International Journal of Molecular Sciences

Article Interleukin-1β Induced Matrix Metalloproteinase Expression in Human Periodontal Ligament-Derived Mesenchymal Stromal Cells under In Vitro Simulated Static Orthodontic Forces

Christian Behm 1,2,† , Michael Nemec 1,†, Alice Blufstein 2,3, Maria Schubert 2, Xiaohui Rausch-Fan 3, Oleh Andrukhov 2,* and Erwin Jonke 1

1 Division of Orthodontics, University Clinic of Dentistry, Medical University of Vienna, 1090 Vienna, Austria; [email protected] (C.B.); [email protected] (M.N.); [email protected] (E.J.) 2 Competence Center for Periodontal Research, University Clinic of Dentistry, Medical University of Vienna, 1090 Vienna, Austria; [email protected] (A.B.); [email protected] (M.S.) 3 Division of Conservative Dentistry and Periodontology, University Clinic of Dentistry, Medical University of Vienna, 1090 Vienna, Austria; [email protected] * Correspondence: [email protected] † These authors contributed equally to this work.

Abstract: The periodontal ligament (PDL) responds to applied orthodontic forces by extracellular matrix (ECM) remodeling, in which human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) are largely involved by producing matrix metalloproteinases (MMPs) and their local



 inhibitors (TIMPs). Apart from orthodontic forces, the synthesis of MMPs and TIMPs is influenced by the aseptic inflammation occurring during orthodontic treatment. Interleukin (IL)-1β is one of Citation: Behm, C.; Nemec, M.; the most abundant inflammatory mediators in this process and crucially affects the expression Blufstein, A.; Schubert, M.; of MMPs and TIMPs in the presence of cyclic low-magnitude orthodontic tensile forces. In this Rausch-Fan, X.; Andrukhov, O.; study we aimed to investigate, for the first time, how IL-1β induced expression of MMPs, TIMPs Jonke, E. Interleukin-1β Induced and how IL-1β in hPDL-MSCs was changed after applying in vitro low-magnitude orthodontic Matrix Metalloproteinase Expression tensile strains in a static application mode. Hence, primary hPDL-MSCs were stimulated with IL-1β in Human Periodontal Ligament-Derived Mesenchymal in combination with static tensile strains (STS) with 6% elongation. After 6- and 24 h, MMP-1, Stromal Cells under In Vitro MMP-2, TIMP-1 and IL-1β expression levels were measured. STS alone had no influence on the basal Simulated Static Orthodontic Forces. expression of investigated target genes, whereas IL-1β caused increased expression of these genes. Int. J. Mol. Sci. 2021, 22, 1027. In combination, they increased the gene and protein expression of MMP-1 and the gene expression https://doi.org/10.3390/ijms22031027 of MMP-2 after 24 h. After 6 h, STS reduced IL-1β-induced MMP-1 synthesis and MMP-2 gene expression. IL-1β-induced TIMP-1 gene expression was decreased by STS after 6- and 24-h. At both Received: 31 December 2020 time points, the IL-1β-induced gene expression of IL-1β was increased. Additionally, this study Accepted: 16 January 2021 showed that fetal bovine serum (FBS) caused an overall suppression of IL-1β-induced expression of Published: 20 January 2021 MMP-1, MMP-2 and TIMP-1. Further, it caused lower or opposite effects of STS on IL-1β-induced expression. These observations suggest that low-magnitude orthodontic tensile strains may favor a Publisher’s Note: MDPI stays neutral more inflammatory and destructive response of hPDL-MSCs when using a static application form with regard to jurisdictional claims in and that this response is highly influenced by the presence of FBS in vitro. published maps and institutional affil- iations. Keywords: orthodontic tooth movement; static orthodontic forces; human periodontal ligament derived mesenchymal stromal cells; matrix metalloproteinases; tissue inhibitor of matrix metallopro- teinases; interleukin-1β

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and conditions of the Creative Commons Orthodontic tooth movement (OTM) plays a pivotal role in the treatment of maloc- Attribution (CC BY) license (https:// clusions and is ultimately induced by applying mechanical strain [1,2]. According to the creativecommons.org/licenses/by/ classic “pressure-tension” hypothesis, mechanical load induces a shift of the teeth, causing 4.0/). a compression zone in the direction of force application and a tension zone at the opposite

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site [3,4]. This creates a local aseptic inflammation, resulting in bone resorption at the compression site and bone formation at the tension site [2]. The fundamental structure for sensing and translating orthodontic forces from teeth to the alveolar bone is the periodontal ligament (PDL) [5], a highly organized connective tissue [6]. Besides bone resorption and formation, the PDLs’ extracellular matrix (ECM) undergoes continuous remodeling during OTM. This ongoing remodeling is mainly orchestrated by the cellular component of the PDL [5,7–10], which consists of a heterogeneous fibroblast-like population of mesenchymal stromal cells (hPDL-MSCs). These non-hematopoietic, multipotent progenitor cells [11–13] were first isolated from the PDL by Seo et al. in 2004 [14,15]. They are characterized by their self-renewal and tri-lineage differentiation potential, as well as by a specific surface marker expression profile and, consequently, fulfil the minimal criteria for MSCs [11–13]. Residing in the perivascular area of the PDL [16,17], hPDL-MSCs are highly involved in PDL homeostasis and directly respond to applied orthodontic forces [18]. It is well established that hPDL-MSCs produce and secrete multiple growth factors, prostaglandins and ECM remodeling enzymes during OTM [5,7–10]. Various proteolytic enzymes, such as matrix metalloproteinases (MMPs) are involved in remodeling ECM proteins within the PDL [5,8,19]. The MMPs belong to a zinc-dependent proteolytic enzyme family which is divided into six different groups depending on their substrate specificity [20–22]. Numerous in vitro studies already investigated the effect of mechanical forces on the expression of various MMPs in the human PDL, mainly focusing on the collagenase MMP-1 and the gelatinase MMP-2 [5,23–27]. These studies mainly showed increased MMP-1 expression in PDL fibroblasts, independently of force type and its static or cyclic application [5,8,24,28,29]. MMP-2 expression, however, is increased in PDL cells after applying tensile forces [5,24,30], whereas compression forces cause no effect or a decrease in its expression [31,32]. Several in vivo studies detected increased MMP-1 and MMP-2 levels in the gingival crevicular fluid (GCF) at the tension as well as compression side shortly after applying orthodontic forces [19,33–35]. Additionally, studies demonstrated changes in the constitutive expression of various local tissue inhibitors of metalloproteinases (TIMPs) in human PDL cells after applying mechanical forces. TIMP-1 expression mainly increased in human PDL cells when applying tensile forces [23,30], whereas compressive strain seems to have a declining effect on TIMP-1 expression [36,37]. In vivo studies mainly showed an overall increase in TIMP-1 levels in the GCF during orthodontic treatment [33,34,38]. Interleukin (IL)-1β is an important pro-inflammatory cytokine having several roles in the human periodontium. During orthodontic treatment-induced aseptic inflammation, IL-1β is one of the most abundant cytokines in both the tension as well as the compression zone [38,39]. It directly contributes to extensive alveolar bone remodeling during OTM [40]. Additionally, IL-1β directly influences the continuing turnover of the PDLs’ ECM by reg- ulating the expression of MMPs and TIMPs [41–43]. Together with other inflammatory cytokines, such as IL-6 [44], IL-1β is also essential in the bacteria-dependent chronic inflam- mation of the periodontium [45]. Hence, it is crucial to have a closer look on the combined effects of orthodontic forces and IL-1β on the MMPs and TIMPs expression in PDL cells. Two studies already demonstrated a significant effect of tensile strain on the production of IL-1β-triggered production of MMPs and TIMPs in PDL cells, using cyclic strain applica- tion [43,46]. However, no data exist showing the influence of static tensile strain (STS) on IL-1β-dependent introduction of MMPs and TIMPs in PDL cells. The discrimination be- tween these two force application forms is essential since they imitate different orthodontic forces applied to patients by different orthodontic appliances [25,47–49]. Therefore, the main aim of the first part of this in vitro study was to investigate the influence of STS on IL-1β-induced expression of MMPs and TIMPs in hPDL-MSCs. In particular, we evaluated the IL-1β-triggered production of MMP-1, MMP-2, TIMP-1 and IL-1β itself in hPDL-MSCs after applying equibiaxial STS with 6% elongation for 6 and 24 h. Secondly, we directly compared the influence of STS on the IL-1β-induced expression of these genes in the absence and presence of fetal bovine serum (FBS). It is essential to Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 18

particular, we evaluated the IL-1β-triggered production of MMP-1, MMP-2, TIMP-1 and IL-1β itself in hPDL-MSCs after applying equibiaxial STS with 6% elongation for 6 and 24 h. Secondly, we directly compared the influence of STS on the IL-1β-induced expression of these genes in the absence and presence of fetal bovine serum (FBS). It is essential to evaluate to what extent FBS affects in vitro simulated orthodontic forces on hPDL-MSCs, Int. J. Mol. Sci. 2021, 22, 1027 3 of 18 since it is known that FBS influences these cells during in vitro cultivation [50].

