Characterization of a periodontal-inflammatory microRNA profile during long-term multibracket orthodontic treatment in adolescents - a split-mouth design study

Characterization of a periodontal-inflammatory microRNA profile during long-term multibracket orthodontic treatment in adolescents - a split-mouth design study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Characterization of a periodontal-inflammatory microRNA profile during long-term multibracket orthodontic treatment in adolescents - a split-mouth design study Florian Rolfes, Johannes Heck, Isabelle Riedel, Christian Bär, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4934654/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract This study aimed to identify functional microRNAs (miRNAs) and their respective targets as central regulatory factors of tooth movement during orthodontic treatment. Gingival crevicular fluid (GCF) of 24 adolescent patients (< 18 years) treated with a full-mouth multibracket appliance (MBA; Thermal Copper Nickel Titanium archwire) was analyzed for miRNAs-21, -29b, -34a, -126, -132, -146a, and -221 in a split-mouth study design. GCF samples were taken from either jaw (second maxillary, mandibular premolar) using non-invasive sampling before, 7 days, 5 weeks, and 3 months after application of orthodontic force (8 samples per patient). Validated miRNA targets and regulated pathways were identified using the miRTarBase database (release 9.0) and Reactome (version 87). All analyzed miRNAs were consistently detected in the GCF (Ct value < 35) and a moderate to high correlation was found between samples taken from the mandible and maxilla before treatment (r = 0.42 to 0.71, all p ≤ 0.041). All miRNAs showed changes in their expression levels with orthodontic tooth movement compared to baseline (significant time effect, all p < 0.001). The general profile indicated an increase in miRNA expression in both jaws with time with the exeption of miR-21, which showed reduced levels one week after MBA application (p = 0.046). For miR-34, a significant interaction effect was observed (time × jaw, p = 0.0396) in that lower levels were found after five weeks and three months of treatment in the mandible compared to the maxilla. The medium to late treatment phase was characterized by an increase in miR-146 and miR-221. Gene signaling pathway analysis suggested regulation of cellular response to stress including hypoxia, matrix reorganization and vascular remodeling. Since the identified miRNA profile was linked to targets involved in the remodeling process of the alveolar supporting apparatus and alveolar bone, GCF-derived miRNAs may represent diagnostic biomarkers to monitor cellular processes during orthodontic tooth movement and potentially optimize individual treatment outcomes. Health sciences/Biomarkers Health sciences/Medical research multi-bracket appliance molecular orthodontics alveolar bone remodeling gingival crevicular fluid mechanical loading extracellular matrix Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Mechanical forces during orthodontic treatment induce a multitude of different, time-dependent physiological pathways. These pathways are related to bone resorption and periodontal ligament (PDL) degeneration in the direction of tooth movement (on the compressive side), and bone apposition and PDL regeneration on the tensile side of the moved tooth (Krishnan and Davidovitch 2006). On the compressive side, the tooth approaches the alveolar bone, causing disruption of blood circulation leading to local hypoxia. The volume of the PDL decreases and fluid is moved from the compressed area via bone canaliculi into the surrounding regions with PDL fibers relaxing. On the tensile side, PDL fibers tighten and the PDL volume increases with fluid influx. This involves not only the direct mechanical pressure of hard tissue as a stimulus but also the fluid pressure in the PDL, evenly distributed due to poroviscoelastic behavior (Maltha and Kuijpers-Jagtman 2023, Najafidoust et al. 2020). This process exposes osteocytes in their lacunae to fluid shear stress, a stimulus that has been suggested as key to activating the osteocyte system (Burger et al. 1995). Regarding to associated bone remodeling, including the resorption of alveolar bone and extracellular matrix, osteoclasts need to be recruited under proinflammatory conditions and angiogenesis is induced for the transport of metabolic products (Bruzzaniti and Baron 2006, Wang et al. 2022). A crucial force-dependent factor for osteoclast function and enhanced recruitment is the Receptor Activator of Nuclear Factor-κB ligand (RANKL). Since the competitive antagonist of RANKL, osteoprotegerin (OPG), inhibits osteoclastogenesis, the RANKL/OPG ratio determines osteoclast formation and the activation of the remodeling process (Theoleyre et al. 2004). On the compressive side, an increase in RANKL levels can be observed in osteoblasts and PDL fibroblasts as early as three hours after force application, while on the tensile side, an increase in OPG expression occurs (Brooks et al. 2009, Garlet et al. 2007, Nishijima et al. 2006, Oshiro et al. 2002). Within twenty-four hours, mechanical stress on the compressive side induces Vascular Endothelial Growth Factor (VEGF), a key angiogenic factor, in fibroblasts and osteoblasts promoting the formation of new blood vessels through proliferation and migration of endothelial progenitor cells (Hu and Olsen 2016, Miyagawa et al. 2009). It has been shown that VEGF in osteoblasts is induced by hypoxia through the Hypoxia-Inducible-Factor-1 (HIF-1), which also directly stimulates RANKL expression in PDL fibroblasts (Kim et al. 2002, Park et al. 2011). As an inflammatory response to mechanical stress, various cytokines are released from PDL cells, including interleukin (IL)-1α, -4, -6, -10, -12, and Tumor Necrosis Factor-alpha (TNF-α), with increased concentrations within 24 hours. Matrix-metalloproteases (MMPs) and the antagonist of MMPs, Tissue Inhibitor of MMP (TIMP), are also released. Together, TNF-α and MMPs, contribute to osteoclast differentiation and, consequently, the direct resorption of alveolar bone (Andrade Jr et al. 2007, Garlet et al. 2007, Uematsu et al. 1996). The time-dependent regulation and overall orchestration of the different involved factors is so far only incompletely understood. Recent research has shown that non-coding RNAs such as microRNAs (miRNAs) play a pivotal role in the signal transduction of mechanical forces induced by tooth movement during orthodontic treatments, as they can act locally and in an endocrine manner, potentially orchestrating changes on the compressive and tensile side (Chang et al. 2015, Pi et al. 2016, Chen and Zhang 2023). miRNAs are small non-coding RNAs (19–25 nucleotides in length) that regulate gene expression at the post-transcriptional level. miRNAs are detectable in virtually all bodily fluids and are expressed in complex networks that enable control of the cellular phenotype (de Gonzalo-Calvo et al. 2019). It has been shown that mechanical stress, orthodontic forces, and hypoxia in the PDL can lead to the regulation of specific miRNAs involved in bone remodeling and inflammatory processes in the periodontium (Asa’ad et al. 2020, Wang et al. 2018). For example, miR-21 was increased in PDL cells and PDL stem cells of adolescent patients (15–18 years old) during one month of orthodontic treatment (80–100 g of force), which was associated with increased osteogenesis and alveolar bone remodeling, likely via the IL-12A axis. However, a detailed and long-term profile of miRNAs induced by mechanical forces during orthodontic treatments is currently not available. Thus, this study aimed to identify a specific set of functional periodontal-inflammatory miRNAs during long-term multibracket orthodontic treatment in healthy adolescent patients. We hypothesized that miRNA alterations would differ over time after treatment initiation depending on associated effectors, providing insight into the involved regulatory pathways. METHODS Study design and orthodontic treatment An interventional split-mouth study design with internal control was used to determine the concentration of miRNAs-21, -29b, -34a, -126, -132, -146a, and -221 in the gingival crevicular fluid (GCF) of adolescent patients (< 18 years) treated with a full-mouth multibracket appliance (MBA). GCF samples were collected from one tooth in either jaw (second maxillary, mandibular premolar) at four planned time points, before application of orthodontic force, after 7 days, after 6 weeks, and after 3 months of the procedure (8 samples per patient). The panel of analyzed miRNAs was the result of an in-depth literature search, considering previous reports on miRNAs affected by orthodontic treatments as well as miRNAs known to be regulated by associated signaling pathways including angiogenesis, hypoxia, etc (Atsawasuwan et al. 2018, Chen et al. 2016, Du et al. 2016, Kumar et al. 2014, Schmitz et al. 2021, Seagraves 2020, Schober et al. 2014, Qi and Zhang 2014, Wang et al. 2022, Wu et al. 2019, Yao et al. 2016). Time points for sample collection were chosen to reflect clinical practice, considering the consensus to change archwires or perform orthodontic checks every 6-8 weeks after start of an orthodontic treatment also covering longer continuous exposure to inital orthodontic force and the complete implementation of the first archwire (Santoro et al. 2001, Uematsu et al. 1996). In detail, a 0.014" Thermal Copper Nickel Titanium archwire (Euroform II G&H Orthodontics, Indiana, USA) was used, applying a recovery force of 78 g at mouth temperature. Three types of brackets (22" slot size, MBT prescription) were used. Patients were treated with either a self-ligating MBA (Genius System Metal, ortho Penthin GmbH, Schwanewede, Germany, 4 participants) or one of two conventionally ligated MBAs (SmartTwin, ortho Penthin GmbH, 16 participants; Iconix Aesthetic Braces, American Orthodontics, Wisconsin, USA, 4 participants). The choice of MBA was independent of study participation and solely based on the decision of the legal guardians to cover costs for additional orthodontic service according to German health insurance regulations (e.g. Thermal Copper Nickel Titanium archewires additionally to stainless steel archwires). Participants and eligibility criteria Self-reported healthy male and female patients (12-18 years of age) undergoing orthodontic treatment with the insertion of a full-mouth MBA involving more than six bracketed and/or banded teeth per jaw were eligible to participate. All methods were carried out in accordance with relevant guidelines and regulations in that written informed consent was obtained from patients and/or their legal guardians. Samples were collected anonymously, without documentation of any personal information approved by the by the local ethical review committee (Ethikkommission Universität Witten-Herdecke). Written informed consent was obtained from patients and/or their legal guardians. Samples were collected anonymously, without documentation of any personal information aligned with the ethics review