Archives of Oral Biology 143 (2022) 105527

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Archives of Oral Biology

journal homepage: www.elsevier.com/locate/archoralbio

Cyclic tensile strain-induced yes-associated protein activity modulates the

response of human periodontal ligament mesenchymal stromal cells to
tumor necrosis factor-α
Zhongqi Zhao a, Christian Behm a, b, Zhiwei Tian a, Marco Aoqi Rausch a, b, Xiaohui Rausch-Fan c,
Oleh Andrukhov a, *
a
Competence Center for Periodontal Research, University Clinic of Dentistry, Medical University of Vienna, 1090 Vienna, Austria
b
Division of Orthodontics, University Clinic of Dentistry, Medical University of Vienna, 1090 Vienna, Austria
c
Division of Conservative Dentistry and Periodontology, University Clinic of Dentistry, Medical University of Vienna, 1090 Vienna, Austria

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

Keywords: Objectives: This study aimed to evaluate the role of yes-associated protein (YAP) in the inflammatory processes
Yes-associated protein induced in human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) by cyclic tensile strain
Tumor necrosis factor-α (CTS).
Cyclic tensile strain
Design: hPDL-MSCs from five periodontally healthy individuals were stimulated with 12% CTS and/or TNF-α for
Inflammatory cytokines
24 h. YAP activity was determined by analyzing the YAP nuclear localization and the target genes expression,
using immunofluorescence and qPCR, respectively. Verteporfin was used to inhibit the activation of YAP. The
gene expression of interleukin (IL)-6, IL-8, vascular cell adhesion molecule (VCAM)-1, and intercellular adhesion
molecule (ICAM)-1 was analyzed by qPCR.
Results: In the absence of TNF-α, application of CTS resulted in the nuclear YAP translocation and upregulation of
YAP target genes. Verteporfin inhibited the activation of YAP pathway and upregulated the basal expression of
IL-6 and IL-8. TNF-α induced the activation of YAP pathway, which was inhibited by verteporfin. However,
application of CTS under these conditions diminished TNF-α-induced YAP activation. TNF-α-induced expression
of IL-6, VCAM-1, and ICAM-1 was inhibited after the application of CTS. Inhibition of YAP activation by ver­
teporfin diminished TNF-α-induced gene expression of IL-6, VCAM-1, and ICAM-1, and under these conditions no
inhibitory effect of CTS on these parameters was observed.
Conclusions: YAP is at least partially involved in the CTS-activated mechanotransduction pathway. The effects of
CTS and YAP on the inflammatory responses depend on the inflammatory environment. A better understanding
of the inflammatory modulation by mechanical stress may help improve the orthodontic strategies, especially in
the patient with periodontitis.

1. Introduction mechanical forces initiate an aseptic inflammatory response in peri­

odontal tissues, culminating in bone remodeling and orthodontic tooth
The increasing number of periodontally compromised adults seeking movement (OTM) (Li et al., 2018). Tooth loading causes local hypoxia
orthodontic treatment poses orthodontists with challenges in avoiding and direct strain on matrix and cells, transducing mechanical loading
complications of the periodontium (Liu et al., 2017). The interaction into the biological cues by a cascade of molecular and cellular responses
between periodontology and orthodontics is highly complex and re­ (Krishnan & Davidovitch, 2009; Li et al., 2018). This process is highly
mains elusive at the biological level. During orthodontic treatment, dependent on the cellular components of the periodontal ligament

Abbreviations: YAP, yes-associated protein; CTS, cyclic tensile strain; TNF, tumor necrosis factor; hPDL-MSCs, human periodontal ligament-derived mesenchymal
stromal cells; IL, interleukin; VCAM, vascular cell adhesion molecule; ICAM, intercellular adhesion molecule; CTGF, connective tissue growth factor; CYR61, cysteine-
rich angiogenic inducer 61; OTM, orthodontic tooth movement; PDL, periodontal ligament; MSC, mesenchymal stromal cells; MFI, mean fluorescence intensity; TAZ,
transcriptional co-activator with PDZ-binding motif.
* Correspondence to: University Clinic of Dentistry, Division of Conservative Dentistry and Periodontology, Sensengasse 2 A, 1090 Vienna, Austria.
E-mail address: [email protected] (O. Andrukhov).

https://doi.org/10.1016/j.archoralbio.2022.105527
Received 6 April 2022; Received in revised form 17 August 2022; Accepted 18 August 2022
Available online 20 August 2022
0003-9969/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Z. Zhao et al. Archives of Oral Biology 143 (2022) 105527

