supernatant was
triplets and the fluorescence was measured optometrically at 590 nm
discarded, and the remaining cell pellet resuspended with proliferation
(excitation at 540 nm) (Fig. B1).
medium (DMEM, 10 % FBS, 1 % P/S). The cells have been identified as
ASCs by differentiation towards the chondrogenic, osteogenic and adi­
pogenic lineage, as described earlier (Kim et al., 2017). Cells were

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Fig. E1. Influence of hyperbaric pressure on cell number and osteogenic dif­
Fig. F1. Influence of hypoxia on cell number and osteogenic differentiation of
ferentiation of ASCs. Cells were stimulated with 3 bar hyperbaric pressure for
ASCs. Cells were stimulated with mild hypoxia (8 %) and severe hypoxia (3 %)
90 min every 48 h. After 12 days of treatment, cell number by means of crystal
for 12 h every 48 h. After 12 days of treatment, cell number by means of crystal
violet staining 1 and osteogenic differentiation by means of cresolphtalein
violet staining 1, osteogenic differentiation by means of cresolphtalein staining
staining 2 and ALP activity 3 was assessed. Data of cresolphtalein and ALP
2 and ALP activity 3 was assessed. Data of cresolphtalein and ALP activity were
activity were normalized to the number of cells (OD crystal violet), and are
normalized to the number of cells (OD crystal violet), and are expressed as box
expressed as box blots of the mean. Statistical analyses were performed by
blots of the mean. Statistical analyses were performed by Mann–Whitney U-test;
Mann–Whitney U-test; *p < 0.05 and * *p < 0.01 versus control (0 bar).
*p < 0.05 and * **p < 0.001 versus 20 % oxygenation.

2.4. Crystal violet assay

incubated for 20 min before washing with aquabidest. Then the crystal
violet was eluted by adding 33 % acetic acid during constant shaking for
To assess cell numbers, the cells were washed with PBS, fixed with
12 min (Fig. B1). 70 μl per sample were transferred in triplets to an
isopropanol for 10 min, and washed again with PBS/Tween 0.05 % so­
optical plate and absorbance was quantified at 595 nm (Gillies et al.,
lution. Subsequently, 0.1 % crystal violet solution was added and
1986).