board of the University of Witten/Herdecke (Ethics Commission University Witten/Herdecke). Sample collection and miRNA quantification Samples were collected as described with modifications (Atsawasuwan et al. 2018, Zhang et al. 2020). Before collecting the GCF, patients were instructed to swallow to avoid sample dilution by saliva. The sampling region was isolated vestibular and, in the lower jaw, in the lingual area using cotton rolls. Tooth were carefully cleared of saliva, especially interdentally, using a dental air syringe. No further cleaning procedures were performed. A PerioPaper (Oraflow Inc., Smithtown, NY, USA) was inserted into the buccal gingival sulcus of a second maxillary as well as a mandibular premolar for 60 seconds. If bleeding occured, the sample was discarded, and collection was reperformed on the same tooth. If no second premolar was present, a first premolar was used for collection. Collection sites were randomly varied, and no specific collection from the pressure or tension side was performed. Samples were transferred immediately to 500 μL peqGold TriFast (VWR, Darmstadt, Germany) and frozen. miRNA extraction and quantification were performed at the Institute of Molecular and Translational Therapeutic Strategies (IMTTS) at the Hannover Medical School in a randomized and blinded manner (to avoid systematic extraction errors or handling artifacts). Samples were thawed at room temperature and briefly vortexed. Synthetic Caenorhabditis elegans miR-39-3p (cel-miR-39-3p) was added as an exogenous spike-in control before RNA isolation (1.6 × 10 8 copies/μL) (Qiagen, Hilden, Germany) as described (Schmitz et al. 2018). Total RNA was then isolated using a standardized protocol. In brief, 100 μl of chloroform were added and the mixture was thoroughly vortexed. After incubation at room temperature, samples were centrifuged for 5 minutes (12,000 g, room temperature). An equal volume of isopropanol was added to the supernatant, followed by incubation at -20°C for 10 minutes. After centrifugation, (4°C, 12,000g) the supernatant was removed, and the pellet was washed twice by adding 500 μl of 75% ethanol. The RNA pellet was air-dried and dissolved in 15 μL RNase-free water. Samples were stored at -80°C until analysis. The isolated RNA (2.5 μl) was reversely transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription (RT) Kit (Applied Biosystems, Darmstadt, Germany) following the manufacturer's instructions. The RT reaction was performed for 30 minutes at 16°C, 30 minutes at 42°C, and 5 minutes at 85°C, followed by cooling to 4°C. For RT-qPCR, the cDNA was diluted 1:3 with water, and 2 μL were used in 10 μL qPCR reactions on a Viia7 RT-PCR System (Fisher Scientific, Waltham, Massachusetts, USA) under standard conditions. The expression levels of hsa-miRNA-21-5p, hsa-miRNA-29b-3p, hsa-miRNA-34a-5p, cel-miR-39, hsa-miRNA-126-3p, hsa-miRNA-132-3p, hsa-miRNA-146a-5p, and hsa-miRNA-221-3p were determined using the corresponding TaqMan miRNA assays (Applied Biosystems) and amplification was performed using the ViiA 7 Real-Time PCR System (Applied Biosystems). Relative miRNA levels were determined by first exporting the raw amplification data to the LinRegPCR (11.0) software which was used to perform QC on amplification to calculate the initial concentration (N0) of a given miRNA per sample (Biss et al. 2023, De Ronde et al. 2017). The relative miRNA levels (RQ) were then calculated as RQ = (N0, miRNA-XY/N0, Cel39). The relative miRNA values were log-transformed and normalized to the mean Ct values of the baseline groups. Statistical analyses The statistical analyses were conducted using SPSS, Version 28.0 (IBM, Chicago, USA), and GraphPad PRISM 10.0 (GraphPad Software Inc., La Jolla, USA). Data is presented as mean ± standard deviation (SD) or 95% confidence interval (CI). Data was checked for normal distribution using D'Agostino-Pearson test (Omnibus K2-Test). Differences over time between the upper and lower jaw (interaction effect) were determined using mixed-effects model. Correlation analysis was performed using Pearson correlation coefficient and linear regression. The significance level was set to p < 0.05. The calculation of the required sample size (power calculation) was conducted based on a comparable study (Zhang et al. 2020) suggesting effect sizes (Cohen’s d) between 1.4 and 2.0, one and four weeks after MBA insertion. With seven miRNAs and a corrected α = 0.007 at a power of 1-β = 0.95 in a repeated-measures ANOVA, a minimum sample size of 23 participants was calculated (G*Power 3.1.9.7). Target gene and pathway analysis Functional miRNA targets were identified using the experimentally validated miRNA-target interaction database miRTarBase (Huang et al. 2022) ( https://mirtarbase.cuhk.edu.cn ) as described (Kotewitsch et al. 2023). The database allows the selection of different in vitro validation methods including reporter assays, western blotting, and quantitative polymerase chain reaction (qPCR), all of which were selected to retrieve a validated list of functional miRNAs. Pathway analysis was performed against Reactome Version 87 (December 2023; human targets (Gillespie et al. 2021), submitting the identified targets (Supplemental Table 1) to the online analysis tool ( https://reactome.org ). Reactome provides an overrepresentation analysis using a hypergeometric distribution test that determines whether certain pathways are enriched in the submitted data compared to what is expected by chance. A probability score, corrected for false discovery using the Benjamani-Hochberg method, is provided. RESULTS Twenty-four patients (female, n = 12; male, n = 12) completed the study protocol. After baseline assessment (T1), mean follow-up sampling time points were as follows. One week (8 ± 2.4 days) after the insertion of the MBA (T2), five weeks (37.1 ± 12.1 days; T3) and three months (91.9 ± 8.0 days; T4) after the insertion of the MBA. Only four GCF samples were missing (two patients missed one appointment) and a total of 188 samples were analyzed. Of note, 99.4% of all analyzed miRNA signals were within the defined detection threshold (Ct value < 35). The mean Ct values of each analyzed miRNA are given in table 1. TABLE 1 Mean Ct values by miRNA over all analyzed samples miRNA Assay ID Mean Ct value (min - max) hsa-miRNA-21-5p 000397 19.8 (16.95 - 25.39) hsa-miRNA-29-3p 000413 26.1 (23.39 - 31.79) hsa-miRNA-34a-5p 000426 26.1 (23.04 - 23.23) hsa-miRNA-126-3p 002228 27.5 (22.31 - 34.24) hsa-miRNA-132-3p 000457 26.8 (23.76 - 32.66) hsa-miRNA-146a-5p 000468 24.5 (21.31 - 31.55) hsa-miRNA-221-3p 000524 24.5 (21.89 - 29. 37) To analyze if the levels of the examined miRNAs are comparable between the upper and lower jaw in general, miRNA expression levels before MBA application (baseline, T1), were compared. This analysis suggested moderate to high correlation between GCF samples taken from the mandible and maxilla with correlation coeficients between r = 0.42 and r = 0.71 (all p ≤ 0.041) (Figure 1). Of note, the lowest correlation was detected for vascular miR-126, which might be based on the known vascularization differences between the lower and upper jaw (Suwanapong et al. 2021). All seven analyzed miRNAs showed changes in their expression levels during orthodontic tooth movement compared to baseline (significant time effect, all p < 0.001) (Figure 2). The general profile indicated an increase in miRNA expression levels in both jaws with time except for miR-21-5p, which showed slightly reduced expression levels at T2 one week after MBA application (p = 0.046). For miR-34-5p, a significant interaction effect was observed (time × jaw, p = 0.0396) in that lower levels were found after five weeks and three months of treatment in the mandible compared to the maxilla (Figure 2). The identified functionally validated target genes of regulated miRNAs are provided in Supplemental table 1. Pathway analysis of the combined set of identified miRNA targets revealed several biological pathways and signaling cascades including those recently associated with orthodontic tooth movement (Figure 3). Of note, overrepresented pathways regulated by the identified miRNAs included cellular response to stress including hypoxia as well as extracellular matrix organization. Comparison of pathways including targets regulated early during tooth movement with targets of the late-response miRNAs, miR-146 and miR-221 (Figure 2), suggested that specifically during the medium to late phase of the treatment, regulation of NOD1/2 as well as RHOH GTPAse and FAS/CD95-L might occur (Supplemental Figure 1 and 2). DISCUSSION This study aimed to identify a treatment-dependent profile of functional periodontal-inflammatory miRNAs during long-term MBA application in healthy adolescent patients. Using subsequent target gene analysis, regulatory pathways induced by orthodontic tooth movement involved in bone resorption and apposition over time were identified. Our main findings are 1) miRNA levels determined in pre-treatment GCF samples are largely comparable between the mandible and maxilla in adolescents, 2) while a clear time-dependent miRNA profile in both jaws over three months of MBA was observed, miR-34a-5p expression levels differed significantly between the mandible and maxilla starting after 5 weeks of treatment, 3) elevated levels of miR-146 and miR-221 were only observed during the medium to late phase of MBA application, and 4) a specific set of miRNA targets was identified indicating regulation of multiple tagets including cellular response to stress including hypoxia as well as extracellular matrix organization. Moreover, it was shown that the applied approach allowed for the stable and reliable detection of miRNA levels based on non-invasive GCF samples. Although miRNAs have long been discussed as important and ubiquitous regulators in bone metabolism, tissue regeneration, and as inflammatory mediators, only a limited number of studies on the effects of orthodontic forces on the miRNA composition in GCF and regulated pathways are available. To the best of our knowledge, our study is the first to describe a miRNA profile determined from GCF samples of both jaws during three months of MBA treatment in adolescent patients with subsequent analysis of functional targets and associated pathways. Thus, comparing our results with previous work is limited by the available reports. However, our data can be interpreted in the light of known pathways involved in the remodeling process of alveolar bone. miR-21 has previously been shown to be upregulated in PDLCs of premolars after one month of orthodontic force application (80 – 100 g) (Chen et al. 2016). Here, we found a slight downregulation of miR-21 during the initial treatment phase, which tended to be stronger in the lower jaw and returned to baseline levels after five weeks. Regulation of miR-21 during the early phase of tooth movement may be explained by the observation that inhibiting miR-21 downregulates osteoclastogenesis and alveolar bone resorption during orthodontic tooth movement likely via the PDCD4 and IL-12A axis, regulating the chondroitin/ dermatan