(PDL). Human periodontal ligament-derived mesenchymal stromal cells culture flasks after outgrowth and passaged after reaching confluence.
(hPDL-MSCs), which are a heterogeneous cell population having char­ The phenotype of isolated hPDL-MSCs was confirmed by immuno­
acteristics comparable to bone marrow-derived MSCs, were reported to staining as described in our former study (Behm et al., 2021). Cells were
be sensitive to mechanical loading and play a central role in periodontal positively stained with mesenchymal cell markers (CD29, CD73, CD90,
tissue remodeling during OTM (Feller et al. 2015; Huang et al., 2018). CD105, and CD146) and negatively stained with hematopoietic stem cell
The transcriptional co-activator yes-associated protein (YAP), which markers (CD31, CD34, and CD45), in agreement with the official criteria
is a major effector in the Hippo signaling pathway (Meng et al., 2016; for MSCs from the International Society for Cell and Gene Therapy
Piccolo et al., 2014), recently attracted attention as a key mediator to (ISCT) (Viswanathan et al., 2019). Cells within passages 4-7 were used in
sense mechanical forces and transduce cytoskeletal signals into tran­ all subsequent experiments.
scriptional activation in the nucleus (Panciera et al., 2017). High levels
of mechanical strain induce translocation of YAP in the nucleus, where it 2.2. Cell strain application and TNF-α treatment
induces the transcription of target genes. At low tension levels, YAP
tends to accumulate in the cytoplasm and could be degraded by the hPDL-MSCs (5×104/well) were seeded into 6-well flexible-bottomed
proteasome (Halder et al., 2012). These processes are a determinant BioFlex® culture plates coated with type I collagen (Flexcell® Interna­
factor in cell behaviors and cell fate, for example, regulating cell pro­ tional Corporation, Burlington, NC, USA). The culture plates were
liferation and differentiation (Halder et al., 2012; Panciera et al., 2017). loaded on BioFlex® Cell Seeders (Flexcell® International Corporation,
YAP signaling pathway is an evolutionarily conserved mechanism and is Burlington, NC, USA) until attachment to confine cells to the central
shared by multiple cell types (Panciera et al., 2017; Totaro et al., 2018). region of the bottom, which subjected cells to well-defined equibiaxial
Recently, YAP-related mechanotransduction was also identified in the strains. After one-day incubation and reaching subconfluence, the me­
cells of periodontal tissues (Huelter-Hassler et al., 2017; Sun et al. 2018; dium was replaced by the medium described above but without FBS
Yang et al., 2018). Due to its regulation by the mechanical strain, YAP is (starvation medium). After overnight-starvation, cells were subjected to
considered to play an essential role in the biological processes in or­ CTS using a Flexcell® FX-5000™ Tension System (Flexcell® Interna­
thodontic treatment. tional Corporation, Burlington, NC, USA) with 12% elongation at 0.1 Hz
In vitro studies revealed the influence of mechanical force on in­ for 24 h. The loading unit was placed in a humidified atmosphere of 5%
flammatory responses, and this was considered as a potential interaction CO2 at 37◦ C, and the flexible bottom of the plates was deformed along
between orthodontic tooth movement and periodontal inflammation the surface of the loading post by a vacuum, generating the dynamic
(Agarwal et al. 2003; Long et al., 2002; Rath-Deschner et al., 2009). equibiaxial tensile strain to attached cells. Unstretched cells seeded on
Several cytokines were assumed to be involved in the OTM-induced BioFlex® plates under the same conditions served as control. Simulta­
aseptic inflammation, one of them being tumor necrosis factor (TNF)-α neously, hPDL-MSCs were stimulated with 10 ng/ml human recombi­
(Behm et al., 2022; Chaushu et al., 2022). Our recent study showed that nant TNF-α (Invivogen, San Diego, CA, USA) for 24 h to simulate the
its downstream effectors were affected by in vitro mechanical loading on inflammatory microenvironment in vitro. Unstimulated hPDL-MSCs with
hPDL-MSCs (Zhao et al. 2021). TNF-α plays also a crucial role in the or without applying CTS served as references.
periodontal inflammation (Graves & Cochran, 2003). Moreover, recent
evidence has demonstrated the sophisticated reciprocal actions between 2.3. Inhibition treatment
the Hippo-YAP pathway and TNF-α/NF-κB signaling (Wang et al., 2020).
Therefore, the assumption that Hippo-YAP pathway might play the role YAP activity in hPDL-MSCs was inhibited by the treatment with 1 µM
in the interaction between the mechanical stress and TNF-α-induced verteporfin (Vp, Sigma-Aldrich, St. Louis, MO, USA, dissolved in
inflammation, especially in periodontal cells, is feasible but remains dimethyl sulfoxide (DMSO)) overnight before strain application and
poorly explored to date. stimulation with TNF-α. Our preliminary experiments showed that used
Therefore, the present study investigated the interaction between DMSO concentration (0.35 µl/ml) had no effect on the viability of hPDL-
YAP-related mechanotransduction and TNF-α signaling pathways in MSCs (Supplementary Fig. 1). All the non-inhibition groups were incu­
hPDL-MSCs. Given that YAP plays a significant role in modulating bated with an equal concentration of DMSO.
mechanotransduction and inflammation, we hypothesized that YAP re­
sponds to the application of cyclic tensile strain (CTS) on hPDL-MSCs 2.4. Cell proliferation/viability
and participates in the modulation of TNF-α-induced expression of in­
flammatory mediators by CTS. Cell proliferation/viability of hPDL-MSCs was evaluated in 6-well
plates with the 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium
2. Materials and methods bromide (MTT) colorimetric assay (Sigma Aldrich, St. Louis, MO,
USA). 300 µl of MTT solution (5 mg/ml in PBS) was added per well. After
2.1. Cell culture incubation at 37◦ C for 4 h, the medium was discarded, and 3 ml DMSO
was added per well. Complete dissolution was aided by 10 min incu­
Primary hPDL-MSCs were derived from the periodontal ligament of bation on a shaker. Finally, 100 µl of each cultured solution was trans­
human third molars with healthy periodontium. Tooth extraction from ferred in triplicate to a 96-well plate. The absorbance was measured at a
five individuals was conducted for orthodontic reasons. All participants wavelength of 570 nm using a spectrophotometer (Synergy HTX, Biotek,
gave their informed consent. The experimental procedures were con­ Winooski, VT, USA).
ducted according to the Declaration of Helsinki and the Good Scientific
Practice Guidelines of the Medical University of Vienna. The Ethics 2.5. Reverse transcription-quantitative polymerase chain reaction (RT-
Committee of the Medical University of Vienna approved the study qPCR)
protocol (ethical approval number: 1079/2019, extended in 2021). In
detail, periodontal ligament slices were scraped off from the middle Cell lysates were prepared and reversely transcribed into cDNA using
third of the tooth roots. The tissue fragments were minced and placed in TaqMan Gene expression Cells-to-CT kit (Applied Biosystems, Foster
Petri dishes with Dulbecco’s modified Eagles Medium (DMEM, Sigma- City, CA, USA) following the manufacturer’s instruction. Primus 96
Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine advanced thermocycler (PeqLab/VWR, Darmstadt, Hessen, Germany)
serum (FBS, Gibco, Carlsbad, CA, USA), 100 U/mL penicillin and 100 was used to perform reverse transcription, which was done by heating at
µg/mL streptomycin (P/S, Gibco, Carlsbad, CA, USA) in 95% humidified 37 ◦ C for 1 h and enzyme deactivation at 95 ◦ C for 5 min. qPCR was
atmosphere with 5% CO2 at 37 ◦ C. The cells were transferred into cell performed using QuantStudio 3 device (Applied Biosystems, Foster City,