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2.5. Cresolphtalein assay Scientific, Waltham, USA) with an integrated incubator (EVOS™
Onstage Incubator, ThermoFisher Scientific, Waltham, USA) at either 3
To determine calcification, ASCs were washed with PBS and then % or 8 % oxygen for 12 h every 2 s day after the medium exchange. The
fixed with 4 % PFA for 20 min, which was followed by washing with control group was placed in the same incubator at 20 % oxygen. N2 was
aquabidest. Subsequently, the calcium ions were leached out by the acidic set at 75 %, 87 % or 92 %, respectively. The CO2 level was at 5 %. The
cresolphtalein solution (0.1 % ortho-cresolphthalein-complexon, 1 % temperature was kept at 37 ◦ C. After 12 h, cells were set back into the
8–hydroxychinolin in 30 ml of 37 % HCl, diluted in 500 ml aquabidest) for breeder and cultivated under standard culture conditions. Hypoxic pe­
a 5 min incubation period, following the complex formation by adding riods were repeated 5 times every 48 h for 12 d.
alkaline AMP-buffer (76 ml AMP in 500 ml aquabidest, pH=10.7 with
HCl) (Fig. B1). Finally, the results were quantified by measuring the 2.10. Analyses and statistics
absorbance in triplets of 100 μl at 580 nm (Gitelman, 1967).
Cresolphatein and ALP values were normalized to cell numbers, i.e.
2.6. Alkaline Phosphatase activity optical density (OD) of crystal violet.
Cells stimulated with hypergravity were obtained from 3 donors with
Cells were washed with PBS and treated with lysis buffer solution (1 81 cell samples. The experiments of hyperbaric pressure and hypoxia
% Tween20 in aquabidest, pH=9 with NaOH). Afterwards, 10 mM Phe­ were conducted with cells from 2 donors respectively and 33 and 55 cell
nylmethylsulfonylfluorid (PMSF) was added and the cells were incu­ samples.
bated at RT for 10 min. ALP substrate solution (10 mM pNpp, 100 mM Each experiment was repeated once, and each experiment was per­
AMPED, and 5 mM MgCl2, pH = 9.5) was added and incubated at 37 ◦ C formed in minimum triplicate. The statistical analysis is based on the
for 1 h. The reaction was stopped by adding 2 M NaOH (Fig. B1). Af­ average of the triplicates. Data were averaged and tested for normality
terwards 200 μl supernatant was transferred to a 96 well reader plate in using the Kolmogorov-Smirnov test (SPSS 25, SPSS Inc.). Normally
triplets and absorbance was measured at 405 nm (Bessey et al., 1946). distributed data were expressed by means ( ± SEM), non-normally
ALP values were normalized to cell numbers, i.e. optical density (OD) of distributed data were presented as box plots. The horizontal black line
crystal violet. within each box denotes the median value. The boxes extend from the
lower and upper quartiles (defined as the 25th and 75th percentiles) of
2.7. Hypergravity the data distribution, vertical lines denote adjacent extreme values.
Statistical differences between more than two groups were analyzed
To test whether hypergravity affects cellular viability, whole plates using one-way analysis of variance (ANOVA). Post hoc tests were per­
containing adherent cells in proliferation medium were placed into a formed using the Bonferroni’s multiple comparison test. Differences
centrifuge (Thermo Scientific Heraeus Multifuge 3SR+, Waltham, USA) between an experimental and the control group were examined by the
containing a plate holding swing out rotor (radius 19 cm). the cells were Mann-Whitney-U-test. Statistical significance was defined as p < 0.05.
centrifuged for 3 min at 10x g, 50x g, 100x g, 500x g, 1000x g, 1500x g,
2000x g, 3000x g, 4000x g. The control was cultivated at normogravity 3. Results
(1x g). PrestoBlue assay was performed immediately after the treatment
and 24 h afterwards. Immediately after stimulation, the metabolic ac­ 3.1. Hypergravity enhanced osteogenic differentiation in ASCs
tivity decreased, which indicated the negative impact of the treatment
on cellular viability. 10x g decreased PrestoBlue conversion of 19 %, and ASCs were exposed for 12 days to osteogenic differentiation medium
50x g induced a 33 % reduction. 50xg was significantly different (p = combined with repetitive hypergravity stimulation at either 1x g, 10x g,
0.016) from the control. With an acceleration of 100x g the decrease (45 20x g, 30x g and 40x g. The Ca2+ content evaluated by Cresolphthalein
%) reached a plateau (Fig. C1). The stimulus impact disappeared 24 h staining gradually increased with higher g-forces. 10x g increased it by
afterwards, and all groups were on similar levels (Fig. C1). Based on 115 % (p < 0.0001, U81 =1706.5) and 20x g by 122 % (p < 0.0001, U81
these results, we decided to apply 10–40x g during osteogenic differ­ =1165.5) compared to the control. While 30x g led to a similar value
entiation as these stimuli had the mildest initial effect on cellular (123 %; p = 0.04, U81 =2214). Whereas at 40x g the pro-osteogen effect
vitality. was reduced (107 % increase), but it was still significantly over the
Experimental hypergravity treatment followed the same regime as control (p = 0.04, U81 =2214) (Fig. D1).
described above, and it began two days after initiating ASC osteogenic In the ALP staining, the results were not as distinct (Fig. D1). How­
differentiation. Whole plates were centrifuged every second day at ever, a significant increase of ALP activity was found during intermitting
different g-forces (10x g, 20x g, 30x g and 40x g / respectively 217 rpm, hypergravity treatment with 30x g (1.2-fold, p < 0.0001, U75 =1956.5).
307 rpm, 376 rpm or 434 rpm) for 3 min at RT. The control group was
cultivated at normogravity (0 rpm). Each sample was stimulated a total 3.2. Hyperbaric exposure increased matrix mineralisation during ASC
of 5 times, every 48 h with the corresponding gravitational force. The osteogenesis
complete experiment was performed for 12 d.
Hyperbaric conditions at 3 bar significantly reduced the cell numbers
2.8. Hyperbaric pressure to 47 % (p = 0.007, U33 = 216) compared to the control (Fig. E1).
Furthermore, this treatment increased ASC osteogenic differentiation.
Hyperbaric pressure was applied in a custom-made small scale hy­ The cresolphthalein staining per cell increased significantly by 314 % (p
perbaric chamber supplied by the HBO-Center Euregio Aachen GmbH & = 0.001, U35 =184) (Fig. E1). Also, the ALP activity significantly
Co. KG (Aachen, Germany) as described previously (Yoshinoya et al., increased upon hyperbaric stimulation (p = 0.001, U33 =183) by 214 %
2020). Cells were either treated with 3 bar or at atmospheric pressure (Fig. E1).
(representing the control group) for 90 min. The hyperbaric treatment
was repeated 5-times on every second day for a period of 12 d. 3.3. Hypoxia affects proliferation and osteogenic differentiation of ASCs