sulfate degeneration pathway (Asangani et al. 2008). With respect to alterations of the extracellular matrix, miR-29 may also play a central role. miR-29 has already been reported to be upregulated after 7 days of treatment (powerchain, ~ 250 g) in the GCF of adolescents during canine retraction and was found in both, exosome-depleted and non-depleted supernatants (Atsawasuwan et al. 2018). This is partly in line with our results, even though we detected an initial downregulation after MBA application which may be explained by the lower force applied or the fact that no teeth were removed at the beginning of the treatment. With respect to alterations of the extracellular matrix, analysis suggested that miR-29b targets different collagens (COL-1A1, -3A1, -4A1), as well as MMP2 and TGFβ2, all known to be regulated during matrix remodeling (Chou et al. 2013, Steele et al. 2010). In vitro experiments have suggested that miR-29b inhibits TGFβ as a negative regulator of osteogenic expression, leading to increased RUNX2 levels and subsequently enhanced osteoblastogenesis and COL-1 expression. Furthermore, miRNA-29b is directly involved in the inhibition of COL-1 mRNA in the late phase of mineralization via interaction with its 3'UTR (Li et al. 2009) Thus, miR-29b may potentially prevent bone fibrosis in the late stages of mineralization through COL-1 inhibition (Garlet et al. 2007, Li et al. 2009, Uematsu et al. 1996). It thus seems conceivable that miR-29b is downregulated in the early phase of orthodontic tooth movement, where the elimination of the hyaline phase and the resorption of alveolar bone by osteoclasts are prominent. In this phase, increased TGFβ expression dampens osteocytogenesis, and increased COL-1 expression in PDLCs contributes to the regeneration capacity of the PDL, especially on the tension side (Ignotz et al. 1987). Later in tooth movement, miR-29b levels increase, potentially activating the expression of RUNX2 through TGFβ inhibition, promoting osteoblast differentiation for bone formation and protecting against fibrosis by directly inhibiting COL-1. Another miRNA known to be involved in matrix reorganization, miR-34a, has been reported to be significantly downregulated during orthodontic tooth movement after 24 h for up to 4 weeks (Zhang et al. 2020). This partly contradicts the results of the present study, where an initial upregulation was observed for up to 5 weeks after MBA insertion. Of note, a significant difference in the expression levels of miR-34a over time was found between the upper and lower jaw, a previously unreported finding. On the molecular level, a negative correlation of miR-34 and MMP-2, -9, and -14 in PDLCs has been demonstrated, affecting the degradation of the extracellular matrix and osteoclast differentiation, and thus resorption of alveolar bone (Zhang et al. 2020). The target analysis for miR-34a revealed lactate dehydrogenase (LDH) A, which is present in the cell plasma and enters the extracellular matrix upon tissue destruction (Xiao et al. 2016). An increased LDH level in the GCF, which has been demonstrated after force application to canine teeth of orthodontic patients (Alfaqeeh and Anil 2011), indicates increased inflammatory potential of the gingiva, which may potentially trigger a counterregulation of miR-34a. To this extent, it has been reported that miR-34a was upregulated during osteoblast differentiation and miR-34a overexpression inhibited late osteoblast differentiation through LDHA-controlled cellular anaerobic glycolysis (Hong et al. 2020). It can be assumed that miR-34a inhibits late osteoblast differentiation through LDHA or LDA during orthodontic tooth movement, while affecting extracellular matrix degradation and osteoclast differentiation through MMPs. The observed expression differences between the upper and lower jaw could be attributed to the reported differences in bone density, PDL composition, or differential blood supply of the maxilla and mandible (Suwanapong et al. 2021) but warrants further investigation. With respect to the regulation of local vascularization changes during orthodontic tooth movement, it is known that miR-126a plays a central role in vasculogenesis (Schmitz et al. 2021). During MBA application, miR-126 was significantly increased over the entire treatment period. Subseqeunt target analysis identified known targets of miR-126, including chemokine receptor CXCR4 and VEGFA (Liu et al. 2014, Zhu et al. 2011). Of note, miR-126 has been discussed as potentially upregulated under hypoxic conditions (Schmitz et al. 2021) and overexpression of miR-126 in vitro has been shown to reduce the expression of IL-1α, IL-6, and TNFα, which are increased in the periodontium after orthodontic force application (Uematsu et al. 1996). Consistently, injection of miRNA-126 mimics into inflamed periodontal tissue in rats has led to reduced bone resorption and osteoclastogenesis (Andrade Jr et al. 2007, Garlet et al. 2007, Uematsu et al. 1996). In addition to the anti-inflammatory effect, miRNA-126 may possibly prevent excessive angiogenesis and osteoclast recruitment by directly inhibiting VEGF. We also observed elevated miR-132 levels over the entire treatment period, with no differences between the upper and lower jaw. While there is little evidence regarding miR-132 activity in the PDL or alveolar bone, it has been described that miR-132 expression in PDLCs is mechanosensitive and shear stress-dependent (Qi and Zhang 2014). Of note, direct targets of miR-132 include sirtuin 1 (SIRT1) (Strum et al. 2009), which regulates bone metabolism and bone mass (Qu et al. 2019) and downregulation of SIRT1 by miR-132 may potentially involve an osteogenesis-inhibiting effect on the pressure side of the PDL. Moreover, SIRT1 is expressed in the vasculature during blood vessel growth, controlling angiogenic activity (Potente et al. 2007). miR-132 may thus control angiogenic activity during tooth movement by targeting the SIRT1 axis. During the late phase of the MBA treatment (> 5 weeks), two miRNAs (miR-146 and -221), which had remained unchanged during the beginning of the treatment, were upregulated. miR-146 has been shown to promote angiogenesis via a comparable route as miR-126, targeting the angiostatic chemokine CXCR4 (Liu et al. 2014). Of note, a study using mini-implant-supported canine retraction after premolar extraction (150 cN), also did not detect upregulation during the early phase of treatment (Seagraves 2020), suggesting that miR-146 is induced as part of a late response in orthodontic tooth movement, potentially balancing the anti-angiogenic activity of miR-132. miR-146a may also be part of an anti-inflammtory response since the validated targets include IL-1-receptor-associated kinase-1 (IRAK1) (Huang et al. 2012), and IRAK1 downregulation may inhibit the proinflammatory cytokines IL-6, IL-8, and TNF-α in gingival fibroblasts. For miR-221, a compression-dependent upregulation in bovine joint cartilage has been demonstrated and in vitro studies showed that miR-221 attenuated the bone-forming potential of osteoblasts, probably by downregulating TIMP-3 (Stadnik et al. 2021, Shang et al. 2021). Furthermore, platelet-derived growth factor A (PDGFA) was identified as a target of miRNA-221, which stimulates fibroblast proliferation during bone fracture healing, leading to improved bone regeneration (Wei et al. 2020). Comparison of the pathways regulated early during tooth movement and pathways regulated by the late-response miR-146a and -221 revealed that NOD1/2 as well as RhoH GTPAse and FAS/CD95 may be regulated specifically during this phase of orthodontic tooth movement. It has been reported that Rho family GTPases such as Rho and Rac are involved in actin assembly and stress fiber formation (Li et al. 2021) and that different members of the Rho family are activated by tensile stress and compressive stress (Chen and Zhang 2023). A change in Rho family member regulation might thus indicate a difference in the predominant stressors over time. The FAS ligand (FASL) induces programmed cell death upon binding to the FAS receptor (FASR), building the death-inducing signaling complex (DISC). The FAS ligand/receptor interactions thus play an important role in the regulation of the immune system (Strasser et al. 2009) and an overactivation of the DISC in tooth movement might lead to the expression of miR-146a to prevent excessive tissue damage by apoptosis. The role of NOD receptors during tooth movements is currently not clear, however, NOD1 and 2 are intracellular pattern recognition receptors functionally expressed in PDLCs (Jeon et al. 2012) and mediate innate and acquired immunity by recognizing molecules including bacterial peptidoglycan. It is thus conceivable that the upregulation of miR-146a and -221 during the later phase of MBA treatment controls NOD signaling to reduce inflammatory processes. Limitations Even though this study had a relatively long observational period, changes in miRNA profiles beyond 3 months of orthodontic tooth movement may need to be investigated to provide a full picture of regulatory processes over the entire treatment period. Also, miRNA profiles in this study were investigated in adolescent patients. Thus, results may not be generalized to orthodontic tooth movement in adults where factors such as periodontitis are known to affect the miRNA profile. Conclusion and Practical implications We conclude that a specific periodontal-inflammatory profile of functional miRNAs involved in orthodontic tooth movement can be determined using non-invasive sampling of GCF. MBA treatment-induced changes in miRNA levels were time-dependent and largely comparable in both jaws, indicating an early and medium to late phase in tooth movement, which was marked by an increase of miR-146 and miR-221. The identified miRNA profile was linked to known targets involved in the remodeling process of the alveolar supporting apparatus and alveolar bone including cellular response to stress including hypoxia, vascularization, osteoclastogenesis as well as extracellular matrix organization. GCF-derived miRNAs may thus represent diagnostic biomarkers to monitor cellular processes induced by orthodontic tooth movement over time and could be used to optimize individual treatment outcomes. The local pharmacological modulation of the miRNAs identified in this study may represent an option to accelerate tooth movement leading to reduced treatment time. This option warrants further investigation in future studies. Declarations Funding No funding was received for this study. CB is supported by the Federal Ministry of Education and Research (BMBF, Germany, ERA-CVD JTC2018 INNOVATION, 01KL1903). BS is supported by the European Commission within the Horizon 2020 framework program (grant number: 101017424). Acknowledgements We greatly acknowledge the cooperation of all participants without whom this study could not have been realized. Competing interests statement BS filed a patent in the field of noncoding RNAs (US Patent App. 17/622,149, 2022) unrelated to orthodontic applications. The other authors delacre that they have no competing interests. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Author contributions FR and BS designed and coordinated the study. FR enrolled participants and took samples. CB, JH and IR performed sample preparation and miRNA measurements and analyzed and interpreted miRNA data. BS, FR analyzed data, interpreted results, and drafted the manuscript. All authors read and approved the final version of the manuscript. References Alfaqeeh SA, Anil S. 2011. Lactate dehydrogenase activity in gingival crevicular fluid as a marker in orthodontic tooth movement. Open Dent J. 5: 105-9 Andrade Jr I, Silva, TA, Silva GAB, Teixeira AL, Teixeira MM. 2007. The role of tumor necrosis factor receptor type 1 in orthodontic tooth movement. J Dent Res. 86(11):1089-94. Asa’ad F, Garaicoa-Pazmino C, Dahlin C, Larsson L. 2020. 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Gillespie M, Jassal B, Stephan R, Milacic M, Rothfels K, Senff-Ribeiro A, Griss J, Sevilla C, Matthews L, Gong C et al.. 2022. The reactome pathway knowledgebase 2022. Nucleic Acids Res. 50(D1):D687-D692. Hong M, Zhang XB, Xiang F, Fei X, Ouyang X-L, Peng X-C. 2020. MiR-34a suppresses osteoblast differentiation through glycolysis inhibition by targeting lactate dehydrogenase-A (LDHA). In Vitro Cell Dev Biol Anim. 56(6):480-487. Hu K, Olsen BR. 2016. Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair. J Clin Invest. 126(2):509-26. Huang HY, Lin YC, Cui S, Huang Y, Tang Y, Xu J, Bao J, Li Y, Wen J, Zuo H et al.. 2022. miRTarBase update 2022: An informative resource for experimentally validated miRNA-target interactions. Nucleid Acids Res. 50(D1):D222-D230. Huang Y, Crawford M, Higuita-Castro N, Nana-Sinkam P, Ghadiali SN. 2012. miR-146a regulates mechanotransduction and pressure-induced inflammation in small airway epithelium. 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Role of flow-sensitive microRNAs in endothelial dysfunction and atherosclerosis: mechanosensitive athero-miRs. Arterioscler Thromb Vasc Biol. 34(10):2206-16. Li Y, Zhan Q, Bao M, Yi J, Li Y. 2021 Biomechanical and biological responses of periodontium in orthodontic tooth movement: up-date in a new decade. Int J Oral Sci. 13(1):20. Li Z, Hassen MQ, Jafferji M, Aqeilan RI, Garzon R, Croce CM, van Wijnen AJ, Stein JL, Stein GS Lian JB. 2009.. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem. 284(23):15676-84. Liu Y, Zhou Y, Feng X, An P, Quan X, Wang H, Ye S, Yu C, He Y, Luo H. 2014. MicroRNA-126 functions as a tumor suppressor in colorectal cancer cells by targeting CXCR4 via the AKT and ERK1/2 signaling pathways. Int J Oncol. 44(1):203-10. Maltha JC, Kuijpers-Jagtam AM. 2023. Mechanobiology of orthodontic tooth movement: An update. J World Fed Orthod. 12(4):156-160. Miyagawa A, Chiba M, Hayashi H, Igarashi K. 2009. Compressive force induces VEGF production in periodontal tissues. J Dent Res. 88(8):752-6. Najafidoust M, Hashemi A, Oskui IZ.2020. Dynamic viscoelastic behavior of bovine periodontal ligament in compression. J Periodontal Res. 55(5):651-659. Nishijima Y, Yamaguchi M, Kojima T, Aihara N, Nakajima R, Kasai K. 2006. Levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro. Orthod Craniofac Res. 9(2):63-70. Oshiro T, Shiotani A, Shibasaki Y, Sasaki T. 2002. Osteoclast induction in periodontal tissue during experimental movement of incisors in osteoprotegerin-deficient mice. Anat Rec. 266(4):218-25. Park HJ, Baek KH, Lee HL, Kwon A, Hwang HR, Qadir AS, Woo KY, Ryoo H-M, Baek J-H. 2011. Hypoxia inducible factor-1α directly induces the expression of receptor activator of nuclear factor-κB ligand in periodontal ligament fibroblasts. Mol Cells. 31(6):573-8. Pi C, Li Y-P, Zhou X, Gao B. 2015. The expression and function of microRNAs in bone homeostasis. Front Biosci (Landmark Ed). 20(1):119-38. Potente M, Ghaeni L, Baldessari D, Mostoslavsky R, Rossig L, Dequiedt F, Haendeler J, Mione M, Dejana E, Alt FW et al.. 2007. SIRT1 controls endothelial angiogenic functions during vascular growth. Genes Dev. 21(20):2644-58. Qi L, Zhang Y. 2014. The microRNA 132 regulates fluid shear stress-induced differentiation in periodontal ligament cells through mTOR signaling pathway. Cell Physiol Biochem. 33(2):433-45. Qu H, Li T, Jin H, Zhang S, He B. 2019. Silent Mating Type Information Regulation 2 Homolog (SIRT1) Influences Osteogenic Proliferation and Differentiation of MC3T3-E1 Cells via Regulation of miR-132-3p. Med Sci Monit. 25:2289-2295. Santoro M, Nicolay OF, Cangialosi TJ. 2001. Pseudoelasticity and thermoelasticity of nickel-titanium alloys: a clinically oriented review. Part II: Deactivation forces. AM J Orthod Dentofacial Orthop. 119(6):594-603. Schmitz B, Rolfes F, Schelleckes K, Mewes M, Thorwesten L, Krüger M, Klose A, Brand S-M. 2018. Longer Work/Rest Intervals During High- Intensity Interval Training (HIIT) Lead to Elevated Levels of miR-222 and miR-29c. Front Physiol. 9:395. Schmitz B. 2021. Regulation of antiatherogenic mir-126 by physical exercise. Am J Physiol Heart Circ Physiol. 321(4):H663-H664. Schober A, Nazari-Jahantigh M, Wei Y, Bidzhekov K, Gremse F, Grommes J, Megens RTA, Heyll K, Noels H, Hristov M. 2014. MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1. Nat Med. 20(4):368-76. Seagraves, AL. 2020. Circulatory MicroRNA-27, -146, and -214 in Gingival Crevicular Fluid During Orthodontic Tooth Movement [master’s thesis]. [Chicago (IL)]: University of Chicago Shang X, Böker KO, Taheri S, Lehmann W, Schilling AF. 2021. Extracellular Vesicles Allow Epigenetic Mechanotransduction between Chondrocytes and Osteoblasts. Int J Mol Sci. 22(24):13282. Stadnik PS, Gilbert SJ, Tarn J, Charlton S, Skelton AJ, Barter MJ, Duance VC, Young DA, Blain EJ. 2021. Regulation of microRNA-221, -222, -21 and -27 in articular cartilage subjected to abnormal compressive forces. J Physiol. 599(1):143-155. Steele R, Mott JL, Ray RB. 2010. MBP-1 Upregulates miR-29b, Which Represses Mcl- 1, Collagens, and Matrix Metalloproteinase-2 in Prostate Cancer Cells. Genes Cancer. 1(4):381-387. Strasser A, Jost PJ, Nagata S. 2009. The many roles of FAS receptor signaling in the immune system. Immunity. 30(2):180-92. Strum JC, Johnson JH, Ward J, Xie H, Feild J, Hester A, Alford A, Waters KM. 2009. MicroRNA 132 regulates nutritional stress-induced chemokine production through repression of SirT1. Mol Endocrinol. 23(11):1876-84. Suwanapong T, Waikakul A, Boonsiriseth K, Ruangsawasdi N. 2021. Pre- and peri-operative factors influence autogenous tooth transplantation healing in insufficient bone sites. BMC Oral Health. 21(1):325. Theoleyre S, Wittrant Y, Tat SK, Fortun Y, Redini F, Heymann D. 2004. The molecular triad OPG/RANK/RANKL: involvement in the orchestration of pathophysiological bone remodeling. Cytokine Growth Factor Rev. 15(6):457-75. Uematsu S, Mogi M, Deguchi T. 1996. Interleukin (IL)-1 beta, IL-6, tumor necrosis factor-alpha, epidermal growth factor, and beta 2-microglobulin levels are elevated in gingival crevicular fluid during human orthodontic tooth movement. J. Dent. Res. 75(1):562-7. Uematsu S, Mogi M, Deguchi. 1996. Increase of transforming growth factor-beta 1 in gingival crevicular fluid during human orthodontic tooth movement. Arch Oral Biol. 41(11):1091-5. Wang Y, Jia L, Zheng Y, Li W. 2018. Bone remodeling induced by mechanical forces is regulated by miRNAs. Biosci Rep. 38(4):BSR20180448. Wang Y, Zheng Y, Li W. 2022. Compression loading of osteoclasts attenuated microRNA-146a-5p expression, which promotes angiogenesis by targeting adiponectin. Sci China Life Sci. 65(1):151-166. Wei J, Chen H, Fu Y, Zhang B, Zhang L, Tao S, Lin F. 2020. Experimental study of expression profile and specific role of human microRNAs in regulating atrophic bone nonunion. Medicine (Baltimore). 99(36):e21653. Wu Y, Ou Y, Liao C, Liang S, Wang Y. 2019. High-throughput sequencing analysis of the expression profile of microRNAs and target genes in mechanical force-induced osteoblastic/cementoblastic differentiation of human periodontal ligament cells. Am J Transl Res. 11(6):3398-3411. Xiao X, Huang X, Ye F, Chen B, Song C, Wen J, Zhang Z, Zheng G, Tang H, Xie X. 2016. The miR-34a-LDHA axis regulates glucose metabolism and tumor growth in breast cancer. Sci Rep. 6:21735. Yao C, Shi X, Zhang Z, Zhou S, Qian T, Wang Y, Ding F, Gu X, Yu B. 2016. Hypoxia-Induced Upregulation of miR-132 Promotes Schwann Cell Migration After Sciatic Nerve Injury by Targeting PRKAG3. Mol Neurobiol. 53(8):5129-39. Zhang B, Yang L, Zheng W, Lin T. 2020. MicroRNA-34 expression in gingival crevicular fluid correlated with orthodontic tooth movement. Angle Orthod. 90(5):702-706. Zhu N, Zhang D, Xie H, Zhou Z, Chen H, Hu T, Bai Y, Shen Y, Yuan W, Jing Q, Qin Y. 2011. Endothelial-specific intron-derived miR-126 is down-regulated in human breast cancer and targets both VEGFA and PIK3R2. Mol CellBiochem. 351(1-2):157-64. Additional Declarations Competing interest reported. BS filed a patent in the field of noncoding RNAs (US Patent App. 17/622,149, 2022) unrelated to orthodontic applications. The other authors delacre that they have no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4934654","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":359063963,"identity":"53a84ee4-3bec-4382-b26e-7fb1b5f31a82","order_by":0,"name":"Florian Rolfes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIie3RMWrDMBTG8ScEnZx0dTBUV7Dp0C6lt+hsY3CWZvcQjKaMmQMO9ArOWOjwCUGmRy6QxV06ZWguEGq3tFPtNFsJ+k9C6If0EJHL9R+DeAWR3y4FcqKrz12vl8jwmxCY6PqvhH5Ioo+R4dYS9i+36iawAPJi/PTAIe3ybjLapGQWb370PM9igO1ktc1iseRuEvIlrAdfVOyFdj/DZFWmkINZH5HUkvuWwByKcVQmWg4Ox0nyRbSMVdDeontmaYhZwE8rvmhmWduoCrLYLNfdZMhS1O8o7iqWpsa0UKp8jOrdtJv88lQ0H3QKIFL6tPMul8t1/n0AdStf7MjR3wgAAAAASUVORK5CYII=","orcid":"","institution":"ALL DENTE MVZ","correspondingAuthor":true,"prefix":"","firstName":"Florian","middleName":"","lastName":"Rolfes","suffix":""},{"id":359063964,"identity":"60c9e12b-048e-4f7b-8fd0-d658a40c28d8","order_by":1,"name":"Johannes Heck","email":"","orcid":"","institution":"Institute of Molecular and Translational Therapeutic Strategies, Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Johannes","middleName":"","lastName":"Heck","suffix":""},{"id":359063965,"identity":"233d1eb3-05e7-443f-91cd-c44e68aeb3b8","order_by":2,"name":"Isabelle Riedel","email":"","orcid":"","institution":"R-CUBE Center of Translational Regenerative Medicine, Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Isabelle","middleName":"","lastName":"Riedel","suffix":""},{"id":359063966,"identity":"9dfebf43-667a-458b-adfd-94ca590bd23f","order_by":3,"name":"Christian Bär","email":"","orcid":"","institution":"Institute of Molecular and Translational Therapeutic Strategies, Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Bär","suffix":""},{"id":359063967,"identity":"5dc410d0-4b44-4e59-9c32-46331b7b03df","order_by":4,"name":"Boris Schmitz","email":"","orcid":"","institution":"Department of Rehabilitational Sciences, Faculty of Health, University of Witten/Herdecke","correspondingAuthor":false,"prefix":"","firstName":"Boris","middleName":"","lastName":"Schmitz","suffix":""}],"badges":[],"createdAt":"2024-08-18 19:56:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4934654/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4934654/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-01794-6","type":"published","date":"2025-06-03T15:57:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66948959,"identity":"779eb3c0-86a9-41bd-86ea-96ce4694a55e","added_by":"auto","created_at":"2024-10-18 09:59:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":199886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiRNA levels are largely comparable between the maxilla and mandible. \u003c/strong\u003emiRNA levels derived from the gingival crevicular fluid (GCF) of the 24 patients were compared before treatment start. Linear regression of individual data ponts is shown with 95% confidence interval. Relative miRNA levels are shown after log2-transformation and normalization. Pearson correlation coefficient is given. All p values ≤ 0.041.