2. Results 2.1.evaluate Immunostaining to what extent of Certain FBS affectsMesenchymalin vitro andsimulated Hematopoietic orthodontic Surface forces Marker on hPDL-MSCs, in hPDL-MSCs since it is known that FBS influences these cells during in vitro cultivation [50]. Supplementary Table S1 shows the percentage of hPDL-MSCs which expressed certain2. Results mesenchymal and hematopoietic surface markers. MSCs surface markers CD29, CD73,2.1. CD90 Immunostaining and CD105 of Certain were Mesenchymalexpressed in and more Hematopoietic than 95% Surface of analysed Marker in hPDL-MSCs. hPDL-MSCs The percentageSupplementary of CD146-positive Table S1 shows hPDL-MSCs the percentage was of about hPDL-MSCs 63.63%. which Hematopoietic expressed certain surface markersmesenchymal CD31, andCD34 hematopoietic and CD45 surface were markers. observed MSCs in surface less markersthan 2.5% CD29, of CD73,analysed hPDL-MSCs.CD90 and CD105 were expressed in more than 95% of analysed hPDL-MSCs. The percent- age of CD146-positive hPDL-MSCs was about 63.63%. Hematopoietic surface markers CD31, CD34 and CD45 were observed in less than 2.5% of analysed hPDL-MSCs. 2.2. Cell Viability 2.2.Figure Cell Viability 1 shows representative pictures of live/dead stained and IL-1β treated hPDL-MSCsFigure which1 shows were representative exposed picturesto STS for of live/dead 24 h. The stained appearance and IL-1 ofβ treatedgreen fluorescent hPDL- livingMSCs hPDL-MSCs which were was exposed comparable to STS for through 24 h. The all appearance used stimulation of green conditions. fluorescent livingIn the ab- sencehPDL-MSCs of STS, IL-1 wasβ comparable caused an through increased all usednumber stimulation of red conditions.fluorescent In dead the absence hPDL-MSCs of fromSTS, 1 ng/mL IL-1β caused to 5 ng/mL, an increased independently number ofred from fluorescent FBS concentration. dead hPDL-MSCs In the from presence 1 ng/mL of STS, to 5 ng/mL, independently from FBS concentration. In the presence of STS, a lower number a lower number of dead hPDL-MSCs is observed, even in the presence of different IL-1β of dead hPDL-MSCs is observed, even in the presence of different IL-1β concentrations. In concentrations.the presence ofIn 2% the FBS, presence STS caused of 2% a slightlyFBS, ST higherS caused number a slightly of dead higher hPDL-MSCs number in theof dead hPDL-MSCsabsence and in the the presence absence of and 1 ng/mL the presence IL-1β compared of 1 ng/mL to hPDL-MSCs IL-1β compared incubated to hPDL-MSCs without incubatedFBS. Nevertheless, without FBS. the majorityNevertheless, of hPDL-MSCs the majority were viableof hPDL-MSCs and no detrimental were viable effect and of no detrimentalexperimental effect conditions of experimental on cell viability conditions was observed. on cell viability was observed.

Figure 1. Cont.

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Figure 1. Representative pictures of live/dead staining of hPDL-MSCs after applying static orthodontic tensile forces Figure 1. Representative pictures of live/dead staining of hPDL-MSCs after applying static orthodontic tensile forces under under IL-1β-induced inflammatory conditions. After stimulating hPDL-MSCs with 1 ng/mL or 5 ng/mL IL-1β, STS with IL-1β-induced inflammatory conditions. After stimulating hPDL-MSCs with 1 ng/mL or 5 ng/mL IL-1β, STS with 6% 6% equibiaxial elongation was applied in the absence or presence of 2% FBS. Unstimulated and non-stretched equibiaxial elongation was applied in the absence or presence of 2% FBS. Unstimulated and non-stretched hPDL-MSCs hPDL-MSCs served as references. 24 h after applying STS, hPDL-MSCs were doubled-stained with green fluorescent served as references. 24 h after applying STS, hPDL-MSCs were doubled-stained with green fluorescent Live-Dye™ Live-Dye™ (Ex/Em = 488/518 nm) and propidium iodide (Pi) (Ex/Em = 488/515) to discriminate between living (green (Ex/Em = 488/518 nm) and propidium iodide (Pi) (Ex/Em = 488/515) to discriminate between living (green fluorescent) fluorescent) and dead (red fluorescent) hPDL-MSCs. Red fluorescent dead cells are indicated by white circles. Fluores- and dead (red fluorescent) hPDL-MSCs. Red fluorescent dead cells are indicated by white circles. Fluorescence was detected cence was detected by ECHO Revolve fluorescence microscope using the FITC and Texas Red channels, a 4-fold magni- by ECHO Revolve fluorescence microscope using the FITC and Texas Red channels, a 4-fold magnification and an exposure fication and an exposure time of 670 ms in both fluorescence channels. time of 670 ms in both fluorescence channels. 2.3. MMP-1 Expression 2.3. MMP-1 Expression Figure 2 shows the MMP-1 gene and protein expression in hPDL-MSCs in the ab- Figure2 shows the MMP-1 gene and protein expression in hPDL-MSCs in the absence senceof FBS,of FBS, 6 and 6 and 24 h 24 after h applyingafter applying STS in theSTS absence in the orabsence presence or ofpresence different of IL-1 differentβ concen- IL-1β concentrations.trations. STS aloneSTS al didone not did affect notMMP-1 affectgene MMP-1 expression gene expression levels after 6- levels and 24-h after incubation 6- and 24-h incubation(Figure2 A,B).(Figure On 2A,B). protein On level, protein MMP-1 level, was MMP-1 also not was affected also by not STS affected in the absence by STS of in the absenceIL-1β of(Figure IL-1β2 (FigureC,D). After 2C,D). 6- and After 24-h 6- incubation, and 24-h incubation, 1 and 5 ng/mL 1 and IL-1 5β ng/mLsignificantly IL-1β in-signif- icantlycreased increasedMMP-1 MMP-1gene expression gene expression in the absence in the of STS absence (Figure of2A,B). STS In(Figure conditioned 2A,B). media, In condi- tioned5 ng/mL media, IL-1 5 ng/mLβ caused IL-1 aβ non-significant caused a non-significant increase in MMP-1 increase protein in MMP-1 levels protein at both timelevels at bothpoints time (Figurepoints2 (FigureC,D). 2C,D). Applying STS caused no effect on IL-1β induced MMP-1 gene expression after 6 h. After 24 h, however, applying STS significantly increased IL-1β induced MMP-1 gene expression levels (Figure2A,B). On protein level, STS non-significantly ( p-value 0.171) counteracted IL-1β-induced protein production after 6 h incubation. After 24 h incubation, IL-1β-induced MMP-1 protein production was further increased by STS, however without any significance (p-value 0.248, Figure2C,D).