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USA) and the following TaqMan Gene Expression Assays (Applied Bio­ Smirnov test. In the absence of normal distribution, data were loga­
systems, Foster City, CA, USA): connective tissue growth factor (CTGF), rithmically transformed for analysis. All data are presented as mean ±
Hs00170014; cysteine-rich angiogenic inducer 61 (CYR61), standard deviation. Partial comparisons used dot plots to visualize the
Hs00155479; interleukin-6 (IL6), Hs00985639_m1; interleukin-8 differences. Statistical analysis was performed using GraphPad Prism
(CXCL8), Hs00174103_m1; vascular cell adhesion molecule-1 software (version 8, La Jolla, CA, USA). P-values < 0.05 were considered
(VCAM1), Hs00365486_m1; intercellular adhesion molecule-1 statistically significant.
(ICAM1), Hs00164932_m1; and glyceraldehyde-3-phosphate dehydro­
genase (GAPDH), Hs99999905_m1. Samples were heated to 95◦ C for 10 3. Results
min followed by 50 cycles of 95◦ C for 15 s and 60◦ C for 1 minute. Each
sample was analyzed in duplicates. The n-fold expression of target genes 3.1. Cell proliferation/viability
compared to the appropriate controls was calculated using the 2− ΔΔCt
method (Blufstein et al., 2018). GAPDH was used as the endogenous Fig. 1 shows the proliferation/viability of hPDL-MSCs measured by
reference gene. MTT after treatment with CTS and TNF-α in the absence or presence of
verteporfin. No statistically significant difference was detected between
2.6. Indirect immunofluorescence and semi-quantitative image analysis CTS- and TNF-α-stimulated cells and the untreated control. Verteporfin
had no significant effect on the proliferation/viability of hPDL-MSCs
The nuclear localization of YAP was measured by indirect immu­ (Fig. 1).
nofluorescence analysis. hPDL-MSCs were fixed on the culture plate
membrane with 4% paraformaldehyde (PFA) for 15 min. After washing,
cells were permeabilized with 0.1% Triton X-100 for 5 min, and then 3.2. Effects of CTS, TNF-α and verteporfin on YAP activation in hPDL-
incubated in a blocking solution containing 1% bovine serum albumin MSCs
(BSA) in 1× PBS for 30 minutes at room temperature (RT). The primary
antibody (anti-YAP, ThermoFisher Scientific, Waltham, MA, USA) was Fig. 2 shows the effect of CTS and TNF-α with or without verteporfin
diluted in blocking solution (1:500) and applied on cells for 1 h at RT. on the gene expression of YAP-regulated genes CTGF and CYR61. In the
After washing three times, cells were incubated for 1 h at RT with sec­ absence of verteporfin, a significant increase was observed in the mRNA
ondary Alexa Fluor 488-conjugated antibody (1:250 in 1× PBS; goat levels of CTGF and CYR61 in hPDL-MSCs after applying CTS with 12%
anti-rabbit IgG, Thermo Fisher, Waltham, MA, USA). After three elongation or treating with TNF-α for 24 h (Figs. 2A and 2C). CTS-
washing steps, nucleus counterstain was performed with DAPI (1:1000 induced expression of CYR61 gene was significantly lower than TNF-
in 1× PBS; Sigma-Aldrich, St. Louis, MO, USA) for 5 min at RT. The α-induced expression of this gene (Fig. 2C), whereas no difference in
images were acquired with ECHO Revolve fluorescence microscope CTS- and TNF-α-induced expression of CTGF gene was observed
(Echo, San Diego, CA, USA) with 10× objective and an identical expo­ (Fig. 2A). The simultaneous stimulation with CTS and TNF-α did not
sure time and gain for each experiment. result in a further increase in the expression of CTGF and CYR61
Semi-quantitative analysis of fluorescence microscopy images was (Figs. 2A and 2B). However, the combined stimulation of CTS and TNF-α
performed using ImageJ software (ImageJ 1.53, National Institutes of caused a significant decrease in CTGF expression (Figs. 2A and 2B), and
Health, Bethesda, MD, USA) with an optimized procedure according to also non-significantly decreased the expression of CYR61 gene (Figs. 2C
previous studies (Eberwein et al. 2015; Hulter-Hassler et al., 2017; and 2D), compared to stimulation with TNF-α alone. Moreover, the
Jensen, 2013). The specific channels of acquired images corresponding expression level of CYR61 in cells treated with CTS and TNF-α were
to DAPI (blue) or YAP (green) were split, followed by converting to significantly higher compared to the cells treated with CTS alone
grayscale. The region of interest (ROI) of cell nuclei was selected in the (Fig. 2C). Inhibition of YAP by verteporfin caused decreases in the
DAPI channel using the threshold tool and optimized using binary tools. expression of CTGF and CYR61 genes (Figs. 2A and 2C), and the dif­
The selected ROI was applied in the YAP channel; therefore, the mean ferences mentioned above between groups caused by stimulation were
fluorescence intensity (MFI) of YAP was analyzed only in nuclei. The abrogated (Figs. 2B and 2D).
MFI of YAP in the cytoplasm was measured in the same way after The effect of CTS and/or TNF-α on the nuclear localization of YAP
excluding the nuclear ROI in the YAP channel. Three different micro­ evaluated by immunofluorescence microscopy in the absence or pres­
scopic fields were analyzed for each sample, and the nuclear to cyto­ ence of verteporfin is presented in Fig. 3. In the absence of verteporfin,
plasmic ratio of MFI was calculated consequently. both CTS and TNF-α enhanced YAP concentrating in nuclei. Simulta­
neous stimulation of hPDL-MSCs with CTS and TNF-α did not result in
2.7. Enzyme-linked immunosorbent assay any further visible increase in the nuclear distribution of YAP (Figs. 3A