\u003c/p\u003e","description":"","filename":"ScientificReportsFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4934654/v1/b1d46c244d8b4716ca09265d.png"},{"id":66948958,"identity":"2d8b7b67-a27b-483a-a16b-f9d113cf737b","added_by":"auto","created_at":"2024-10-18 09:59:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":396292,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiRNA alterations during full-mouth multibracket appliance (MBA) treatment can be classified as early- and late-response. \u003c/strong\u003emiRNA levels from the gingival crevicular fluid (GCF) of the 24 patients are presented by jaw (UJ, upper jaw [maxilla]; LJ, lower jaw [mandible]) at the respective visits: T1, pre-treatment assessment, T2, after one week, T3 after five weeks, T4 after three months. The red box indicates the late treatment response marked by increased levels of miR-146a-5p and miR-221-3p. Data is given as mean and 95% confidence interval. Mixed-model analysis was used to identify differences over time between the upper and lower jaw (interaction effect). Relative miRNA levels are shown after log2-transformation and normalization. Significant interaction effect (time × jaw) is indicated by \u003csup\u003e#\u003c/sup\u003e; significant differences compared to pre-treatment level (T1) are indicated by asterisks, *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"ScientificReportsFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4934654/v1/08c1a53ab7cd5e2e2733d7c5.png"},{"id":66948960,"identity":"43b0a14c-e655-474e-95e9-90687e8cb03a","added_by":"auto","created_at":"2024-10-18 09:59:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6329864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathways regulated by the analyzed miRNAs. \u003c/strong\u003eVisualization of overrepresented pathways (yellow) was performed using the Reactome online analysis tool (Version 87, human targets). \u003cem\u003eIn vitro\u003c/em\u003e validated targets of miRNAs were submitted to determine pathway enrichment. Darker shades indicate lower p values.\u003c/p\u003e","description":"","filename":"ScientificReportsFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4934654/v1/6291f78ae2f75d9d542a64bc.png"},{"id":84242678,"identity":"ba631779-dd48-477f-8b12-0c8addafd45b","added_by":"auto","created_at":"2025-06-09 16:11:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6105083,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934654/v1/b9fd11b3-e219-448f-bdb0-0ab81e93d973.pdf"},{"id":66949720,"identity":"e1f7483c-8437-4d37-a1b2-2ab20679f2bc","added_by":"auto","created_at":"2024-10-18 10:07:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2891195,"visible":true,"origin":"","legend":"","description":"","filename":"ScientificReportsSupplement.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934654/v1/93efae0c9d5ea03cd3b0ff6e.pdf"},{"id":66948957,"identity":"ec309a71-cfa5-49f4-b06e-a926c7abf051","added_by":"auto","created_at":"2024-10-18 09:59:00","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":35476,"visible":true,"origin":"","legend":"","description":"","filename":"ScientificReportsSupplementalData.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4934654/v1/6e76cec2b8711ceb972a8353.xlsx"}],"financialInterests":"Competing interest reported. BS filed a patent in the field of noncoding RNAs (US Patent App. 17/622,149, 2022) unrelated to orthodontic applications. The other authors delacre that they have no competing interests.","formattedTitle":"Characterization of a periodontal-inflammatory microRNA profile during long-term multibracket orthodontic treatment in adolescents - a split-mouth design study","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMechanical forces during orthodontic treatment\u0026nbsp;induce a multitude of different, time-dependent physiological pathways. These pathways are related to bone resorption and periodontal ligament (PDL) degeneration in the direction of tooth movement (on the compressive side), and bone apposition and PDL regeneration on the tensile side of the moved tooth (Krishnan and Davidovitch 2006). On the compressive side, the tooth approaches the alveolar bone, causing disruption of blood circulation leading to local hypoxia. The volume of the PDL decreases and fluid is moved from the compressed area via bone canaliculi into the surrounding regions with PDL fibers relaxing. On the tensile side, PDL fibers tighten and the PDL volume increases with fluid influx. This involves not only the direct mechanical pressure of hard tissue as a stimulus but also the fluid pressure in the PDL, evenly distributed due to poroviscoelastic behavior (Maltha and Kuijpers-Jagtman 2023, Najafidoust et al. 2020). This process exposes osteocytes in their lacunae to fluid shear stress, a stimulus that has been suggested as key to activating the osteocyte system (Burger et al. 1995). Regarding to associated bone remodeling, including the resorption of alveolar bone and extracellular matrix, osteoclasts need to be recruited under proinflammatory conditions and angiogenesis is induced for the transport of metabolic products (Bruzzaniti and Baron 2006, Wang et al. 2022). A crucial force-dependent factor for osteoclast function and enhanced recruitment is the Receptor Activator of Nuclear Factor-\u0026kappa;B ligand (RANKL). Since the competitive antagonist of RANKL, osteoprotegerin (OPG), inhibits osteoclastogenesis, the RANKL/OPG ratio determines osteoclast formation and the activation of the remodeling process (Theoleyre et al. 2004). On the compressive side, an increase in RANKL levels can be observed in osteoblasts and PDL fibroblasts as early as three hours after force application, while on the tensile side, an increase in OPG expression occurs (Brooks et al. 2009, Garlet et al. 2007, Nishijima et al. 2006, Oshiro et al. 2002). Within twenty-four hours, mechanical stress on the compressive side induces Vascular Endothelial Growth Factor (VEGF), a key angiogenic factor, in fibroblasts and osteoblasts promoting the formation of new blood vessels through proliferation and migration of endothelial progenitor cells (Hu and Olsen 2016, Miyagawa et al. 2009). It has been shown that VEGF in osteoblasts is induced by hypoxia through the Hypoxia-Inducible-Factor-1 (HIF-1), which also directly stimulates RANKL expression in PDL fibroblasts (Kim et al. 2002, Park et al. 2011). As an inflammatory response to mechanical stress, various cytokines are released from PDL cells, including interleukin (IL)-1\u0026alpha;, -4, -6, -10, -12, and Tumor Necrosis Factor-alpha (TNF-\u0026alpha;), with increased concentrations within 24 hours. Matrix-metalloproteases (MMPs) and the antagonist of MMPs, Tissue Inhibitor of MMP (TIMP), are also released. Together, TNF-\u0026alpha;\u0026nbsp;and MMPs, contribute to osteoclast differentiation and, consequently, the direct resorption of alveolar bone (Andrade Jr et al. 2007, Garlet et al. 2007, Uematsu et al. 1996).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe time-dependent regulation and overall orchestration of the different involved factors is so far only incompletely understood. Recent research has shown that\u0026nbsp;non-coding\u0026nbsp;RNAs such as microRNAs (miRNAs) play a pivotal role in the signal transduction of mechanical forces induced by tooth movement during orthodontic treatments, as they can act locally and in an endocrine manner, potentially orchestrating changes on the compressive and tensile side (Chang et al. 2015, Pi et al. 2016, Chen and Zhang 2023). miRNAs are small non-coding RNAs (19\u0026ndash;25 nucleotides in length) that regulate gene expression at the post-transcriptional level. miRNAs are detectable in virtually all bodily fluids and are expressed in complex networks that enable control of the cellular phenotype (de Gonzalo-Calvo et al. 2019).\u0026nbsp;It has been shown that mechanical stress, orthodontic forces, and hypoxia in\u0026nbsp;the PDL can lead to the regulation of specific miRNAs involved in bone remodeling and inflammatory processes in the periodontium (Asa\u0026rsquo;ad et al. 2020, Wang et al. 2018).\u0026nbsp;For example, miR-21 was increased in PDL cells and PDL stem cells of adolescent patients (15\u0026ndash;18 years old) during one month of orthodontic treatment (80\u0026ndash;100 g of force), which was associated with increased osteogenesis and alveolar bone remodeling, likely via the IL-12A axis. However, a detailed and long-term profile of miRNAs induced by mechanical forces during orthodontic treatments is currently not available.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThus, this study aimed to identify a specific set of functional periodontal-inflammatory miRNAs during long-term multibracket orthodontic treatment in healthy adolescent patients. We hypothesized that miRNA alterations would differ over time after treatment initiation depending on associated effectors, providing insight into the involved regulatory pathways.\u0026nbsp;\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eStudy design and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eorthodontic treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn interventional split-mouth study design with internal control was used to determine the concentration of miRNAs-21, -29b, -34a, -126, -132, -146a, and -221 in the gingival crevicular fluid (GCF) of adolescent patients (\u0026lt; 18 years) treated with a full-mouth multibracket appliance (MBA). GCF samples were collected from one tooth in either jaw (second maxillary, mandibular premolar) at four planned time points, before application of orthodontic force, after 7 days, after 6 weeks, and after 3 months of the procedure (8 samples per patient). The panel of analyzed miRNAs was the result of an in-depth literature search, considering previous reports on miRNAs affected by orthodontic treatments as well as miRNAs known to be regulated by associated signaling pathways including angiogenesis, hypoxia, etc (Atsawasuwan et al. 2018, Chen et al. 2016, Du et al. 2016, Kumar et al. 2014, Schmitz et al. 2021, Seagraves 2020, Schober et al. 2014, Qi and Zhang 2014, Wang et al. 2022, Wu et al. 2019, Yao et al. 2016).