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FigureFigure 2. Effect 2. Effect of ofstatic static orthodontic orthodontic tensiletensile forces forces on theon IL-1the βIL-1-inducedβ-induced gene andgene protein and expressionprotein expression of MMP-1 inof hPDL-MMP-1 in hPDL-MSCsMSCs in thein absencethe absence of FBS. of STS FBS. with STS 6% equibiaxialwith 6% equi elongationbiaxial was elongation applied to was untreated applied or IL-1 to βuntreated-treated hPDL-MSCs or IL-1β-treated in hPDL-MSCsthe absence in ofthe FBS. absence 6 (A) and of FBS. 24 (B )6 hours(A) and after 24 stimulation (B) hours with after or stimulation without STS, with qPCR or was without performed STS, showing qPCR was the nperformed-fold showingexpression the n levels-fold expression of MMP-1 compared levels of to MMP-1 the appropriate compared controls to the (n-fold appropriate expression controls = 1). GAPDH (n-foldserved expression as an endogenous = 1). GAPDH servedcontrol. as an Corresponding endogenous MMP-1control. protein Corresponding levels were determinedMMP-1 protein in pg/mL levels by were enzyme-linked determined immunosorbent in pg/mL by assay enzyme-linked (ELISA) immunosorbentin conditioned assay media, (ELISA) which werein conditio harvestedned 6 media, (C) and which 24 (D) hourswere afterharvested stimulation 6 (C) withand or24 without (D) hours STS. after All data stimulation are withpresented or without as meanSTS. ±Allstandard data are error presented of mean as (S.E.M.). mean *±p -valuestandard≤ 0.05 error significantly of mean higher(S.E.M.). compared * p-value to the≤ 0.05 appropriate significantly highercontrol; compared # p-value to ≤the0.05 appropriate significantly control; higher compared # p-value to ≤ the 0.05 appropriate significantly IL-1β higherstimulated compared cells without to the static appropriate STS. IL-1β stimulated cells without static STS. Figure3 shows the MMP-1 gene and protein expression in hPDL-MSCs in the presence of 2% FBS, 6 and 24 h after applying STS in the absence and presence of different IL-1β Applying STS caused no effect on IL-1β induced MMP-1 gene expression after 6 h. concentrations. Applying STS in the absence of IL-1β had no significant effect on MMP- After1 gene 24 h, and however, protein expression applying levelsSTS significantly (Figure3A–D). increased In the absence IL-1βof induced STS, both MMP-1 IL-1 β gene expressionconcentrations levels significantly (Figure 2A,B). increased On MMP-1proteingene level, expression STS non-significantly after 6- and 24 h incubation(p-value 0.171) counteracted(Figure3A,B). IL-1 MMP-1β-induced protein protein levels wereproduction also enhanced after 6 by h IL-1incubation.β in the absenceAfter 24 of h STS, incuba- tion,showing IL-1β-induced a significant MMP-1 increase protein after 6 hproduction incubation (Figurewas further3C,D). increased by STS, however withoutApplying any significance STS to IL-1 (pβ-value-treated 0.248, hPDL-MSCs Figure 2C,D). significantly increased MMP-1 gene ex- pressionFigure levels3 shows after the 6 h incubation.MMP-1 gene 24 hand treatment protein with expression STS also strengthenedin hPDL-MSCs IL-1 βin the presenceinduced ofMMP-1 2% FBS,gene 6 and expression, 24 h after only applying showing STS a significantin the absence increase and in presence the presence of different of 5 ng/mL IL-1β (Figure3A,B). On protein level, STS significantly inhibited IL-1 β induced IL-1β concentrations. Applying STS in the absence of IL-1β had no significant effect on MMP-1 after 6 h stimulation. Applying STS for 24 h causes a further increase in IL-1β MMP-1triggered gene MMP-1 and protein protein expression expression; however,levels (Figure without 3A–D). any (p -valueIn the 0.198)absence significance of STS, both IL-1(Figureβ concentrations3C,D). significantly increased MMP-1 gene expression after 6- and 24 h incubation (Figure 3A,B). MMP-1 protein levels were also enhanced by IL-1β in the ab- sence of STS, showing a significant increase after 6 h incubation (Figure 3C,D).

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Figure 3. Effect of static orthodontic tensile forces on the IL-1β-induced gene and protein expression of MMP-1 in hPDL- Figure 3. MScsEffect in theof static presence orthodontic of 2% FBS. STS tensile with 6% forces equibiaxial on the elongation IL-1β was-induced applied gene to untreated and protein or IL-1β-treated expression hPDL-MSCs of MMP-1 in hPDL-MScsin thein presencethe presence of 2% FBS.of 2% 6 (A FBS.) and STS 24 (B )with hours 6% after eq stimulationuibiaxial withelongation and without was STS, applied qPCR wasto untreated performed showingor IL-1β-treated hPDL-MSCsthe nin-fold the expression presence levels of 2% of MMP-1FBS. 6 compared(A) and to24 the (B appropriate) hours after controls stimulation (n-fold expression with and = 1). withoutGAPDH STS,served qPCR as an was per- formed showingendogenous the control.n-fold Correspondingexpression levels MMP-1 of protein MMP-1 levels compared were determined to the inappropriate pg/mL by ELISA controls in conditioned (n-fold expression media, = 1). GAPDH servedwhich were as an harvested endogenous 6 (C) and control. 24 (D) hours Corresponding after stimulation MMP-1 with or withoutprotein STS. levels All data were are determined presented as mean in pg/mL± S.E.M. by ELISA * p-value ≤ 0.05 significantly higher compared to the appropriate control; # p-value ≤ 0.05 significantly higher compared to in conditioned media, which were harvested 6 (C) and 24 (D) hours after stimulation with or without STS. All data are the appropriate IL-1β stimulated cells without STS; & p-value ≤ 0.05 significantly lower compared to the appropriate Il-1β presentedstimulated as mean cells± S.E.M. without * p STS.-value ≤ 0.05 significantly higher compared to the appropriate control; # p-value ≤ 0.05 significantly higher compared to the appropriate IL-1β stimulated cells without STS; & p-value ≤ 0.05 significantly lower compared to the appropriate Il-1β stimulated cells without STS. 2.4. MMP-2 Expression ApplyingFigure4 showsSTS to theIL-1MMP-2β-treatedgene hPDL-MSCs expression in significantly hPDL-MSCs increased in the absence MMP-1 of FBS, gene 6 ex- pressionand 24 levels h after after applying 6 h incubation. STS in the presence 24 h treatment of different with IL-1 STSβ concentrations. also strengthened Applying IL-1β in- ducedSTS MMP-1 in the absence gene expression, of IL-1β had only no significant showing effecta significant on MMP-2 increasegene expression in the presence levels. of 5 ng/mLAfter IL-1 bothβ6 (Figure and 24-h 3A,B). incubation, On proteinMMP-2 genelevel, expression STS significantly in hPDL-MSCs inhibited was significantly IL-1β induced MMP-1enhanced after by 6 IL-1h stimulation.β, leading to Applying an overall higherSTS forMMP-2 24 h causesgene expression a further level increase after 24in h IL-1β incubation (Figure4A,B). On protein level, MMP-2 was not detectable. triggered MMP-1 protein expression; however, without any (p-value 0.198) significance Applying STS for 6 h caused a decrease in IL-1β induced MMP-2 gene expression, (Figureshowing 3C,D). a significant decline in the presence of 5 ng/mL IL-1β (Figure4A). After 24 h, STS strengthened IL-1β induced MMP-2 gene expression in a concentration-dependent manner, 2.4.showing MMP-2 aExpression significant rise in the presence of 1 ng/mL IL-1β (Figure4B). Figure 4 shows the MMP-2 gene expression in hPDL-MSCs in the absence of FBS, 6 and 24 h after applying STS in the presence of different IL-1β concentrations. Applying STS in the absence of IL-1β had no significant effect on MMP-2 gene expression levels. After both 6 and 24-h incubation, MMP-2 gene expression in hPDL-MSCs was signifi- cantly enhanced by IL-1β, leading to an overall higher MMP-2 gene expression level after 24 h incubation (Figure 4A,B). On protein level, MMP-2 was not detectable.