Conditioned media were harvested to analyze the protein levels of IL-

6 and IL-8 using IL-6 Human Uncoated ELISA kit and IL-8 Human Un­
coated IL-8 ELISA kits (both from ThermoFisher Scientific, Waltham,
USA), respectively, according to the manufacturer’s instruction. The
optical density (OD) values were measured at 450 nm (Synergy HTX
multi-mode reader, BioTek Instruments, Winooski, USA), those
measured at 570 nm were used for the correction. The final concentra­
tions were calculated based on the appropriate standard curve con­
structed with Gen5 All-In-One Microplate Reader Software version 2.09
(BioTek Instruments, Winooski, USA). The detection limit was 2 pg/ml
for both IL-6 and IL-8.
Fig. 1. Effects of CTS, TNF-α and verteporfin on hPDL-MSCs proliferation/
viability. hPDL-MSCs were treated with CTS and/or TNF-α in the absence or
2.8. Statistical analysis presence of verteporfin. The proliferation/viability of hPDL-MSCs was
measured with MTT (n=3). Untreated cells served as control. The y-axis shows
Differences between groups were assessed by one-way analysis of the means ± SD of the optical density (OD) measured at 570 nm. The com­
variance (ANOVA) for repeated measures followed by Sidak’s multiple parison was performed by repeated measured one-way ANOVA (RM one-way
comparison tests. Normal distribution was proved by the Kolmogorov- ANOVA) followed by Sidak’s post-hoc test.

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Fig. 2. Effects of CTS, TNF-α, and verteporfin on the gene expression of CTGF and CYR61 in hPDL-MSCs. hPDL-MSCs were treated with CTS and/or TNF-α in the
absence or presence of verteporfin. The gene expression of CTGF and CYR61 was measured with RT-qPCR (n=5). Y-axes show the n-fold expression levels compared
to the untreated control (n-fold expression = 1). The data of CTGF (A) and CYR61 (C) expression are presented as the mean ± standard deviation and analyzed using
RM one-way ANOVA followed by Sidak’s post-hoc test. † p-value <0.05 significantly different compared to the corresponding cells without verteporfin; * p-value
<0.05 significantly different compared to the appropriate control without any mechanical and inflammatory stimulation; # p-value <0.05 between groups as
indicated. The individual values of CTGF (B) and CYR61 (D) expression in the cells stimulated with (w.) or without (w.o.) TNF-α are shown as dot-blots. P-values in
their corresponding bar graphs are shown as indicated.

and 3B). Quantification of the immunofluorescent data confirmed these Data of CXCL8 gene expression showed that TNF-α led to an overall
observations, showing that the ratio of MFI of nuclear/cytoplasm YAP higher CXCL8 expression compared to the appropriate control (Fig. 4E).
was significantly increased by CTS and TNF-α (Figs. 3C and 3D). TNF-α CTS had no significant effect on the basal and TNF-α-induced CXCL8
treated group had a significantly higher nuclear to cytoplasmic ratio of gene expression both in the absence and presence of verteporfin
MFI compared to the CTS-treated group (Fig. 3C). The simultaneous (Figs. 4E and 4F). Verteporfin significantly increased the basal IL-8 gene
stimulation with CTS and TNF-α caused a non-significant decrease in expression level but did not affect the TNF-α-induced CXCL8 expression
this MFI ratio, compared to the cells stimulated with TNF-α alone independently of the presence of CTS (Fig. 4E). The protein production
(Fig. 3E). Verteporfin excluded YAP from the nucleus and kept it in the of IL-8 in the absence of TNF-α was below the detection limit. In contrast
cytoplasm under all conditions (Figs. 3A and 3B). The ratios of nuclear to mRNA data, CTS significantly increased TNF-α-induced IL-8 produc­
MFI to cytoplasmic MFI were lower in verteporfin treated groups tion in the absence of verteporfin (Fig. 4G and H).
compared to the corresponding groups without verteporfin treatment,
although a significant decrease was only found in the cells treated with
CTS (Fig. 3C). 3.4. Effects of verteporfin on VCAM-1 and ICAM-1 expression in hPDL-
MSCs after treatment with CTS and/or TNF-α

3.3. Effects of verteporfin on IL-6 and IL-8 expression in hPDL-MSCs Fig. 5 shows the gene expression of VCAM1 and ICAM1 in hPDL-
after treatment with CTS and/or TNF-α MSCs after treatment with TNF-α and CTS in the absence or presence
of verteporfin. TNF-α induced significantly higher VCAM1 and ICAM1
Fig. 4 shows the gene and protein expression of IL-6 and IL-8 in expression compared to the appropriate control (Figs. 5A and 5C). In the
hPDL-MSCs after treatment with CTS and TNF-α in the absence or absence of verteporfin, CTS applied on hPDL-MSCs did not affect the
presence of verteporfin. TNF-α led to significantly higher IL6 expression basal gene expression of VCAM1 and ICAM1 (Figs. 5A and 5C) but
compared to the appropriate control (Fig. 4A). In the absence of verte­ significantly decreased TNF-α-induced expression of these genes
porfin, CTS applied on hPDL-MSCs had no significant effect on the basal (Figs. 5C and 5D). Verteporfin had no effect on the basal VCAM1 and
expression (i.e., the expression in the absence of TNF-α) of IL6 (Fig. 4A), ICAM1 gene expression but significantly decreased TNF-α-induced
but significantly inhibited the TNF-α-induced IL6 expression (Figs. 4A VCAM1 and ICAM1 expression independently on the presence of CTS
and 4B). Verteporfin caused a significant increase in basal IL6 gene (Figs. 5A and 5C). In the presence of verteporfin, CTS exerted no effect
expression but significantly inhibited TNF-α-induced IL6 expression, on TNF-α-induced gene expression of VCAM1 and ICAM1 (Figs. 5B and
independently on the application of CTS (Fig. 4A). In the presence of 4D).
verteporfin, no further inhibition caused by CTS on TNF-α-induced IL6
expression was observed (Figs. 4A and 4B). The protein production of IL- 4. Discussion
6 in the absence of TNF-α was below the detection limit. In the presence
of TNF-α, verteporfin significantly decreased IL-6 production indepen­ It has been known that orthodontic treatment in periodontal patients
dently on the application of CTS (Fig. 4C). CTS exerted a relatively requires adequate inflammation control to prevent the aggravation of
pronounced inhibition on IL-6 production in the no-verteporfin group, tissue destruction (Liu et al., 2017; Lucchese & Bondemark, 2021). Or­
compared to the verteporfin-treated group (Fig. 4D). thodontic forces were considered to play a role in worsening clinical