\u0026nbsp;Time points for sample collection were chosen to reflect clinical practice, considering the consensus to change archwires or perform orthodontic checks every 6-8 weeks after start of an orthodontic treatment also covering longer continuous exposure to inital orthodontic force and the complete implementation of the first archwire (Santoro et al. 2001, Uematsu et al. 1996). In detail, a 0.014\u0026quot; Thermal Copper Nickel Titanium archwire (Euroform II G\u0026amp;H Orthodontics, Indiana, USA) was used, applying a recovery force of 78 g at mouth temperature. Three types of brackets (22\u0026quot; slot size, MBT prescription) were used. Patients were treated with either a self-ligating MBA (Genius System Metal, ortho Penthin GmbH, Schwanewede, Germany, 4 participants) or one of two conventionally ligated MBAs (SmartTwin, ortho Penthin GmbH, 16 participants; Iconix Aesthetic Braces, American Orthodontics, Wisconsin, USA, 4 participants). The choice of MBA was independent of study participation and solely based on the decision of the legal guardians to cover costs for additional orthodontic service according to German health insurance regulations (e.g. Thermal Copper Nickel Titanium archewires additionally to stainless steel archwires).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticipants and eligibility criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSelf-reported healthy male and female patients (12-18 years of age) undergoing orthodontic treatment with the insertion of a full-mouth MBA involving more than six bracketed and/or banded teeth per jaw were eligible to participate. All methods were carried out in accordance with relevant guidelines and regulations in that written informed consent was obtained from patients and/or their legal guardians. Samples were collected anonymously, without documentation of any personal information approved by the by the local ethical review committee (Ethikkommission Universit\u0026auml;t Witten-Herdecke). Written informed consent was obtained from patients and/or their legal guardians. Samples were collected anonymously, without documentation of any personal information aligned with the ethics review board of\u0026nbsp;the\u0026nbsp;University of Witten/Herdecke (Ethics Commission University Witten/Herdecke).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample collection and miRNA quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples were collected as described with modifications (Atsawasuwan et al. 2018, Zhang et al. 2020). Before collecting\u0026nbsp;the GCF, patients were instructed to swallow to avoid sample dilution by saliva. The sampling region was isolated vestibular and, in the lower jaw, in the lingual area using cotton rolls. Tooth were carefully cleared of saliva, especially interdentally, using a dental air syringe. No further cleaning procedures were performed. A PerioPaper (Oraflow Inc., Smithtown, NY, USA) was inserted into the buccal gingival sulcus of a second maxillary as well as a mandibular premolar for 60 seconds. If bleeding occured, the sample was discarded, and collection was reperformed on the same tooth. If no second premolar was present, a first premolar was used for collection. Collection sites were randomly varied, and no specific collection from the pressure or tension side was performed. Samples were transferred immediately to 500\u0026nbsp;\u0026mu;L peqGold TriFast (VWR, Darmstadt, Germany) and frozen.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003emiRNA extraction and quantification were performed at the Institute of Molecular and Translational Therapeutic Strategies (IMTTS) at the Hannover Medical School in a randomized and blinded manner (to avoid systematic extraction errors or handling artifacts). Samples were thawed at room temperature and briefly vortexed. Synthetic Caenorhabditis elegans miR-39-3p (cel-miR-39-3p) was added as an exogenous spike-in control before RNA isolation (1.6 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e copies/\u0026mu;L) (Qiagen, Hilden, Germany) as described (Schmitz et al. 2018). Total RNA was then isolated using a standardized protocol. In brief, 100\u0026nbsp;\u0026mu;l of chloroform were added and the mixture was thoroughly vortexed. After incubation at room temperature, samples were centrifuged\u0026nbsp;for 5 minutes (12,000 g, room temperature). An equal volume of isopropanol was added to the supernatant, followed by incubation at -20\u0026deg;C for 10 minutes. After centrifugation, (4\u0026deg;C, 12,000g) the supernatant was removed, and the pellet was washed twice by adding 500\u0026nbsp;\u0026mu;l of 75% ethanol. The RNA pellet was air-dried and dissolved in 15\u0026nbsp;\u0026mu;L RNase-free water. Samples were stored at -80\u0026deg;C until analysis. The isolated RNA (2.5\u0026nbsp;\u0026mu;l) was reversely transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription (RT) Kit (Applied Biosystems, Darmstadt, Germany) following the manufacturer\u0026apos;s instructions. The RT reaction was performed for 30 minutes at 16\u0026deg;C, 30 minutes at 42\u0026deg;C, and 5 minutes at 85\u0026deg;C, followed by cooling to 4\u0026deg;C. For RT-qPCR, the cDNA was diluted 1:3 with water, and 2\u0026nbsp;\u0026mu;L were used in 10\u0026nbsp;\u0026mu;L qPCR reactions on a Viia7 RT-PCR System (Fisher Scientific, Waltham, Massachusetts, USA) under standard conditions.\u003c/p\u003e\n\u003cp\u003eThe expression levels of hsa-miRNA-21-5p, hsa-miRNA-29b-3p, hsa-miRNA-34a-5p, cel-miR-39, hsa-miRNA-126-3p, hsa-miRNA-132-3p, hsa-miRNA-146a-5p, and hsa-miRNA-221-3p were determined using the corresponding TaqMan miRNA assays (Applied Biosystems)\u0026nbsp;and amplification was performed using the ViiA 7 Real-Time PCR System (Applied Biosystems). Relative miRNA levels were determined by first exporting the raw amplification data to the LinRegPCR (11.0) software which was used to perform QC on amplification to calculate the initial concentration (N0) of a given miRNA per sample (Biss et al. 2023, De Ronde et al. 2017).\u0026nbsp;The relative miRNA levels (RQ) were then calculated as RQ = (N0, miRNA-XY/N0, Cel39). The relative miRNA values were log-transformed and normalized to the mean Ct values of the baseline groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe statistical analyses were conducted using SPSS, Version 28.0 (IBM, Chicago, USA), and GraphPad PRISM 10.0 (GraphPad Software Inc., La Jolla, USA). Data is presented as mean \u0026plusmn; standard deviation (SD) or 95% confidence interval (CI). Data was checked for normal distribution using D\u0026apos;Agostino-Pearson test (Omnibus K2-Test). Differences over time between the upper and lower jaw (interaction effect) were determined using mixed-effects model. Correlation analysis was performed using Pearson correlation coefficient and linear regression. The significance level was set to p \u0026lt; 0.05. The calculation of the required sample size (power calculation) was conducted based on a comparable study (Zhang et al. 2020) suggesting\u0026nbsp;effect sizes (Cohen\u0026rsquo;s d) between 1.4 and 2.0, one and four weeks after MBA insertion. With seven miRNAs and a corrected\u0026nbsp;\u0026alpha;\u0026nbsp;= 0.007 at a power of 1-\u0026beta;\u0026nbsp;= 0.95 in a repeated-measures ANOVA, a minimum sample size of 23 participants was calculated (G*Power 3.1.9.7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTarget gene and pathway analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional miRNA targets were identified using the experimentally validated miRNA-target interaction database miRTarBase (Huang et al. 2022) (\u003cspan lang=\"EN-US\"\u003ehttps://mirtarbase.cuhk.edu.cn\u003c/span\u003e) as described (Kotewitsch et al. 2023). The database\u0026nbsp;allows the selection of different \u003cem\u003ein vitro\u003c/em\u003e validation methods including reporter assays, western blotting, and quantitative polymerase chain reaction (qPCR), all of which were selected to retrieve a validated list of functional miRNAs. Pathway analysis was performed against Reactome\u0026nbsp;Version 87 (December 2023; human targets (Gillespie et al. 2021), submitting the identified targets (Supplemental Table 1) to the\u0026nbsp;online analysis tool (\u003cspan lang=\"EN-US\"\u003ehttps://reactome.org\u003c/span\u003e). Reactome\u003cem\u003e\u0026nbsp;\u003c/em\u003eprovides an overrepresentation analysis using a hypergeometric distribution test that determines whether certain pathways are enriched in the submitted data compared to what is expected by chance. A probability score, corrected for false discovery using the Benjamani-Hochberg method, is provided. \u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eTwenty-four patients (female, n = 12; male, n = 12) completed the study protocol. After baseline assessment (T1), mean follow-up sampling time points were as follows. One week (8 \u0026plusmn; 2.4 days) after the insertion of the MBA (T2), five weeks (37.1 \u0026plusmn; 12.1 days; T3) and three months (91.9 \u0026plusmn; 8.0 days; T4) after the insertion of the MBA. Only four GCF samples were missing (two patients missed one appointment) and a total of 188 samples were analyzed. Of note, 99.4% of all analyzed miRNA signals were within the defined detection threshold (Ct value \u0026lt; 35). The mean Ct values of each analyzed miRNA are given in table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTABLE 1 \u003cstrong\u003eMean Ct values by miRNA over all analyzed samples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"444\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34.009%;\"\u003e\n \u003cp\u003e\u003cstrong\u003emiRNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.3964%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAssay ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.5946%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Ct value\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(min - max)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34.009%;\"\u003e\n \u003cp\u003ehsa-miRNA-21-5p\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.3964%;\"\u003e\n \u003cp\u003e000397\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.5946%;\"\u003e\n \u003cp\u003e19.8 (16.95 - 25.39)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34.009%;\"\u003e\n \u003cp\u003ehsa-miRNA-29-3p\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.3964%;\"\u003e\n \u003cp\u003e000413\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.5946%;\"\u003e\n \u003cp\u003e26.1 (23.39 - 31.79)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34.009%;\"\u003e\n \u003cp\u003ehsa-miRNA-34a-5p\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.3964%;\"\u003e\n \u003cp\u003e000426\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.5946%;\"\u003e\n \u003cp\u003e26.1 (23.04 - 23.