2.9. Hypoxia The ASC cell number was not affected under mild hypoxic (8 %)
conditions (p = 0.15, U51 =951.5). Meanwhile, it was reduced under
To simulate hypoxia, the cells were placed in a live cell imaging strong (3 %) hypoxic conditions (p = 0.043, U51 =871.5) (Fig. F1). Mild
microscope (EVOS™ FL Auto 2 Imaging System, ThermoFisher and strong hypoxia reduced the matrix mineralisation significantly.

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Cells under mild conditions showed over 57 % reduction (p < 0.0001, vivo (Chen et al., 2010). In vitro contradicting results have been reported,
U46 =154) and strong hypoxic conditions reduced it by 85 % (p < showing enhanced osteogenesis of osteoblasts (Al Hadi et al., 2015) and
0.0001, U46 =127) (Fig. F1). Measurement of ALP activity showed that it no effect of hyperbaric oxygen therapy on osteogenic differentiation of
was increased significantly under both conditions. Mild hypoxia con­ hASCs (Yoshinoya et al., 2020). Regarding hyperbaric pressure alone it
ditions led to a significant increase of 413 % (p = 0.024, U46 =586) and has been shown, that continuous hyperbaric pressure can lead to
strong hypoxia significantly increased it by 320 % (p = 0.02, U36 =442) osteonecrosis in vivo (Lehner et al., 1994), while in vitro hyperbaric
(Fig. F1). pressure applied daily in previous studies showed barely any effect on
the osteogenic differentiation of osteoblast (Al Hadi et al., 2015) and
4. Discussion hASCs (Gardin et al., 2020). Our results however indicate that inter­
mitting hyperbaric pressure under normoxic conditions lead to an
Reconstructing large bone defects with tissue engineered bone based improved osteogenic differentiation of hASCs. It was observed that the
on adult stem cells has already shown promising results. However, using level of calcium deposition and the ALP activity was significantly higher
adult stem cells raises obstacles such as harvesting, expansion and dif­ when hASCs were stimulated with 3 bar of atmospheric pressure every
ferentiation. In the present study, we investigated several experimental 48 h for 12d. Furthermore, we found that cell proliferation was slower at
approaches to improve osteogenic differentiation of ASCs in vitro. Based stimulation with intermitting hyperbaric pressure stimulation after 12
on the physiology of bone in vivo to remodel in response to the local days. The findings of the present study lead to the assumption that the
stimuli it is exposed to, one of the most efficient ways to improve mechanical load caused by hyperbaric pressure can be used for
osteogenesis of adult stem cells is to stimulate with biomechanical forces improving bone tissue engineering when a suitable experimental design
(Huang et al., 2009; Lin et al., 2014; Prè et al., 2011). We investigated is used, hence the hASCs are given time do regenerate in between
hypergravity, hyperbaric pressure and hypoxia additional to the sup­ stimulations. Further studies will be needed to investigate the details of
plementing osteogenic medium to improve in vitro osteogenesis of this phenomena. To our best knowledge, this study is the first one to
hASCs. show the supporting effect of intermitting hyperbaric pressure on
We found that hypergravity of 10x g, 20x g, 30x g and 40x g osteogenesis of hASCs.
enhanced calcification of the extracellular matrix and 40x g increased
the ALP activity. In addition, our study showed that hyperbaric pressure 4.3. Hypoxia inhibited osteogenesis in ASCs
of 3 bar, applied intermittingly for 90 min every 48 h, reduced the cell
number after 12 d while it increased markers of osteogenesis in ASCs. Healthy bone cells reside in a physiologically hypoxic niche in vivo.
Intermitting severe hypoxia of 3 % reduced the cell number while mild Therefore, we investigated the impact of hypoxic conditions similar to
hypoxia reduced the calcification of osteogenic stimulated ASCs, the physiological oxygen levels of bone tissue (5.4–7 %) (Mas-Bargues
whereas ALP activity is increased by both severe and mild hypoxia. et al., 2019) during osteogenic differentiation of hASCs. To our best
knowledge the effect intermitting stimulation of hypoxia levels similar
4.1. Hypergravity increased osteogenic differentiation in ASCs to the oxygen levels in the physiological niche of ASCs on the osteogenic
differentiation has not been investigated. Our results show a signifi­
The impact of gravitational forces on the osteogenic differentiation cantly lower calcium deposition after 12 days indicating a low osteo­