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Figure 4. Effect of static orthodontic tensile forces on the IL-1β-induced gene expression of MMP-2 in hPDL-MSCs in the absence of FBS. STS with 6% equibiaxial elongation was applied to untreated or IL-1β-treated hPDL-MSCs in the absence of FBS. 6 (A) and 24 (B) hours after stimulation with or without STS, qPCR was performed showing the n-fold expression levels of MMP-2 compared to the appropriate controls (n-fold expression = 1). GAPDH served as an en- dogenous control. All data are presented as mean ± S.E.M. * p-value ≤ 0.05 significantly higher compared to the ap- propriate control; # p-value ≤ 0.05 significantly higher compared to the appropriate IL-1β stimulated cells without STS; Figure 4. Effect of static orthodontic tensile forces on the IL-1β-induced gene expression of MMP-2 in hPDL-MSCs in the Figure& p-value 4. absenceEffect ≤ 0.05 of ofsignificantly FBS.static STS orthodontic with lower 6% equibiaxial compared tensile elongation forces to the on was appropriate the applied IL-1 toβ-induced untreated Il-1β stimulated or gene IL-1β -treatedexpression cells hPDL-MSCs without of MMP-2 STS. in the absencein hPDL-MSCs of in the absence of FBS. STS with 6% equibiaxial elongation was applied to untreated or IL-1β-treated hPDL-MSCs in the FBS. 6 (A) and 24 (B) hours afterApplying stimulation STS with for or 6 withouth caused STS, a qPCR decrease was performed in IL-1β showing induced the MMP-2n-fold expression gene expression, MMP-2 n GAPDH absence oflevels FBS. of 6 (A) andcompared 24 (Bshowing) hours to the appropriate aftera significant stimulation controls decline ( with-fold in expressionor the without presence = 1).STS, of qPCR 5 ng/mLserved was asperformedIL-1 an endogenousβ (Figure showing 4A). control. After the n 24-fold h, All data are presented as mean ± S.E.M. * p-value ≤ 0.05 significantly higher compared to the appropriate control; # p-value expression levels of MMP-2 comparedSTS strengthened to the appropriate IL-1β induced controls MMP-2 (n-fold gene expression expression = in1). a GAPDHconcentration-dependent served as an en- dogenous≤ control.0.05 significantly All data higher are comparedpresented to theas mean appropriate ± S.E.M. IL-1β stimulated* p-valuecells ≤ 0.05 without significantly STS; & p-value higher≤ 0.05 compared significantly to the ap- lower compared to the appropriatemanner, showing Il-1β stimulated a significant cells without rise STS. in the presence of 1 ng/mL IL-1β (Figure 4B). propriate control; # p-value ≤ 0.05 significantlyFigure 5 shows higher the comparedMMP-2 gene to the expression appropriate in hPDL-MSCsIL-1β stimulated in the cells presence without of STS; 2% & p-value ≤ 0.05 significantly lowerFBS, 6 compared andFigure 245 hshows after to the theapplying appropriateMMP-2 STSgene Il-1in expression theβ stimulated presence in hPDL-MSCs of cells different without in theIL-1 STS. presenceβ concentrations. of 2% FBS, STS 6 and 24 h after applying STS in the presence of different IL-1β concentrations. STS alone aloneApplying had no effectSTS for on 6 hMMP-2 caused gene a decrease expression in IL-1 (Figureβ induced 5A,B). MMP-2 After gene 6 h expression,incubation, MMP-2had no gene effect expression on MMP-2 genewas expressionsuppressed (Figure by both5A,B). IL-1 Afterβ 6concentrations, h incubation , MMP-2 showinggene a sig- showingexpression a significant was suppressed decline by bothin the IL-1 presenceβ concentrations, of 5 ng/mL showing IL-1 aβ significant (Figure suppression4A). After 24 h, nificant suppression in the presence of 1 ng/mL IL-1β (Figure 5A). No effect of IL-1β on STSin strengthened the presence of IL-1 1 ng/mLβ induced IL-1β (Figure MMP-25A). gene No effect expression of IL-1 β inon aMMP-2 concentration-dependentgene expression manner,MMP-2was observed gene showing expression after a significant 24 h was incubation observed rise in (Figure the after presence5B). 24 Onh in protein ofcubation 1 ng/mL level, (Figure IL-1 ββ -induced5B).(Figure On 4B).protein MMP-2 level, IL-1productionβFigure-induced 5 wasshows MMP-2 not detectable.the production MMP-2 gene was expressionnot detectable. in hPDL-MSCs in the presence of 2% FBS, 6 and 24 h after applying STS in the presence of different IL-1β concentrations. STS alone had no effect on MMP-2 gene expression (Figure 5A,B). After 6 h incubation, MMP-2 gene expression was suppressed by both IL-1β concentrations, showing a sig- nificant suppression in the presence of 1 ng/mL IL-1β (Figure 5A). No effect of IL-1β on MMP-2 gene expression was observed after 24 h incubation (Figure 5B). On protein level, IL-1β-induced MMP-2 production was not detectable.

FigureFigure 5. Effect 5. Effect of static of static orthodontic orthodontic tensile tensile forces forces on on MMP-2MMP-2 geneexpression expression in in hPDL-MSCs hPDL-MSCs in the in presencethe presence of IL-1 ofβ IL-1andβ and 2% FBS.2% FBS.STS STSwith with 6% 6%equibiaxial equibiaxial elongation elongation waswas applied applied to to untreated untreated or IL-1 or βIL-1-treatedβ-treated hPDLSCs hPDLSCs in the presence in the presence of 2% FBS. of 2% FBS. 66 (A)) andand 24 24 (B ()B hours) hours after after stimulation stimulation with orwith without or without STS, qPCR STS, was qPCR performed was performed showing the showingn-fold expression the n-fold levels expression of levelsMMP-2 of MMP-2compared compared to the to appropriate the appropriate controls controls (n-fold expression (n-fold expression = 1). GAPDH = 1).served GAPDH as an served endogenous as an control.endogenous All data control. are presented as mean ± S.E.M. * p-value ≤ 0.05 significantly higher compared to the appropriate control; # p-value ≤ 0.05 significantly higher compared to the appropriate IL-1β stimulated cells without STS.

Figure 5. Effect of static orthodontic tensile forces on MMP-2 gene expression in hPDL-MSCs in the presence of IL-1β and 2% FBS. STS with 6% equibiaxial elongation was applied to untreated or IL-1β-treated hPDLSCs in the presence of 2% FBS. 6 (A) and 24 (B) hours after stimulation with or without STS, qPCR was performed showing the n-fold expression levels of MMP-2 compared to the appropriate controls (n-fold expression = 1). GAPDH served as an endogenous control.

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All data are presented as mean ± S.E.M. * p-value ≤ 0.05 significantly higher compared to the appropriate control; #

pInt.-value J. Mol. ≤ Sci.0.052021 significantly, 22, 1027 higher compared to the appropriate IL-1β stimulated cells without STS. 8 of 18

STS counteracted the suppressive effect of IL-1β on MMP-2 gene expression, causing a significant increase after 6 h incubation (Figure 5A). After 24 h incubation, STS caused β MMP-2 no effectsSTS on counteracted IL-1β-induced the suppressive MMP-2 gene effect expression of IL-1 on (Figure 5B).gene expression, causing a significant increase after 6 h incubation (Figure5A). After 24 h incubation, STS caused no effects on IL-1β-induced MMP-2 gene expression (Figure5B). 2.5. TIMP-1 Expression 2.5.Figure TIMP-1 6 shows Expression the TIMP-1 gene expression in hPDL-MSCs in the absence of FBS, 6 and 24 Figureh after6 applyingshows the STSTIMP-1 in thegene absence expression or presence in hPDL-MSCs of different in the IL-1 absenceβ concentrations. of FBS, In the6 and absence 24 h after of applyingIL-1β, STS STS did in thenotabsence affect TIMP-1 or presence gene of expression different IL-1 afterβ concentrations. both 6- and 24-h incubationIn the absence (Figure of IL-16A,B).β, STS IL-1 didβ alone not affect enhancedTIMP-1 TIMP-1gene expression gene expression after both after 6- and 6- 24-hand 24-h incubation (Figure6A,B). IL-1 β alone enhanced TIMP-1 gene expression after 6- and 24-h incubation, causing a significant increase in the presence of 5 ng/mL IL-1β at both time incubation, causing a significant increase in the presence of 5 ng/mL IL-1β at both time pointspoints and and a concentration-dependent a concentration-dependent riserise 24 24 h h after after starting starting the the treatment treatment (Figure (Figure6A,B). 6A,B). OnOn protein protein level, level, IL-1 IL-1ββ-induced-induced TIMP-1 production production was was not not detectable. detectable.