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34.009%;\"\u003e\n \u003cp\u003ehsa-miRNA-126-3p\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.3964%;\"\u003e\n \u003cp\u003e002228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.5946%;\"\u003e\n \u003cp\u003e27.5 (22.31 - 34.24)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34.009%;\"\u003e\n \u003cp\u003ehsa-miRNA-132-3p\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.3964%;\"\u003e\n \u003cp\u003e000457\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.5946%;\"\u003e\n \u003cp\u003e26.8 (23.76 - 32.66)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34.009%;\"\u003e\n \u003cp\u003ehsa-miRNA-146a-5p\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.3964%;\"\u003e\n \u003cp\u003e000468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.5946%;\"\u003e\n \u003cp\u003e24.5 (21.31 - 31.55)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 34.009%;\"\u003e\n \u003cp\u003ehsa-miRNA-221-3p\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.3964%;\"\u003e\n \u003cp\u003e000524\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.5946%;\"\u003e\n \u003cp\u003e24.5 (21.89 - 29. 37)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;To analyze if the levels of the examined miRNAs are comparable between the upper and lower jaw in general, miRNA expression levels before MBA application (baseline, T1), were compared. This analysis suggested moderate to high correlation between GCF samples taken from the mandible and maxilla with correlation coeficients between r = 0.42 and r = 0.71 (all p \u0026le; 0.041) (Figure 1). Of note, the lowest correlation was detected for vascular miR-126, which might be based on the known vascularization differences between the lower and upper jaw (Suwanapong et al. 2021). All seven analyzed miRNAs showed changes in their expression levels during orthodontic tooth movement compared to baseline (significant time effect, all p \u0026lt; 0.001) (Figure 2). The general profile indicated an increase in miRNA expression levels in both jaws with time except for miR-21-5p, which showed slightly reduced expression levels at T2 one week after MBA application (p = 0.046). For miR-34-5p, a significant interaction effect was observed (time \u0026times; jaw, p = 0.0396) in that lower levels were found after five weeks and three months of treatment in the mandible compared to the maxilla (Figure 2).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The identified functionally validated target genes of regulated miRNAs are provided in Supplemental table 1. Pathway analysis of the combined set of identified miRNA targets revealed several biological pathways and signaling cascades including those recently associated with orthodontic tooth movement (Figure 3). Of note, overrepresented pathways regulated by the identified miRNAs included cellular response to stress including hypoxia as well as extracellular matrix organization. Comparison of pathways including targets regulated early during tooth movement with targets of the late-response miRNAs, miR-146 and miR-221 (Figure 2), suggested that specifically during the medium to late phase of the treatment, regulation of NOD1/2 as well as RHOH GTPAse and FAS/CD95-L might occur (Supplemental Figure 1 and 2).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study aimed to identify a treatment-dependent profile of functional periodontal-inflammatory miRNAs during long-term MBA application in healthy adolescent patients. Using subsequent target gene analysis, regulatory pathways induced by orthodontic tooth movement involved in bone resorption and apposition over time were identified. Our main findings are 1) miRNA levels determined in pre-treatment GCF samples are largely comparable between the mandible and maxilla in adolescents, 2) while a clear time-dependent miRNA profile in both jaws over three months of MBA was observed, miR-34a-5p expression levels differed significantly between the mandible and maxilla starting after 5 weeks of treatment, 3) elevated levels of miR-146 and miR-221 were only observed during the medium to late phase of MBA application, and 4) a specific set of miRNA targets was identified indicating regulation of multiple tagets including cellular response to stress including hypoxia as well as extracellular matrix organization. Moreover, it was shown that the applied approach allowed for the stable and reliable detection of miRNA levels based on non-invasive GCF samples.\u003c/p\u003e\n\u003cp\u003eAlthough miRNAs have long been discussed as important and ubiquitous regulators in bone metabolism, tissue regeneration, and as inflammatory mediators, only a limited number of studies on the effects of orthodontic forces on the miRNA composition in GCF and regulated pathways are available. To the best of our knowledge, our study is the first to describe a miRNA profile determined from GCF samples of both jaws during three months of MBA treatment in adolescent patients with subsequent analysis of functional targets and associated pathways. Thus, comparing our results with previous work is limited by the available reports. However, our data can be interpreted in the light of known pathways involved in the remodeling process of alveolar bone. miR-21 has previously been shown to be upregulated in PDLCs of premolars after one month of orthodontic force application (80 \u0026ndash; 100 g) (Chen et al. 2016). Here, we found a slight downregulation of miR-21 during the initial treatment phase, which tended to be stronger in the lower jaw and returned to baseline levels after five weeks. Regulation of miR-21 during the early phase of tooth movement may be explained by the observation that inhibiting miR-21 downregulates osteoclastogenesis and alveolar bone resorption during orthodontic tooth movement likely via the PDCD4 and IL-12A axis, regulating the chondroitin/ dermatan sulfate degeneration pathway (Asangani et al. 2008). With respect to alterations of the extracellular matrix, miR-29 may also play a central role. miR-29 has already been reported to be upregulated after 7 days of treatment (powerchain, ~ 250 g) in the GCF of adolescents during canine retraction and was found in both, exosome-depleted and non-depleted supernatants (Atsawasuwan et al. 2018). This is partly in line with our results, even though we detected an initial downregulation after MBA application which may be explained by the lower force applied or the fact that no teeth were removed at the beginning of the treatment. With respect to alterations of the extracellular matrix, analysis suggested that miR-29b targets different collagens (COL-1A1, -3A1, -4A1), as well as MMP2 and TGF\u0026beta;2, all known to be regulated during matrix remodeling (Chou et al. 2013, Steele et al. 2010). \u003cem\u003eIn\u0026nbsp;\u003c/em\u003e\u003cem\u003evitro\u003c/em\u003e experiments have suggested that miR-29b inhibits TGF\u0026beta;\u0026nbsp;as a negative regulator of osteogenic expression, leading to increased RUNX2 levels and subsequently enhanced osteoblastogenesis and COL-1 expression. Furthermore, miRNA-29b is directly involved in the inhibition of COL-1 mRNA in the late phase of mineralization via interaction with its 3\u0026apos;UTR\u0026nbsp;(Li et al. 2009)\u0026nbsp;Thus, miR-29b may potentially prevent bone fibrosis in the late stages of mineralization through COL-1 inhibition (Garlet et al. 2007, Li et al. 2009, Uematsu et al. 1996). It thus seems conceivable that miR-29b is downregulated in the early phase of orthodontic\u0026nbsp;tooth movement, where the elimination of the hyaline phase and the resorption of alveolar bone by osteoclasts are prominent. In this phase, increased TGF\u0026beta;\u0026nbsp;expression dampens osteocytogenesis, and increased COL-1 expression in PDLCs contributes to the regeneration capacity of the PDL, especially on the tension side (Ignotz et al. 1987). Later in tooth movement, miR-29b levels increase, potentially activating the expression of RUNX2 through TGF\u0026beta;\u0026nbsp;inhibition, promoting osteoblast differentiation for bone formation and protecting against fibrosis by directly inhibiting COL-1. Another miRNA known to be involved in matrix reorganization, miR-34a, has been reported to be significantly downregulated during orthodontic tooth movement after 24 h for up to 4 weeks (Zhang et al. 2020). This partly contradicts the results of the present study, where an initial upregulation was observed for up to 5 weeks after MBA insertion. Of note, a significant difference in the expression levels\u0026nbsp;of miR-34a over time was found between the upper and lower jaw, a previously unreported finding. On the molecular level, a negative correlation of miR-34 and MMP-2, -9, and -14 in PDLCs has been demonstrated, affecting the degradation of the extracellular matrix and osteoclast differentiation, and thus resorption of alveolar bone (Zhang et al. 2020). The target analysis for miR-34a revealed lactate dehydrogenase (LDH) A, which is present in the cell plasma and enters the extracellular matrix upon tissue destruction (Xiao et al. 2016). An increased LDH level in the GCF, which has been demonstrated after force application to canine teeth of orthodontic patients (Alfaqeeh and Anil 2011), indicates\u0026nbsp;increased inflammatory potential of the gingiva, which may potentially trigger a counterregulation of miR-34a. To this extent, it has been reported that miR-34a was upregulated during osteoblast differentiation and miR-34a overexpression inhibited late osteoblast differentiation through LDHA-controlled cellular anaerobic glycolysis (Hong et al. 2020). It can be assumed that miR-34a inhibits late osteoblast differentiation through LDHA or LDA during orthodontic tooth movement, while affecting extracellular matrix degradation\u0026nbsp;and osteoclast differentiation through MMPs. The observed expression differences between the upper and lower jaw could be attributed to the reported differences in bone density, PDL composition, or differential blood supply of the maxilla and mandible (Suwanapong et al. 2021)\u0026nbsp;but warrants further investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith respect to the regulation of local vascularization changes during orthodontic tooth movement, it is known that miR-126a plays a central role in vasculogenesis (Schmitz et al. 2021). During MBA application, miR-126 was significantly increased over the entire treatment period. Subseqeunt target analysis identified known targets of miR-126, including chemokine receptor CXCR4 and VEGFA (Liu et al. 2014, Zhu et al. 2011). Of note, miR-126 has been discussed as potentially upregulated under hypoxic conditions (Schmitz et al. 2021) and overexpression of miR-126 \u003cem\u003ein vitro\u003c/em\u003e has been shown to reduce the expression of IL-1\u0026alpha;, IL-6, and TNF\u0026alpha;, which are increased in the periodontium after orthodontic force application (Uematsu et al. 1996). Consistently, injection of miRNA-126 mimics into inflamed periodontal tissue in rats has led to reduced bone resorption and osteoclastogenesis (Andrade Jr et al. 2007, Garlet et al. 2007, Uematsu et al. 1996). In addition to the anti-inflammatory effect, miRNA-126 may possibly prevent excessive angiogenesis and osteoclast recruitment by directly inhibiting VEGF. We also observed elevated miR-132 levels over the entire treatment period, with no differences between the upper and lower jaw. While there is little evidence regarding miR-132 activity in the PDL or alveolar bone, it has been described that miR-132 expression in PDLCs is mechanosensitive and shear stress-dependent (Qi and Zhang 2014). Of note, direct targets of miR-132 include sirtuin 1 (SIRT1) (Strum et al. 2009), which regulates bone metabolism and bone mass (Qu et al. 2019) and downregulation of SIRT1 by miR-132 may potentially involve an osteogenesis-inhibiting effect on the pressure side of the PDL. Moreover, SIRT1 is expressed in the vasculature during blood vessel growth, controlling angiogenic activity (Potente et al. 2007). miR-132 may thus control angiogenic activity during tooth movement by targeting the SIRT1 axis.