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Fig. 3. Effects of CTS, TNF-α, and verteporfin on the nuclear localization of YAP in hPDL-MSCs. hPDL-MSCs were treated with CTS and/or TNF-α in the absence or
presence of verteporfin. The localization of YAP (green) in the absence (A) or presence (B) of verteporfin was analyzed by immunofluorescence followed by semi-
quantitative analysis (n=5). Bars correspond to 50 µm. Quantitative analysis of YAP distribution within cells was performed by calculating the ratio of MFI in nuclei
to that in the cytoplasm (ratio of MFI, N/C). The data are presented as the mean ± standard deviation (C) and analyzed using RM one-way ANOVA followed by
Sidak’s post-hoc test. † p-value <0.05 significantly different compared to the corresponding cells without verteporfin; * p-value <0.05 significantly different
compared to the appropriate control without any mechanical and inflammatory stimulation; # p-value <0.05 between groups as indicated. The individual values of
MFI N/C ratio in the cells of different donors and experiments stimulated with (w., E) or without (w.o., D) TNF-α are shown as dot-blots. P-values in their corre­
sponding bar graphs are shown as indicated.

conditions by activating periodontal cells and influencing cellular re­ when simulating the application of super-elastic nickel-titanium springs,
sponses to the inflammatory microenvironment (Agarwal et al., 2003; while others believed cyclic force correlates better when occlusal force is
Gkantidis et al., 2010; Lucchese et al., 2018). However, the exact also taken into account (Behm et al., 2021; Jacobs et al., 2014). In any
mechanisms of biological interplay between mechanical loading and case, in vitro loading cannot translate the orthodontic force from a tooth
inflammatory responses remain unclear. Our study investigated to the monolayer cells fairly well. Moreover, the force parameters of
hPDL-MSCs responses to 12% CTS (Chang et al., 2015; Chen et al., 2015; CTS, such as magnitudes and durations, have not been standardized and
Natali et al., 2004; Shen et al., 2014) and 10 ng/ml TNF-α (Oyama et al. may influence the results significantly. 12% in our study is a commonly
2000; Yao et al. 2017; Yu et al. 2021) in vitro, which were used to imitate used magnitude based on the finite element analysis of PDL strains on a
the orthodontic force and periodontal inflammation condition, maxillary central incisor (Chang et al., 2015; Chen et al., 2015; Natali
respectively. et al., 2004; Shen et al., 2014). The force duration of 24 hours was
Noteworthy, although CTS is often used to simulate the strain on the chosen to reflect the initial stage after force application, in which
tension side of the PDL (Sun et al. 2021), its clinical relevance is force-induced inflammation plays a major role in the biological re­
disputable. Some researchers considered that static force is predominant sponses of OTM.

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Fig. 4. The gene and protein expression of IL-6 and IL-8 in hPDL-MSCs treated with CTS and/or TNF-α in the absence or presence of verteporfin. hPDL-MSCs were
treated with CTS and/or TNF-α in the absence or presence of verteporfin. The gene expression of IL6 (A) and CXCL8 (E) was measured with RT-qPCR (n=5). Y-axes
shows the n-fold expression levels compared to the untreated control (n-fold expression = 1). The protein production of IL-6 (C) and IL-8 (G) in the cells treated with
(w.) TNF-α was measured with ELISA (n=5). Y-axes shows the concentration in conditioned media. All data are presented as the mean ± standard deviation and
analyzed using RM one-way ANOVA followed by Sidak’s post-hoc test. † p-value <0.05 significantly different compared to the corresponding cells without verte­
porfin; * p-value <0.05 significantly different compared to the appropriate control without any mechanical and inflammatory stimulation; # p-value <0.05 compared
between groups as indicated. The individual values of assayed levels of IL6 mRNA (B), CXCL8 mRNA (F), IL-6 protein (D), and IL-8 protein (H) in the cells stimulated
with (w.) TNF-α are shown as dot-blots. P-values in their corresponding bar graphs are shown as indicated.

TNF-α is a primary inflammatory cytokine that is highly correlated activation of TNF-α signaling pathway and induce the expression of
with not only the development of periodontitis but also the initial stage endogenous TNF-α (Agarwal et al., 2003; Lee et al. 2012). However, the
of OTM (Liu et al., 2016; Yucel-Lindberg & Bage, 2013). Previous studies levels of endogenous TNF-α produced by hPDL-MSCs is usually in pg/ml
showed that biomechanical cues on periodontal cells could regulate the range (Zhao et al., 2021) and is markedly lower than the exogenous

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Fig. 5. The gene expression of VCAM-1 and ICAM-1 in hPDL-MSCs treated with CTS and/or TNF-α in the absence or presence of verteporfin. hPDL-MSCs were treated
with CTS and/or TNF-α in the absence or presence of verteporfin. The gene expression of VCAM1 (A) and ICAM1 (C) was measured with RT-qPCR (n=5). Y-axes
shows the n-fold expression levels compared to the untreated control (n-fold expression = 1). The data are presented as the mean ± standard deviation and analyzed
using RM one-way ANOVA followed by Sidak’s post-hoc test. † p-value <0.05 significantly different compared to the corresponding cells without verteporfin; * p-
value <0.05 significantly different compared to the appropriate control without any mechanical and inflammatory stimulation; # p-value <0.05 compared between
groups as indicated. The individual values of the VCAM1 (B) and ICAM1 (D) mRNA levels in the cells stimulated with (w.) TNF-α are shown as dot-blots. P-values in
their corresponding bar graphs are shown as indicated.