Figure 6. Effect of static orthodontic tensile forces on the IL-1β-induced gene expression of TIMP-1 in hPDL-MSCs in the Figure 6. Effectabsence of of FBS.static STS orthodontic with 6% equibiaxial tensile elongation forces on was the applied IL-1 toβ-induced untreated or gene IL-1β -treatedexpression hPDL-MSCs of TIMP-1 in the absencein hPDL-MSCs of in the absenceFBS. of 6 FBS. (A) and STS 24 (Bwith) hours 6% after equibiaxial stimulation elongation with or without was STS, applied qPCR was to performed untreated showing or IL-1 theβn-treated-fold expression hPDL-MSCs levels in the absence ofof FBS.TIMP-1 6 (comparedA) and 24 to ( theB) appropriatehours after controls stimulation (n-fold expression with or =without 1). GAPDH STS,served qPCR as an was endogenous performed control. showing All data the n-fold expressionare levels presented of TIMP-1 as mean ± comparedS.E.M. * p-value to the≤ 0.05 appropriate significantly highercontrols compared (n-fold to theexpression appropriate = control;1). GAPDH & p-value served≤ 0.05 as an en- β dogenoussignificantly control. All lower data compared are presented to the appropriate as mean Il-1 ± S.E.M.stimulated * p cells-value without ≤ 0.05 STS. significantly higher compared to the ap- propriate control; & p-value ≤ 0.05 significantlyIn the presence lower of IL-1 comparedβ, applying to the STS appropriate significantly decreased Il-1β stimulatedTIMP-1 gene cells expression without STS. after 6 h incubation. A significant suppression was also observed in the presence of In the presence of IL-1β, applying STS significantly decreased TIMP-1 gene expres- 5 ng/mL IL-1β after 24 h incubation (Figure6A,B). sion afterFigure 6 h 7incubation. shows the TIMP-1A significantgene expression suppressi inon hPDL-MSCs was also observed in the presence in the of presence 2% FBS, of 5 ng/mL6 and IL-1 24 hβ afterafter applying 24 h incubation STS in the (Figure absence 6A,B). or presence of different IL-1β concentrations. InFigure the absence 7 shows of IL-1 theβ, STSTIMP-1 had no gene significant expression effect onin TIMP-1hPDL-MSCsgene expression in the presence in hPDL- of 2% FBS,MSCs 6 and at both24 h timeafter points applying (Figure STS7A,B). in the Both absence IL-1 β concentrationsor presence of showed different no influenceIL-1β concen- trations.on TIMP-1 In thegene absence expression of IL-1 afterβ, STS 6 hincubation had no significant (Figure7A). effect After on 24 TIMP-1 h incubation, gene IL-1expressionβ in hPDL-MSCsalone enhanced atTIMP-1 both timegene points expression, (Figure causing 7A,B). a significant Both IL-1 increaseβ concentrations with 1 ng/mL showed IL-1β no influence(Figure 7onB). TIMP-1 On protein gene level, expression IL-1 β-induced after 6 TIMP-1 h incubation production (Figure was not7A). detectable. After 24 h incuba- Applying STS in the presence of IL-1β caused a significant increase in TIMP-1 gene tion, IL-1β alone enhanced TIMP-1 gene expression, causing a significant increase with 1 expression in the presence of both IL-1β concentrations after 6 h incubation (Figure7A). 24 h ng/mLafter applyingIL-1β (Figure STS to 7B). IL-1 βOntreated protein hPDL-MSCs, level, IL-1TIMP-1β-inducedgene expression TIMP-1 wasproduction significantly was not detectable.enhanced at 5 ng/mL IL-1β (Figure7B).

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 18

Int.Int. J. J. Mol. Mol. Sci. Sci. 20212021,, 2222,, x 1027 FOR PEER REVIEW 9 9of of 18 18

Figure 7. Effect of static orthodontic tensile forces on TIMP-1 gene expression in hPDL-MSCs in the presence of IL-1β and 2% FBS. STS with 6% equibiaxial elongation was applied to untreated or IL-1β-treated hPDL-MSCs in the presence of 2% FBS. 6 (A) and 24 (B) hours after stimulation with or without STS, qPCR was performed showing the n-fold expression levels of TIMP-1 compared to the appropriate controls (n-fold expression = 1). GAPDH served as an endogenous control. All data are presented as mean ± S.E.M. * p-value ≤ 0.05 significantly higher compared to the appropriate control; # pFigure-valueFigure ≤7. 7. 0.05 EffectEffect significantly of of static static orthodontic orthodontic higher compared tensile tensile forces forcesto the on onappropriate TIMP-1TIMP-1 genegene IL-1 expression expressionβ stimulated in cellshPDL-M hPDL-MSCs withoutSCs inSTS. the presence of IL-1 β andand 2%2% FBS. FBS. STS STS with with 6% 6% equibiaxial equibiaxial elonga elongationtion was was applied applied to to untreated untreated or or IL-1 IL-1ββ-treated-treated hPDL-MSCs hPDL-MSCs in in the the presence presence of of 2% 2% FBS.FBS. 6 (A)) andand 2424 ( B(B)) hours hours after after stimulationApplying stimulation STS with with in or or withoutthe without presence STS, STS, qPCR of qPCR IL-1 was βwas performed caused performed a showingsignificant showing the nincrease -foldthe n-fold expression in expression TIMP-1 levels gene levelsof TIMP-1 of TIMP-1compared compared to theexpression appropriateto the appropriate in controls the presencecontrols (n-fold ( expressionn of-fold both expression IL-1 = 1).βGAPDH concentrations = 1). GAPDHserved asserved after an endogenous as6 han incubation endogenous control. (Figure control. All data 7A). Allare presenteddata are presented as mean ± asS.E.M. 24mean h *after ±p -valueS.E.M. applying≤ * 0.05p-value significantlySTS ≤ to0.05 IL-1 significantly higherβ treated compared hPDL-MSCs,higher to compar the appropriate TIMP-1ed to the gene control; appropriate expression # p-value control;≤ was0.05 # sig- p-value ≤ 0.05 significantly higher compared to the appropriate IL-1β stimulated cells without STS. significantly higher comparednificantly to the appropriateenhanced at IL-1 5 βng/mLstimulated IL-1β cells (Figure without 7B). STS. Applying STS in the presence of IL-1β caused a significant increase in TIMP-1 gene 2.6. IL-1β Expression expression2.6. IL-1β Expression in the presence of both IL-1β concentrations after 6 h incubation (Figure 7A). Figure 8 shows the IL-1β gene expression in hPDL-MSCs in the absence of FBS, 6 and 24 h afterFigure applying8 shows theSTS IL-1to IL-1β geneβ treated expression hPDL-MSCs, in hPDL-MSCs TIMP-1 in gene the absence expression of FBS, was 6 sig- and 24 h after applying STS in the absence and presence of different IL-1β concentrations. nificantly24 h after enhanced applying at STS 5 ng/mL in the IL-1 absenceβ (Figure and 7B). presence of different IL-1β concentrations. Applying STS alone caused no detectable IL-1β gene expression in hPDL-MSCs, neither Applying STS alone caused no detectable IL-1β gene expression in hPDL-MSCs, neither after 6- nor after 24-h incubation. After 6- and 24-h stimulation, IL-1β alone caused a 2.6.after IL-1 6-β nor Expression after 24-h incubation. After 6- and 24-h stimulation, IL-1β alone caused a concentration-dependent increase in IL-1β gene expression (Figure 8A,B). Changes in concentration-dependentFigure 8 shows the IL-1 increaseβ gene in expressionIL-1β gene in expression hPDL-MSCs (Figure in the8A,B). absence Changes of FBS, in IL-16 andβ IL-1β induced IL-1β protein expression were not possible to detect due to the high IL-1β 24induced h after IL-1 applyingβ protein STS expression in the absence were notand possible presence to detectof different due to IL-1 theβ high concentrations. IL-1β levels levels used for cell stimulation. Applyingused for cell STS stimulation. alone caused no detectable IL-1β gene expression in hPDL-MSCs, neither after 6- nor after 24-h incubation. After 6- and 24-h stimulation, IL-1β alone caused a concentration-dependent increase in IL-1β gene expression (Figure 8A,B). Changes in IL-1β induced IL-1β protein expression were not possible to detect due to the high IL-1β levels used for cell stimulation.