of adult mesenchymal stem cells was often examined with regards to genic differentiation both under mild (8 %) and sever (3 %) hypoxic
bone loss during space flight. Hence, in vitro microgravity was applied to conditions. Contradicting to the calcium deposition ALP activity was
mesenchymal stem cells and caused an inhibition of osteogenic differ­ significantly higher in both groups. The contradicting results between
entiation (Stavnichuk et al., 2020; Zayzafoon et al., 2004). The opposing high ALP activity and low matrix mineralization in our study under mild
force of microgravity is hypergravity, which showed opposing results and severe hypoxic conditions could possibly be explained by a
regarding the osteogenic differentiation of rodent bmMSCs and goat time-shift of osteogenic induction caused by hypoxia, since matrix
ASCs (Huang et al., 2009; Versari et al., 2007). These promising previous mineralization is known to be a marker for late osteogenic differentia­
results indicated that hypergravity is useful for improvement of osteo­ tion of mesenchymal stem cells, whereas ALP is upregulated earlier in
genic differentiation and therefore could be viable as an additional the processes of osteogenic differentiation (Hoemann et al., 2009).
stimulus for osteogenic differentiation in the field of bone tissue engi­ Altogether osteogenesis of ASCs is not impaired by hypoxia indifferent if
neering. It was shown before that stems cells harvested from animals can in the experimental design hypoxia is chosen to be close to the hypoxic
have different responses to stimuli than human stem cells (Scuteri et al., niche or severe hypoxia (1–3 %) is applied (Choi et al., 2014; Fotia et al.,
2014). Furthermore, bmMSCs cannot be set equal in their response to 2015; Merceron et al., 2010). Thus, it is possible that the favourable
stimuli and need to be considered a separate cell type (Bourin et al., environment for osteogenesis in a hypoxic niche seems of little impor­
2013). We therefore aimed to investigate the influence of hyper­ tance in vitro. A well oxygenated environment is needed for bone tissue
gavitation on the osteogenic differentiation of human ASCs and to find engineering and fracture heeling in vivo (Kimelman-Bleich et al., 2009).
the optimal gravitational force to stimulate human ASCs towards an Consequently, the need of oxygen for the osteogenic differentiation
osteogenic lineage. In addition, we chose human cells for a better seems to overweigh the hypoxic niche as a favourable condition in vitro
application in regenerative medicine by eradicating a possible immune as well.
response to xenogenic cells. In our study mineralization was signifi­ We also found a significantly lower relative cell number under severe
cantly stronger at 10x g, 20x g, 30x g, and 40x g indicating a hypoxic conditions of 3 % O2 while they were stable at intermitting mild
pro-osteogenic effect on ASC differentiation with best results at 30x g. hypoxia of 8 % O2. Previous studies using permanent hypoxia of 1 % on
Regarding the impact of hypergravity on the metabolic activity of hASCs osteonectin transfected rabbit ASCs, hASCs and 2 % on hASCs in contrast
we saw immediately after the treatment a decrease of the metabolic show a higher cell viability and proliferation (Choi et al., 2014; Fotia
activity at ≥ 50x g, which was reversable after 24 h.These findings of the et al., 2015; Xu et al., 2014). It is possible that the experimental design, i.
present study correspond well with the previous results (Tavakolinejad e., continuously vs. intermitting hypoxia, and various hypoxia-levels
et al., 2015). cause the different results. We assume that the time of stimulation
(12 h) in our study could be too short for an adequate production of
4.2. Hyperbaric stimulation increased osteogenic differentiation in ASCs Hif-1α. Hif-1a is a master regulator of hypoxia mediated cell effects,
which could be involved in this case as well and therefore may cause a
Hyperbaric pressure in combination with high oxygenation (100 % slower cell proliferation. Additionally, the hypoxia levels in our study
O2) has shown promising results for bone regeneration and formation in were chosen close to the physiological hypoxic niche (Lempesis et al.,

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L.F. Lingens et al. Tissue and Cell 78 (2022) 101886

2020; Mas-Bargues et al., 2019), which also might not be sufficient to References
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