TNF-α level used in our experiments (10 ng/ml), implying that endog­ of pro-inflammatory cytokines, whereas low magnitudes (2–8%) exerted
enous TNF-α production has a minor influence on the results. TNF-α anti-inflammatory effects in PDL cells (Agarwal et al., 2003; Long et al.,
activates the production of diverse inflammatory mediators in 2001; Nogueira et al., 2014; Nokhbehsaim et al., 2012). CTS with 12%
hPDL-MSCs, and in our study we focused on IL-6, IL-8, VCAM-1, and elongation, applied in our study, is a commonly used magnitude for
ICAM-1 (Baud & Karin, 2001; Choi et al., 2018; Liu et al., 2016). IL-6 is human PDL cells in vitro and is based on a finite element model (Chen
highly involved in the destruction of periodontal tissues through its et al., 2015; Natali et al., 2004; Saminathan et al., 2012; Sun et al., 2017;
contribution to osteoclastogenesis (Irwin & Myrillas, 1998; Kurihara Wescott et al. 2007). However, 12% performed various effects on in­
et al., 1990). IL-8 promotes the recruitment and activation of neutro­ flammatory responses in different studies, generating aggravation or no
phils to the lesion site (Baggiolini et al., 1989). VCAM-1 and ICAM-1 aggravation of inflammation (Agarwal et al., 2003; Lee et al., 2012).
play important roles in leukocyte recruitment and mediate the immu­ Another possible explanation for this anti-inflammatory effect of CTS
nomodulatory function of MSCs (Choi et al., 2018; Ren et al., 2010). observed in our study is that orthodontic loading exerts only a transient
More key mediators in inflammatory modulation, especially effect on periodontal inflammation. Particularly, Nokhbehsaim et al.
anti-inflammatory cytokines, should be further investigated in future reported that even the CTS with a magnitude up to 20% upregulated the
studies. IL-1β-induced IL-1β only at day 1 but downregulated it after 6 days
Although it is well known that orthodontic forces generate a local (Nokhbehsaim et al., 2010). Summarizing, our findings showed that the
aseptic inflammation, which initiates OTM (Li et al., 2018), our data impact of CTS on the inflammatory response of hPDL-MSCs seems to
showed that CTS with 12% elongation alone had no influence on the depend on the inflammatory environments. The force magnitude and
basal IL6, CCXL8, VCAM1, and ICAM1 expression in hPDL-MSCs. Our the investigation time seem to play important roles in CTS functions and
data are in accordance with the investigation on IL-6 and IL-8 from should be considered in further studies.
several studies (Jia et al., 2020; Long et al., 2001; Nazet et al. 2020), Our data showed that 12% CTS had no effect on TNF-α-induced
which used approached force magnitudes but with different force CXCL8 mRNA expression but significantly increased IL-8 protein pro­
application forms, including cyclic or static tension or compression, and duction. This differential expression pattern of the gene and protein
our recent study using similar conditions (Zhao et al., 2021). We further might indicate some post-transcriptional modulation of IL-8 (Zhao et al.,
found that the response of hPDL-MSCs to TNF-α is modulated by 2021). Previous in vivo and in vitro studies showed some consistent re­
biomechanical forces. 12% CTS significantly suppressed the gene sults regarding the promoted IL-8 protein levels by mechanical forces in
expression of IL6, VCAM1, and ICAM1 in TNF-α-treated hPDL-MSCs, the inflammatory microenvironment. An animal study reported that
suggesting its anti-inflammatory effect. It is well known that applying OTM increased IL-8 production, especially in the tension area (Tuncer
orthodontic forces on inflamed periodontal tissues will aggravate the et al., 2005). Another study on human PDL cells showed an elevated IL-8
periodontal state and result in iatrogenic damage (Kalemaj et al., 2017). protein level by CTS when cells were stimulated with F. nucleatum
However, many in vitro studies on hPDL-MSCs established a (Rath-Deschner et al., 2021). These findings implied the role of IL-8 in
dose-dependent manner of the effects of CTS under inflammatory con­ early sensing of the cell stretching under pathological conditions.
ditions. Generally, high magnitudes (15–20%) enhanced the expression To unveil the potential mechanism of such interaction between CTS