FigureFigure 8. 8. EffectEffect of of static static orthodontic orthodontic tensile tensile forces forces on on the the IL-1 IL-1ββ-induced-induced gene gene expression expression of of IL-1IL-1ββ inin hPDL-MSCshPDL-MSCs in in the the absence absence of ofFBS. FBS. STS STS with with 6% 6% equibiaxial equibiaxial elongation elongation was was applied applied to untreated to untreated or IL-1orβ IL-1-treatedβ-treated hPDL-MSCs hPDL-MSCs in the in absence the absence of FBS. of FBS.6 (A 6) and (A) and24 (B 24) hours (B) hours after after stimulation stimulation with with or or without STS, qPCR was performed showing the relative IL-1β expression levels normalized to the internal reference gene GAPDH. All data are presented as mean ± S.E.M. # p-value ≤ 0.05 Figure 8. Effect of static orthodontic tensile forces on the IL-1β-induced gene expression of IL-1β in significantly higher compared to the appropriate IL-1β stimulated cells without STS; § p-value ≤ 0.05 hPDL-MSCs in the absence of FBS. STS with 6% equibiaxial elongation was applied to untreated or β IL-1significantlyβ-treated higherhPDL-MSCs compared in the to 1absence ng/mL of IL-1 FBS.in 6 the(A) presenceand 24 (B of) STS.hours after stimulation with or

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 18

without STS, qPCR was performed showing the relative IL-1β expression levels normalized to the internal reference gene GAPDH. All data are presented as mean ± S.E.M. # p-value ≤ 0.05 signifi- Int. J. Mol. Sci. 2021, 22, 1027 10 of 18 cantly higher compared to the appropriate IL-1β stimulated cells without STS; § p-value ≤ 0.05 sig- nificantly higher compared to 1 ng/mL IL-1β in the presence of STS.

6 6h h after after applying applying STS inin thethe presencepresence of of IL-1 IL-1ββ, a, a strengthening strengthening of ofIL-1 IL-1β geneβ gene expres- ex- pressionsion in a in concentration-dependent a concentration-dependent manner ma wasnner observed, was observed, causing a causing significant a highersignificantIL-1β highergene expressionIL-1β geneat expression 5 ng/mL IL-1at 5 βng/mLcompared IL-1β to compared 1 ng/mL (Figureto 1 ng/mL8A). (Figure After 24 8A). h incubation, After 24 hSTS incubation, caused a STS significant caused increasea significant in IL-1 increaseβ induced in IL-1IL-1ββ inducedgene expression, IL-1β gene independently expression, independentlyfrom the present from IL-1 theβ concentrationspresent IL-1β concentrations (Figure8B). (Figure 8B). FigureFigure 99 showsshows the the IL-1IL-1β βgenegene expression expression in in hPDL-MSCs hPDL-MSCs in in the the presence presence of of 2% 2% FBS, FBS, 6-6- and and 24-h 24-h afterafter applying applying STS STS in in the the absence absence or or the the presence presence of of different different IL-1 IL-1ββ concen-concen- trations.trations. STS STS alone alone caused caused no no detectable detectable IL-1IL-1β βgenegene expression expression in in hPDL-MSCs, hPDL-MSCs, neither neither afterafter 6- 6- nor nor after after 24-h 24-h incubation incubation (Figure (Figure 9A,B).9A,B). After 6 h stimulation, IL-1 ββ causedcaused aa concentration-dependentconcentration-dependent increase increase in in IL-1IL-1β βgenegene expression expression (Figure (Figure 9A).9A). 24 24 h h incubation withwith IL-1 IL-1ββ alonealone led led to to an an overall overall lower lower IL-1IL-1β βgenegene expression expression levels, levels, showing showing the the highest highest levellevel at at 1 1ng/mL ng/mL IL-1 IL-1β β(Figure(Figure 9B).9B).

FigureFigure 9. 9. EffectEffect of of static static orthodonti orthodonticc tensile tensile forces forces on on the the IL-1 IL-1ββ-induced-induced gene gene expression expression of of IL-1IL-1ββ inin hPDL-MSCshPDL-MSCs in in the the presence ofof 2%2% FBS. FBS. STS STS with with 6% 6% equibiaxial equibiaxial elongation elongation was was applied applied to untreated to un- treatedor IL-1 orβ-treated IL-1β-treated hPDL-MSCs hPDL-MSCs in the in presence the presence of 2% of FBS. 2% FBS. 6 (A )6 and (A) 24and (B 24) hours (B) hours after after stimulation stimu- lation with or without STS, qPCR was performed showing the relative IL-1β expression levels with or without STS, qPCR was performed showing the relative IL-1β expression levels normalized normalized to the internal reference gene GAPDH. All data are presented as mean ± S.E.M. # to the internal reference gene GAPDH. All data are presented as mean ± S.E.M. # p-value ≤ 0.05 p-value ≤ 0.05 significantly higher compared to the appropriate IL-1β stimulated cells without STS; β ≤ § significantlyp-value ≤ 0.05 higher significantly compared higher to the compared appropriate to 1 IL-1 ng/mLstimulated IL-1β in cellsthe presence without STS;of STS. § p -value 0.05 significantly higher compared to 1 ng/mL IL-1β in the presence of STS. Applying STS in the presence of 5 ng/mL IL-1β caused an increase in IL-1β gene ex- Applying STS in the presence of 5 ng/mL IL-1β caused an increase in IL-1β gene pression after 6 h incubation. A significantly higher IL-1β gene expression was observed expression after 6 h incubation. A significantly higher IL-1β gene expression was observed at 5 ng/mL compared to 1 ng/mL IL-1β (Figure 9A). After 24 h incubation, STS increased at 5 ng/mL compared to 1 ng/mL IL-1β (Figure9A). After 24 h incubation, STS increased IL-1β induced IL-1β gene expression, showing a significant increase at 5 ng/mL (Figure IL-1β induced IL-1β gene expression, showing a significant increase at 5 ng/mL (Figure9B). 9B). 3. Discussion 3. Discussion One of the major goals in orthodontic research is to reduce the treatment time by acceleratingOne of the OTM. major Nevertheless, goals in orthodontic the OTM velocityresearch is is limited to reduce by biological the treatment processes time andby acceleratingnumerous possibilitiesOTM. Nevertheless, are currently the OTM under veloci investigationty is limited to by speed biological up OTM processes via affecting and numerousthese biological possibilities pathways are currently [2]. A huge under number investigation of in vitro to speedand inup vivo OTMstudies via affecting already thesedemonstrate biological the pathways involvement [2]. A of hPDL-MSCshuge number in of PDL in vitro ECM and and bonein vivo remodeling studies already during demonstrateOTM (reviewed the involvement in [18,51–53]). of WithinhPDL-MSCs this big in data PDL pool, ECM multiple and bone studies remodeling investigate during the OTMinfluence (reviewed of orthodontic in [18,51–53]). forces Within on MMPs this big and data TIMPs pool, within multiple the studiesPDL [5 investigate,23–27,30,36 the,37 ]. influenceSeveral ofof these orthodontic studies forces already on evaluated MMPs and the TIMPs effect ofwithin orthodontic the PDL forces [5,23–27,30,36,37]. on MMPs and TIMPs under inflammatory conditions within the PDL [36,43,46]. All of these studies used a cyclic tensile strain application in vitro, mimicking cyclic orthodontic forces. Such cyclic forces are generated by multibracket appliance in combination with occlusal forces [47]. However, cyclic tensile strain does not reflect orthodontic forces caused by nickel-titanium Int. J. Mol. Sci. 2021, 22, 1027 11 of 18