\u003c/p\u003e\n\u003cp\u003eDuring the late phase of the MBA treatment (\u0026gt; 5 weeks), two miRNAs (miR-146 and -221), which had remained unchanged during the beginning of the treatment, were upregulated. miR-146 has been shown to promote angiogenesis via a comparable route as miR-126, targeting the angiostatic chemokine CXCR4 (Liu et al. 2014). Of note, a study using mini-implant-supported canine retraction after premolar extraction (150 cN), also did not detect upregulation during the early phase of treatment (Seagraves 2020), suggesting that miR-146 is induced as part of a late response in orthodontic tooth movement, potentially balancing the anti-angiogenic activity of miR-132. miR-146a may also be part of an anti-inflammtory response since the validated targets include IL-1-receptor-associated kinase-1 (IRAK1) (Huang et al. 2012), and IRAK1 downregulation may inhibit the proinflammatory cytokines IL-6, IL-8, and TNF-\u0026alpha; in gingival fibroblasts. For miR-221, a compression-dependent upregulation in bovine joint cartilage has been demonstrated and \u003cem\u003ein vitro\u003c/em\u003e studies showed that miR-221 attenuated the bone-forming potential of osteoblasts, probably by downregulating TIMP-3 (Stadnik et al. 2021, Shang et al. 2021). Furthermore, platelet-derived growth factor A (PDGFA) was identified as a target of miRNA-221, which stimulates fibroblast proliferation during bone fracture healing, leading to improved bone regeneration (Wei et al. 2020). Comparison of the pathways regulated early during tooth movement and pathways regulated by the late-response miR-146a and -221 revealed that NOD1/2 as well as RhoH GTPAse and FAS/CD95 may be regulated specifically during this phase of orthodontic tooth movement. It has been reported that Rho family GTPases such as Rho and Rac are involved in actin assembly and stress fiber formation (Li et al. 2021) and that different members of the Rho family are activated by tensile stress and compressive stress (Chen and Zhang 2023). A change in Rho family member regulation might thus indicate a difference in the predominant stressors over time. The FAS ligand (FASL) induces programmed cell death upon binding to the FAS receptor (FASR), building the death-inducing signaling complex (DISC). The FAS ligand/receptor interactions thus play an important role in the regulation of the immune system (Strasser et al. 2009) and an overactivation of the DISC in tooth movement might lead to the expression of miR-146a to prevent excessive tissue damage by apoptosis. The role of NOD receptors during tooth movements is currently not clear, however, NOD1 and 2 are intracellular pattern recognition receptors functionally expressed in PDLCs (Jeon et al. 2012) and mediate innate and acquired immunity by recognizing molecules including bacterial peptidoglycan. It is thus conceivable that the upregulation of miR-146a and -221 during the later phase of MBA treatment controls NOD signaling to reduce inflammatory processes.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEven though this study had a relatively long observational period, changes in miRNA profiles beyond 3 months of orthodontic tooth movement may need to be investigated to provide a full picture of regulatory processes over the entire treatment period. Also, miRNA profiles in this study were investigated in adolescent patients. Thus, results may not be generalized to orthodontic tooth movement in adults where factors such as periodontitis are known to affect the miRNA profile.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion and Practical implications","content":"\u003cp\u003eWe conclude that a specific periodontal-inflammatory profile of functional miRNAs involved in orthodontic tooth movement can be determined using non-invasive sampling of GCF. MBA treatment-induced changes in miRNA levels were time-dependent and largely comparable in both jaws, indicating an early and medium to late phase in tooth movement, which was marked by an increase of miR-146 and miR-221. The identified miRNA profile was linked to known targets involved in the remodeling process of the alveolar supporting apparatus and alveolar bone including cellular response to stress including hypoxia, vascularization, osteoclastogenesis as well as extracellular matrix organization. GCF-derived miRNAs may thus represent diagnostic biomarkers to monitor cellular processes induced by orthodontic tooth movement over time and could be used to optimize individual treatment outcomes. The local pharmacological modulation of the miRNAs identified in this study may represent an option to accelerate tooth movement leading to reduced treatment time. This option warrants further investigation in future studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for this study. CB is supported by the Federal Ministry of Education and Research (BMBF, Germany, ERA-CVD JTC2018 INNOVATION, 01KL1903). BS is supported by the European Commission within the Horizon 2020 framework program (grant number: 101017424).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe greatly acknowledge the cooperation of all participants without whom this study could not have been realized. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBS filed a patent in the field of noncoding RNAs (US Patent App. 17/622,149, 2022) unrelated to orthodontic applications. The other authors delacre that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFR and BS designed and coordinated the study. FR enrolled participants and took samples. CB, JH and IR performed sample preparation and miRNA measurements and analyzed and interpreted miRNA data. BS, FR analyzed data, interpreted results, and drafted the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlfaqeeh SA, Anil S. 2011. Lactate dehydrogenase activity in gingival crevicular fluid as a marker in orthodontic tooth movement. 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Mol CellBiochem. 351(1-2):157-64.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"multi-bracket appliance, molecular orthodontics, alveolar bone remodeling, gingival crevicular fluid, mechanical loading, extracellular matrix ","lastPublishedDoi":"10.21203/rs.3.rs-4934654/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4934654/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to identify functional microRNAs (miRNAs) and their respective targets as central regulatory factors of tooth movement during orthodontic treatment.\u003c/p\u003e\n\u003cp\u003eGingival crevicular fluid (GCF) of 24 adolescent patients (\u0026lt; 18 years) treated with a full-mouth multibracket appliance (MBA; Thermal Copper Nickel Titanium archwire) was analyzed for miRNAs-21, -29b, -34a, -126, -132, -146a, and -221 in a split-mouth study design. GCF samples were taken from either jaw (second maxillary, mandibular premolar) using non-invasive sampling before, 7 days, 5 weeks, and 3 months after application of orthodontic force (8 samples per patient). Validated miRNA targets and regulated pathways were identified using the miRTarBase database (release 9.0) and Reactome (version 87).\u003c/p\u003e\n\u003cp\u003eAll analyzed miRNAs were consistently detected in the GCF (Ct value \u0026lt; 35) and a moderate to high correlation was found between samples taken from the mandible and maxilla before treatment (r = 0.42 to 0.71, all p ≤ 0.041). All miRNAs showed changes in their expression levels with orthodontic tooth movement compared to baseline (significant time effect, all p \u0026lt; 0.001). The general profile indicated an increase in miRNA expression in both jaws with time with the exeption of miR-21, which showed reduced levels one week after MBA application (p = 0.046). For miR-34, a significant interaction effect was observed (time × jaw, p = 0.0396) in that lower levels were found after five weeks and three months of treatment in the mandible compared to the maxilla. The medium to late treatment phase was characterized by an increase in miR-146 and miR-221. Gene signaling pathway analysis suggested regulation of cellular response to stress including hypoxia, matrix reorganization and vascular remodeling.\u003c/p\u003e\n\u003cp\u003eSince the identified miRNA profile was linked to targets involved in the remodeling process of the alveolar supporting apparatus and alveolar bone, GCF-derived miRNAs may represent diagnostic biomarkers to monitor cellular processes during orthodontic tooth movement and potentially optimize individual treatment outcomes.\u003c/p\u003e","manuscriptTitle":"Characterization of a periodontal-inflammatory microRNA profile during long-term multibracket orthodontic treatment in adolescents - a split-mouth design study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-18 09:58:55","doi":"10.21203/rs.3.rs-4934654/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-06T18:54:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-10T13:06:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170813987365279928942527998091046322028","date":"2024-10-31T10:05:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171716671198277887724022826183513725942","date":"2024-10-27T15:29:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-17T17:23:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276088456332936295392544218428953950428","date":"2024-09-11T12:52:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-09T05:54:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-09T05:50:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-09-03T10:01:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-02T09:52:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-08-18T19:54:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5803882d-7aca-4c52-8ed3-b3bff5fb8926","owner":[],"postedDate":"October 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38207172,"name":"Health sciences/Biomarkers"},{"id":38207173,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-06-09T16:05:16+00:00","versionOfRecord":{"articleIdentity":"rs-4934654","link":"https://doi.org/10.1038/s41598-025-01794-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-06-03 15:57:08","publishedOnDateReadable":"June 3rd, 2025"},"versionCreatedAt":"2024-10-18 09:58:55","video":"","vorDoi":"10.1038/s41598-025-01794-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-01794-6","workflowStages":[]},"version":"v1","identity":"rs-4934654","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4934654","identity":"rs-4934654","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}