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and inflammation, the role of YAP in TNF-α-induced inflammation, CTS- identified to be sensitive to various mechanical signals (Dupont et al.,
induced mechanotransduction, and their interplays were analyzed. The 2011). Mechanical loads change the structure of cell-extracellular ma­
effects of YAP were investigated using verteporfin, which is known to trix (ECM) connections and promote the tension of the cytoskeleton,
suppress the translocation of YAP into the nucleus by increasing levels of leading to the activation and nuclear translocation of YAP (Dupont et al.,
its adapter protein 14-3-3σ in the cytoplasm (Hulter-Hassler et al., 2017; 2011). Previous in vitro and in vivo studies showed that YAP was upre­
Wang et al., 2016). gulated and activated both in the periodontium and PDL cells after
Firstly, we investigated the crosstalk between YAP signaling and applying cyclic tensile forces (He et al., 2019; Sun et al., 2018; Yang
inflammatory modulation. Recently, emerging evidence elucidated that et al., 2018). Our results verified the effects of CTS on YAP and validated
YAP played crucial regulatory roles in innate immunity (Wang et al., our loading protocol of hPDL-MSCs. Interestingly, the effect of CTS on
2020). The Hippo-YAP pathway is a positive regulator of anti-bacterial YAP activation was drastically affected by inflammatory conditions. In
and anti-inflammatory responses in mammals. YAP cytoplasmic reten­ contrast to the basal conditions, the application of CTS to hPDL-MSCs in
tion and degradation is involved in the attenuation of NF-κB signaling the presence of TNF-α significantly decreased the nuclear translocation
(Halder et al., 2012; Lee et al., 2019), whereas long-term expression of of YAP and the expression of YAP-regulated genes CTGF and CYR61.
YAP potently activates the expression of inflammatory factors (Wang This finding suggests that the effect of CTS on YAP activation depends on
et al., 2020). On the other hand, YAP was also demonstrated to play an the inflammatory environment: At basal conditions, YAP is activated by
inhibitory role in NF-κB activation. YAP upon the translocation into CTS, but CTS inhibits TNF-α-activated YAP.
nuclei functions as a transcriptional activator, reducing the production Stimulation of hPDL-MSCs with TNF-α and CTS resulted in differ­
of inflammatory cytokines (Zhang et al., 2018). Therefore, the ential pattern of YAP target genes CTGF and CYR61. The proteins
Hippo-YAP pathway plays a dual role in the modulation of NF-κB encoded by these genes are involved in cell adhesion, migration, pro­
signaling and might be either a positive or negative regulator of it. liferation, and ECM synthesis, and their expression is upregulated in
Our data showed that YAP in hPDL-MSCs was activated by TNF-α, as mechanically challenged organs (Brigstock, 2003; Chaqour &
suggested by YAP nuclear translocation and increased CTGF and CYR61 Goppelt-Struebe, 2006), and is induced by multiple mechanical cues,
gene expression. Previous studies showed some inconsistencies such as stretch, shear stress, and hydrostatic pressure, in cultured cell in
regarding the effect of TNF-α on YAP activity in different cell types. On vitro (Chaqour & Goppelt-Struebe, 2006). Our study showed CTS
the one hand, TNF-α was reported to activate YAP in vascular endo­ induced a more pronounced upregulation of the mRNA level of CTGF
thelial cells (Choi et al., 2018). On the other hand, YAP/TAZ was compared to CYR61 after 24 h. Similar results were found by another
phosphorylated and degraded by TNF-α through modulating the study using 10% CTS on PDL cells for 24 h (Yang et al., 2018). However,
component of NF-κB pathway or Hippo pathway in chondrocytes (Deng other studies showed that the induction of CTGF required a higher strain
et al., 2018). Therefore, the response might be dependent on the cell level than CYR61 (Chaqour & Goppelt-Struebe, 2006). The possible
type. Furthermore, YAP inhibition by verteporfin receded the high explanation for these observations is that the induction of CYR61
expression of IL6, VCAM1, and ICAM1 in the presence of TNF-α, indi­ expression occurred rapidly after the application of CTS and gradually
cating that TNF-α-induced inflammatory responses in hPDL-MSCs at return afterwards (Tamura et al., 2001). Moreover, our data showed
least partially dependent on YAP activation. TNF-α induced a more significant increase in CYR61 than CTGF. CYR61
Interestingly, the effects of verteporfin on the gene expression of was reported to collaborate with cytokines such as TNF-α in inflamma­
inflammatory mediators in hPDL-MSCs depended qualitatively on an tion (Lau, 2011), while the effects of TNF-α on CTGF expression may be
inflammatory environment: verteporfin significantly enhanced the basal dependent on cells type (Cooker et al., 2007; Laug et al., 2012).
expression of IL6 and CCXL8 but exerted opposite effect under TNF-α Thirdly, the contribution of YAP in the effects of CTS on inflamma­
stimulation. Particularly, TNF-α-induced IL6 expression was down- tory responses in hPDL-MSCs was investigated in the experiments with
regulated by verteporfin, whereas no effect on TNF-α-induced CCXL8 verteporfin. We found that in the presence of YAP inhibitor, CTS had no
expression was observed. The reason for the different effects of verte­ inhibitory effect on TNF-α-induced responses in hPDL-MSCs. Thus, it can
porfin on TNF-α-induced IL-6 and IL-8 expression is not clear. One study be assumed that the anti-inflammatory effect of CTS in the presence of
in hepatocellular carcinoma cells found that IL-8 expression was TNF-α could be at least partially associated with YAP-dependent
modulated by a paralog of YAP, transcriptional co-activator with PDZ- mechanisms. In the absence of CTS and the presence of TNF-α, YAP
binding motif (TAZ), while IL-6 was not (Zhang et al., 2020). These inhibition had an anti-inflammatory effect in hPDL-MSCs. CTS inhibited
findings together emphasize the distinct contribution of YAP and TAZ in YAP activation in the presence of TNF-α, as suggested by the decreased
inflammatory modulation, although they share large parts of their reg­ expression of YAP target genes CTGF and CYR61 and lower nuclear YAP
ulatory machinery in Hippo pathway. Our data also showed that YAP translocation, and this inhibition could explain the anti-inflammatory
inhibition did not change the basal expression levels of adhesion mol­ action of CTS in the presence of TNF-α. In contrast, in the absence of
ecules VCAM1 and ICAM1 but caused a decrease in TNF-α-induced TNF-α, the effects of CTS seem not to be mediated by YAP. Under these
VCAM1 and ICAM1. In line with our data, Choi et al. demonstrated conditions, YAP inhibition has a pro-inflammatory effect, and, therefore,
knockdown of YAP/TAZ inhibited TNF-α-induced VCAM1 and ICAM1 YAP activation is assumed to be anti-inflammatory. However, YAP
expression in endothelial cells (Choi et al., 2018), suggesting the activation by CTS has neither pro- nor anti-inflammatory effect in the
pro-inflammatory role of YAP in these conditions. Taken together, these absence of TNF-α. Therefore, it seems that YAP might mediate some CTS
findings suggest a dual role of YAP in the inflammatory response in effects in the presence of TNF-α-induced inflammation but not under
hPDL-MSCs, to be anti-inflammatory in the absence of TNF-α and resting conditions.
pro-inflammatory in the presence of TNF-α. The sophisticated reciprocal
interaction between YAP signaling and the inflammatory response needs 5. Conclusion
further exploration.
Secondly, we analyzed the YAP activation in response to CTS Our study is the first which investigate the role of YAP in the inter­
application. Our study demonstrated that YAP might be involved in the action of TNF-α and CTS in hPDL-MSCs. The presence of inflammation is
complicated networking of mechanotransduction in response to the an essential factor determining the effects of CTS and YAP in hPDL-
external mechanical loading on hPDL-MSCs. After applying CTS with MSCs. TNF-α induces YAP activation, which seems to promote the in­
12% elongation for 24 hours, the nuclear localization of YAP was pro­ flammatory response further. CTS at the magnitude of 12% inhibits TNF-
moted, and the expression of YAP-regulated genes was increased. YAP α-induced responses in hPDL-MSCs, and this effect could be associated
acts as a transcriptional co-activator, regulates multiple aspects of cell with the inhibition of YAP activation. However, in the absence of in­
behaviors like cell proliferation and apoptosis, and was recently flammatory stimuli, the effect of CTS on the inflammatory response