coil springs [25,48,49]. In this case, static tensile strain is a more appropriate model and therefore, we used it in our in vitro study. The discrimination between these two applica- tion forms seems to be essential in vitro, since several studies demonstrated significantly different effects on ECM remodeling enzymes in the human PDL [29,54]. In this study, we demonstrated for the first time that static forces during OTM may favor a more inflamma- tory and destructive local environment at the tension zone within the PDL when applying low-magnitude forces. It is well established that cyclic tensile strain executes its function on hPDL-MSCs under inflammatory conditions in a dose-dependent manner [43,55]. High magnitudes increase the expression of pro-inflammatory cytokines, whereas low magnitudes (3–6%) act in an anti-inflammatory manner. Moreover, the application of cyclic tensile strain at low magnitudes results in a downregulated expression of MMPs under inflammatory condi- tions and an increased expression of TIMPs. This indicates that low force magnitudes could be a reason for bone and PDLs’ ECM formation at the tension site during OTM in the pres- ence of inflammatory stimuli, which normally favor bone and PDL destruction [43,55]. To ensure the comparability of our findings with the two studies of Long et al. [43,55], we used the same low force magnitude (6%). In contrast to these studies, our data demonstrated opposite effects of mechanical forces on MMPs and TIMPs expression in hPDL-MSCs under inflammatory conditions, using tensile forces in a static mode. This indicates that the influence of tensile strains under inflammatory conditions on hPDL-MSCs also depends on its application form. Our data showed that STS with 6% elongation alone had no influence on the hPDL- MSCs viability and on the MMP-1, MMP-2 and TIMP-1 expression. The influence of in vitro simulated orthodontic forces on the expression of multiple MMPs and TIMPs within the human PDL has been investigated in multiple studies, showing rather inconsistent results [5,8,25,28,30,56]. Our data are in accordance with the findings of Long et al. [43], who used the same force magnitude and comparable incubations times, but a cyclic tensile force application form. This indicates that applied orthodontic forces have no effects on the production of ECM remodeling enzymes within the tension zone of the human PDL in the absence of local aseptic inflammation. However, other studies demonstrated clear changes in the expression of MMP-1, MMP-2 and TIMP-1 in both directions when applying STS alone [8,25,30]. This inconsistency may result from the use of variable combinations of different parameters, such as force types (compression versus pressure), force magnitudes, application forms (static versus cyclic) and incubation times. Since the effects of orthodontic forces on hPDL-MSCs are influenced by local aseptic inflammation, which is a prerequisite for OTM [2], we simulated this condition by adding different concentrations of IL-1β. Several clinical studies demonstrated that IL-β is one of the most abundant pro-inflammatory cytokines within the GCF during the initial OTM phase, at both the tension and the compression sites [38,39]. The chosen IL-1β concen- trations and incubation times are in the range of previous studies, in which the authors used concentrations and time points ranging from 0.1 ng/mL to 10 ng/mL and from 4 to 48 h, respectively, to investigate the influence of IL-1β on the production of various MMPs [41–43]. In the absence of STS, our experiments demonstrated that both IL-1β concentrations significantly induce the production of MMP-1 and MMP-2 in hPDL-MSCs in a time- dependent manner. This is in accordance with other studies, which also showed IL-1β induced expression of MMP-1, MMP-2 and MMP-3 [41–43]. These data, together with the fact that STS alone does not influence MMPs expression, suggest that the local aseptic inflammation is the major key for ECM remodeling within the PDL. Our results also showed that MMP-1 expression was markedly stronger induced by both IL-1β concentrations than MMP-2 expression. On protein levels, MMP-2 was not detectable by ELISA. This finding is in accordance with Abe et al. [57], who conducted a study with gingival fibroblasts and observed that IL-1β induces a much higher expression of MMP-1 and that MMP-2 is also not detectable on protein level. Int. J. Mol. Sci. 2021, 22, 1027 12 of 18

IL-1β also regulates the breakdown of PDL collagens by affecting the expression of TIMPs, such as TIMP-1 and TIMP-2 [43], which are inhibitors of MMPs’ enzymatic activities [58]. Our experiments showed a stimulating effect of IL-β on the expression of TIMP-1 by hPDL-MSCs in a concentration of 5 ng/mL, but not of 1 ng/mL. This corresponds to the data of Long et al. [43], who did not detect any effect of 1 ng/mL IL-β on TIMP-1 expression. This indicated that higher local IL-1β levels have to be reached within the PDL to influence TIMP-1 expression, whereas MMPs’ expression can be affected already by lower IL-1β concentrations. Hence, it can be suggested that the aseptic inflammation, especially the presence of IL-1β, possesses a dual role during OTM: low IL-1β levels at the beginning of inflammation favor PDL matrix degradation by increasing MMP-1 and MMP-2, whereas higher IL-1β levels at a later inflammation state additionally induces TIMP-1 expression and counteract MMPs-dependent matrix degradation. More importantly, for the first time, we showed an enhancement of the IL-1β-induced MMP-1 and MMP-2 expression after applying STS for 24 h. It can be excluded that these increased MMPs levels result from an enhanced endogenous IL-1β production since high amounts of exogenous IL-1β were present during the experiment. Hence, it seems that tensile strain with 6% elongation, which was applied statically, favors ECM degradation within the PDL under inflammatory conditions. Our TIMP-1 expression data support this conclusion, showing a negative effect of STS on IL-1β-triggered TIMP-1 expression, which should favor MMPs’ activity. However, these data are different from Long et al. [43], show- ing an opposite effect of cyclic tensile strain with 6% elongation on IL-1β-induced MMPs and TIMPs production. Hence, it can be suggested that besides force magnitudes [43], the force application form could be a critical parameter that determines the effect of tensile strains on the production of MMPs and TIMPs in hPDL-MSCs and consequently on the ECM remodeling. However, it should be kept in mind that ECM remodeling is an inter- action between numerous MMPs, TIMPs and other proteolytic enzymes [5,8,19], and not only the parameters investigated in this study. In the second part of this study, for the first time, we directly compared the influence of 2% FBS on the STS-based effects on IL-1β induced expression of MMP-1, MMP-2, TIMP-1 and IL-1β. This is especially important since FBS is known to affect hPDL-MSCs during in vitro cultivation [50]. Here we demonstrated that the hPDL-MSCs’ viability and the basal expression of investigated target genes are not affected by 2% FBS. In contrast, 2% FBS mainly caused a lower IL-1β-induced expression of MMP-1, MMP-2 and TIMP-1, although IL-1β gene expression was clearly higher than in the absence of FBS. Most importantly, 2% FBS caused, on the one hand, lower and, on the other hand, opposite effects of STS on IL-1β-induced target gene expression. Hence, it is especially important to keep in mind the possible effects of FBS on MMPs and TIMPs synthesis in hPDL-MSCs when interpreting such results. Studies, which investigate MMPs and TIMPs expression in hPDL-MSCs, are very heterogeneous concerning the use of FBS while applying mechanical load on the cells. Numerous studies exist which use a broad range of FBS (1–10%) [24,28,42,46,55,56], whereas others do not use any FBS [5,25,41,43]. Additionally, multiple studies do not specify if FBS is added to cell culture during the experimental procedure [8,30,59,60]. This heterogeneity of FBS usage could be, besides using various combinations of force type, magnitude, application form and duration, another reason for the inconsistent results between several studies. Extrapolating our results into the clinical situation suggests that not only the applied force magnitude but also the force application form (static versus cyclic) seems to be crucial for successful OTM. For example, nickel-titanium coil springs cause static forces during OTM [48,49]. Based on our results, it is possible that such static forces favor a pro-inflammatory environment at the tension site when applying low-magnitudes and, consequently, osteoclastogenesis [2,40]. Our results also indicate that such static forces may favor ECM degradation at the tension zone within the PDL. Hence, it is possible that nickel-titanium coil springs cause, at least at low-magnitude forces, a bone and PDLs’ ECM destruction, rather than the desired composition. On the contrary, multibracket appliance, Int. J. Mol. Sci. 2021, 22, 1027 13 of 18

together with occlusal forces, cause a cyclic orthodontic load [25,47], possibly having anti-inflammatory and osteogenic effects in the tension zone when using low-magnitude forces. Additionally, these cyclic tensile forces may inhibit ECM degradation in the tension area within the PDL [43]. Hence, when choosing materials for malocclusion treatment, orthodontists should be aware of their possible variable effects due to different force magnitudes and static and cyclic tensile forces caused by different materials [25,43,47–49]. Further, the choice of right orthodontic appliances may be especially important in adult patients, in which orthodontic treatment is a risk factor for periodontitis [61,62], since both have comparable molecular mechanisms and cause extensive PDL and alveolar bone remodeling [2,63,64]. However, it should be considered that periodontitis is characterized by a dysbiotic state and impaired host-microbe homeostasis [65–67].