8
Z. Zhao et al. Archives of Oral Biology 143 (2022) 105527

seems to be YAP-independent. This complex interaction between me­ Chaqour, B., & Goppelt-Struebe, M. (2006). Mechanical regulation of the Cyr61/CCN1
and CTGF/CCN2 proteins. Aug FEBS J, 273(16), 3639–3649. https://doi.org/
chanical stress, YAP signaling, and inflammatory response could be
10.1111/j.1742-4658.2006.05360.x.
important in the fine-tuning of biological processes involved in OTM and Chaushu, S., Klein, Y., Mandelboim, O., Barenholz, Y., & Fleissig, O. (2022). Immune
should be further explored in the future. Changes Induced by Orthodontic Forces: A Critical Review. Jan Journal of Dental
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Funding Chen, Y., Mohammed, A., Oubaidin, M., Evans, C. A., Zhou, X., Luan, X.,
Diekwisch, T. G., & Atsawasuwan, P. (2015). Cyclic stretch and compression forces
alter microRNA-29 expression of human periodontal ligament cells. Jul 15 Gene, 566
This study was supported by the Austrian Science Fund (FWF)
(1), 13–17. https://doi.org/10.1016/j.gene.2015.03.055.
(P35037). Zhongqi Zhao is supported by China Scholarship Council Choi, H. J., Kim, N. E., Kim, B. M., Seo, M., & Heo, J. H. (2018). TNF-alpha-induced YAP/
(CSC) (No. 201806170075). TAZ activity mediates leukocyte-endothelial adhesion by regulating VCAM1
expression in endothelial cells. International Journal of Molecular Sciences, 19(11).
https://doi.org/10.3390/ijms19113428
CRediT authorship contribution statement Cooker, L. A., Peterson, D., Rambow, J., Riser, M. L., Riser, R. E., Najmabadi, F.,
Brigstock, D., & Riser, B. L. (2007). TNF-α, but not IFN-γ, regulates CCN2 (CTGF),
collagen type I, and proliferation in mesangial cells: possible roles in the progression
Zhongqi Zhao: Conceptualization, Methodology, Validation, Formal
of renal fibrosis. American Journal of Physiology-Renal Physiology, 293(1), F157–F165.
analysis, Investigation, Data Curation, Writing - Original Draft. Chris­ https://doi.org/10.1152/ajprenal.00508.2006
tian Behm: Methodology, Formal analysis, Validation, Writing - Review Deng, Y., Lu, J., Li, W., Wu, A., Zhang, X., Tong, W., Ho, K. K., Qin, L., Song, H., &
Mak, K. K. (2018). ). Reciprocal inhibition of YAP/TAZ and NF-kappaB regulates
& Editing. Zhiwei Tian: Formal analysis, Data Curation, Visualization,
osteoarthritic cartilage degradation. Nature Communications, 9(1), 4564. https://doi.
Writing - Review & Editing. Marco Aoqi Rausch: Formal analysis, org/10.1038/s41467-018-07022-2
Investigation, Writing - Review & Editing. Xiaohui Rausch-Fan: Dupont, S., Morsut, L., Aragona, M., Enzo, E., Giulitti, S., Cordenonsi, M., Zanconato, F.,
Conceptualization, Resources, Methodology, Writing - Review & Edit­ Le Digabel, J., Forcato, M., Bicciato, S., Elvassore, N., & Piccolo, S. (2011). Role of
YAP/TAZ in mechanotransduction. Nature, 474(7350), 179–183. 〈https://doi.org/
ing, Supervision. Oleh Andrukhov: Conceptualization, Resources, 10.1038/nature10137〉.
Methodology, Writing - Review & Editing, Supervision, Funding Eberwein, P., Laird, D., Schulz, S., Reinhard, T., Steinberg, T., & Tomakidi, P. (2015).
acquisition. Modulation of focal adhesion constituents and their down-stream events by EGF: on
the cross-talk of integrins and growth factor receptors. Biochimica Et Biophysica Acta-
Molecular Cell Research, 1853(10), 2183–2198. https://doi.org/10.1016/j.
bbamcr.2015.06.004
Declaration of Competing Interest Feller, L., Khammissa, R. A., Schechter, I., Moodley, A., Thomadakis, G., & Lemmer, J.
(2015). Periodontal biological events associated with orthodontic tooth movement:
The authors declare that they have no known competing financial the biomechanics of the cytoskeleton and the extracellular matrix.
ScientificWorldJournal, 2015, Article 894123. https://doi.org/10.1155/2015/
interests or personal relationships that could have appeared to influence
894123
the work reported in this paper. Gkantidis, N., Christou, P., & Topouzelis, N. (2010). The orthodontic–periodontic
interrelationship in integrated treatment challenges: A systematic review. Journal of
Oral Rehabilitation, 37(5), 377–390. https://doi.org/10.1111/j.1365-
Acknowledgments 2842.2010.02068.x
Graves, D. T., & Cochran, D. (2003). The contribution of interleukin-1 and tumor necrosis
The authors thank Phuong Quynh Nguyen for the excellent technical factor to periodontal tissue destruction. Journal of Periodontology, 74(3), 391–401.
https://doi.org/10.1902/jop.2003.74.3.391
assistance. Zhongqi Zhao is grateful for the financial support provided
Halder, G., Dupont, S., & Piccolo, S. (2012). Transduction of mechanical and cytoskeletal
by China Scholarship Council (CSC) (No. 201806170075). cues by YAP and TAZ. Nature Reviews Molecular Cell Biology, 13(9), 591–600. https://
doi.org/10.1038/nrm3416
He, Y., Xu, H., Xiang, Z., Yu, H., Xu, L., Guo, Y., Tian, Y., Shu, R., Yang, X., Xue, C.,
Appendix A. Supporting information Zhao, M., He, Y., Han, X., & Bai, D. (2019). YAP regulates periodontal ligament cell
differentiation into myofibroblast interacted with RhoA/ROCK pathway. Journal of
Supplementary data associated with this article can be found in the Cellular Physiology, 234(4), 5086–5096. https://doi.org/10.1002/jcp.27312
Huang, H., Yang, R., & Zhou, Y. H. (2018). Mechanobiology of periodontal ligament stem
online version at doi:10.1016/j.archoralbio.2022.105527.
cells in orthodontic tooth movement. Stem Cells International, 2018, 6531216.
https://doi.org/10.1155/2018/6531216
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