4. Materials and Methods 4.1. Cell Culture Third molars were extracted from periodontally healthy individuals due to orthodon- tic reasons and were used to isolate primary hPDL-MSCs. After the outgrowth of these cells from periodontal ligament slices, which were scrapped off from the mid-third of the tooths’ roots, hPDL-MSCs were cultivated in Dulbecco’s modified Eagle’s Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA), which was supplemented with 100 U/mL penicillin, 50 µg/mL streptomycin (both Sigma-Aldrich, St. Louis, MO, USA) and 10% FBS (Gibco, Carlsbad, CA, USA). hPDL-MSCs were cultured at 95% humidity, 5% carbon dioxide and at 37◦ Celsius. Passage levels five to seven were used for all conducted experiments. The mesenchymal stromal cell character of isolated primary hPDL-MSCs was verified according to the minimal criteria for mesenchymal stromal cells defined by the International Society for Cell and Gene Therapy (ISCT) [11]. The expression of hematopoietic and mesenchymal stromal cell surface markers was evaluated by immunostaining, as described in our previous studies [68,69]. The Ethics Committee of the Medical University of Vienna (EK Nr. 1079/2019, date of approval: 18 March 2019) approved the study protocol. All participating individuals got informed before the extraction procedure and gave their written consent. All experimental methods throughout the study were conducted according to the Declaration of Helsinki and the Good Scientific Practice Guidelines of the Medical University of Vienna.

4.2. Stimulation Protocol 50,000 hPDL-MSCs were seeded per well in 3 mL DMEM medium supplemented with 10% FBS, 100 U/mL penicillin and 50 µg/mL streptomycin at collagen type I coated 6-well BioFlex® plates (FlexCell International Cooperation, Burlington, VT, USA). After 24 h incubation, cell cycles were synchronized by changing medium to DMEM without any FBS, supplemented with only 100 U/mL penicillin and 50 µg/mL streptomycin. Af- ter overnight-starvation, hPDL-MSCs were stimulated with 1 ng/mL or 5 ng/mL IL-1β (Invivogen, San Diego, CA, USA) using DMEM medium supplemented with either only antibiotics or with antibiotics and 2% FBS. Appropriate unstimulated hPDL-MSCs served as control. Simultaneously, STS was applied to hPDL-MSCs using the FlexCell FX-5000T™ Tension System (FlexCell International Cooperation, Burlington, VT, USA) with 6% equib- iaxial elongation. IL-1β stimulated and unstimulated hPDL-MSCs, which were seeded on the same cell culture plate without applying STS served as references. 6 and 24 h later, MMP-1, MMP-2, TIMP-1 and IL-1β gene and protein expression levels were measured by quantitative polymerase chain reaction (qPCR) and ELISA, respectively. Additionally, cell viability was determined by live/dead staining followed by fluorescence microscopy 24 h after stimulation.

4.3. Cell Viability Cell viability was determined 24 h after stimulation by live/dead staining using the Live-Dead Cell Staining Kit from Enzo (Enzo, Framingdale, NY, USA). After washing the Int. J. Mol. Sci. 2021, 22, 1027 14 of 18

adherent cells in cell culture plates with 1xPBS (phosphate buffered saline) three times, 1 mL staining solution (1 µL solution A + 1 µL solution B in 1 mL staining buffer) were added per well. After the incubation at 37 ◦C for 15 min, the fluorescence of green fluorescent Live-Dye™ (Ex/Em = 488/518 nm) and propidium iodide (Ex/Em = 488/515 nm) were determined by ECHO Revolve fluorescence microscope (Echo, San Diego, CA, USA) using four-fold magnification and an exposure time of 670 ms for both fluorescence dyes.

4.4. Quantitative Polymerase Chain Reaction After cell harvesting, TaqMan Gene Expression Cells-to-CT kit (Applied Biosystems, Foster City, CA, USA) was used to prepare cell lysates, to reverse transcribe mRNA into cDNA and to perform qPCR. For reverse transcription, samples were heated to 37 ◦C for 1 h followed by 95 ◦C for 5 min using Primus 96 advanced thermocycler (PeqLab/VWR, Darmstadt, Hessen, Germany). qPCR was performed in paired reactions heating the sam- ples to 1 × 95 ◦C for 10 min followed by 50 cycles of 15 s at 95 ◦C and 1 min at 60 ◦C. Target genes were amplified, and their expression levels were detected by a QuantStudio 3 device (Applied Biosystems, Foster City, CA, USA) using the following TaqMan Gene Expression Assays (all from Applied Biosystems, Foster City, CA, USA): MMP-1, Hs00899658_m1; MMP-2, Hs01548727_m1; TIMP-1, Hs99999139_m1; IL-1β, Hs01555410_m1 and GAPDH, Hs99999905_m1. After determining Ct values, the n-fold expression of MMP-1, MMP- 2 and TIMP-1 compared to the appropriate controls was calculated using the 2-∆∆Ct method. The housekeeping gene GAPDH served as an endogenous reference. IL-1β gene expression levels are shown relative to GADPH expression without normalization to the appropriate control.

4.5. Immunoassay MMP-1 protein levels were determined in conditioned media, which were harvested 6 and 24 h after stimulation. MMP-1 protein levels were evaluated by using Human MMP-1 ELISA Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. Provided standards ranged from 24.69 to 18,000 pg/mL. Absorbance was measured at OD450nm and OD570nm (optical density) followed by plotting OD570-subtracted values against the appropriate standard curve to calculate appropriate protein concentra- tions. MMP-2 and TIMP-1 protein production was not detectable in conditioned media.

4.6. Statistical Analysis Data for each experiment were obtained from at least 4 independent repetitions with hPDL-MSCs isolated from at least 4 different individuals. All measured data are presented as mean values ± S.E.M. Statistical analysis was performed using SPSS (Version 24.0, IBM, Armonk, NY, USA). The normal distribution of the data was tested by the Kolmogorov- Smirnov test. For normally distributed data, statistical differences between groups were investigated by one-way analysis of variance (ANOVA) followed by post-hoc LSD test. In the absence of normal distribution, the analysis was performed using the Friedman Test, followed by the Wilcoxon test for pairwise comparison. P-values ≤ 0.05 were considered to be statistically significant.

5. Conclusions In conclusion, this in vitro study demonstrated that applying STS with 6% elongation on hPDL-MSCs increased the expression of MMP-1, MMP-2 and IL-1β under inflammatory conditions. Furthermore, TIMP-1 expression decreased under this experimental setting. These data suggest that compared to a cyclic application, a static application form of low- magnitude forces may favor a more inflammatory and destructive local environment at the tension zone within the PDL. In further studies, these results have to be verified by directly comparing the static and cyclic application form of low-magnitude forces on hPDL-MSCs. Int. J. Mol. Sci. 2021, 22, 1027 15 of 18

Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006 7/22/3/1027/s1. Author Contributions: Conceptualization, C.B., O.A., E.J.; methodology, C.B., M.N. and M.S.; valida- tion, C.B., M.N. and O.A.; formal analysis, C.B., M.N. and A.B.; resources, E.J. and O.A.; data curation, C.B., M.N. and M.S.; writing—original draft preparation, C.B.; writing—review and editing, O.A., M.N., A.B., M.S., X.R.-F. and E.J.; visualization, C.B. and M.N.; supervision, O.A., E.J. and X.R.-F.; project administration, O.A. and E.J. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the Medical University of Vienna (EK Nr. 1079/2019; date of approval: 18 March 2019). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors acknowledge Phuong Quynh Nguyen for excellent technical assistance. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ECM Extracellular matrix OTM Orthodontic tooth movement PDL Periodontal ligament hPDL-MSCs Human periodontal ligament-derived mesenchymal stromal cells MMPs Matrix metalloproteinases GCF Gingival crevicular fluid TIMPs Tissue inhibitor of matrix metalloproteinases IL Interleukin STS Static tensile strain FBS Fetal bovine serum ELISA Enzyme linked immunosorbent assay S.E.M. Standard error of the mean DMEM Dulbecco’s modified Eagles medium ISCT International Society for Cell and Gene Therapy qPCR Quantitative polymerase chain reaction OD Optical density ANOVA One-way analysis of variance

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