Effect of hydrogen peroxide-induced oxidative stress on simulated orthodontic tooth movement in a cell culture model

Hosseini, Seyedeh Samira (2026): Effect of hydrogen peroxide-induced oxidative stress on simulated orthodontic tooth movement in a cell culture model. Dissertation, LMU München: Medizinische Fakultät

Abstract

The PhD project entitled “Effect of hydrogen peroxide-induced oxidative stress on simulated orthodontic tooth movement in a cell culture model” was conducted at the Department of Orthodontics and Dentofacial Orthopaedics, University Hospital, Ludwig-Maximilians-Universität München, under the supervision of PD Dr. rer. nat. Uwe Baumert, Prof. Dr. med. dent. Andrea Wichelhaus and Prof. Dr. Dr. Matthias Folwaczny. Orthodontic tooth movement: In orthodontics, therapeutic forces are applied to correct abnormally positioned teeth and orthodontic tooth movement (OTM) is the result of this force application (Wichelhaus 2018). Following the application of continuous mechanical forces to teeth, complex aseptic inflammatory cellular and molecular responses are triggered in the surrounding tissues (Davidovitch 1991; Vansant et al. 2018). This effect is particularly evident in the periodontal ligament (PDL) and alveolar bone where human periodontal ligament cells (hPDLCs) and human osteoblasts (hOBs) are recognized as key cells in the mechanosensing process (Janjic et al. 2018). The outcome of mechanical stimulation is bone removal in the direction of the force and bone apposition in the opposite direction (Davidovitch 1991) (Figure 1), with static compression and tension forces playing dominant roles in these processes. Oxidative stress: Oxidative stress (OS) refers to a metabolic condition in cells and tissues which occurs due to an imbalance between the production and accumulation of reactive oxygen species (ROS) on one hand, and the body's capability to either eliminate and neutralize these compounds or repair the resulting cellular damage on the other hand (Hosseini et al. 2024b; Pizzino et al. 2017). ROS, including hydrogen peroxide (H2O2), hydroxyl radicals (•OH), superoxide radicals (O2•−), and singlet oxygen (1O2), are generated as metabolic byproducts in biological systems (Pizzino et al. 2017). These compounds are involved in essential cellular processes such as inflammatory signalling, apoptosis and differentiation (Pizzino et al. 2017). For these mechanisms to operate effectively, the concentrations of ROS must be appropriately regulated (Pizzino et al. 2017). ROS is known to present a dual nature in cellular biology. At moderate physiological concentrations, ROS function as important signalling molecules in various cellular functions, including cell differentiation, proliferation, apoptosis and autophagy pathways (Pizzino et al. 2017). However, high concentrations can be detrimental. When ROS levels become excessive, they can lead to significant harm to cellular components such as lipids, proteins, and nucleic acids, which can compromise cell viability and lead to cell death (Pizzino et al. 2017; Zhu et al. 2021). Influence of oxidative stress on the periodontium: Periodontitis is a widespread chronic inflammatory condition characterized by ongoing alveolar bone resorption. During periodontitis, persistent inflammation in periodontal tissues results in PDL and alveolar bone loss. This is followed by tooth loss and mastication problems, significantly impacting overall well-being and life quality (Durham et al. 2013; Schröder et al. 2021; Tonetti et al. 2017). There have been several studies reporting that oxidative stress (OS) plays an important role in the pathogenesis of different chronic inflammations including periodontitis, particularly in its initiation and progression (Chen et al. 2022; Mittal et al. 2014; Wei et al. 2021). Studies have also demonstrated higher levels of OS were observed in gingival crevicular fluid, in saliva, and in serum of patients with periodontal disease (Almerich-Silla et al. 2015; Chen et al. 2019b; Wang et al. 2017). This pathological condition leads into a direction in which neutrophils become excessively activated. This hyperactivation of neutrophils triggers an increased production of ROS as part of the body's defensive mechanism (Wei et al. 2021). Increased levels of OS can cause cytotoxic effects on periodontal tissue. It is reported that ROS affects the osteogenic differentiation of hPDLCs by stimulating the production of inflammatory mediators, both in vitro and in vivo (Chen et al. 2022). Influence of oxidative stress on OTM: During the recent years, the number of adult patients seeking orthodontic treatment increased remarkably. This leads to a higher prevalence of individuals with periodontal disease in orthodontic practices seeking orthodontic care (Christensen and Luther 2015). Both orthodontic tooth movement and periodontal disease are associated with inflammation and alveolar bone remodelling (Schröder et al. 2021). Establishment of an oxidative stress in vitro loading model: Various biological processes including inflammation, bone remodelling, autophagy, and apoptosis are known to be influenced by either mechanical force or OS (Zhu et al. 2022). However, the specific interplay between OS and OTM remains unexplored and poorly understood. Despite the recognized importance of OS in such physiological mechanisms, its precise role in the context of OTM is still a subject of considerable uncertainty and requires investigation (Hosseini et al. 2024b). Accordingly, in the presented Ph.D. thesis, two projects were conducted in which the two cell types playing a dominant role in OTM, hPDLCs and hOBs, were subjected to mechanical stimulation with either static compression or static tension after OS stimulation using H2O2. ROS stimulation model establishment: Force stimulations were applied using the “weight approach”-based (WAB) model (Janjic et al. 2018) and the static tension in vitro loading model (Sun et al. 2021), mimicking orthodontic forces, with or without preinduction with oxidative stress. For this purpose, we combined an in vitro oxidative stress stimulation method with the two in vitro mechanical stimulation models (Hosseini et al. 2024a; Hosseini et al. 2024b). This method would allow us to study cellular and molecular mechanisms of OTM in an increased OS environment. According to the literature, OS has been induced by adding H2O2 to the cell culture medium (Chen et al. 2019a; Tan et al. 2021; Wei et al. 2021). To determine the optimal concentration of H2O2 not affecting cell proliferation and viability, dose-response experiments was carried out for each cell type. The cells were stimulated with H2O2 concentrations ranging from 50 μM to 500 μM for 24 h. Afterwards, cell viability and proliferation of the cells were assessed to identify “optimal” H2O2 concentrations not affecting cell viability and proliferation (Hosseini et al. 2024a; Hosseini et al. 2024b). These were then used in the next step of the experiment where OS exposure was combined with mechanical stimulation. Herein, the cells were first stimulated 24 h with medium containing the predetermined H2O2 “optimal” concentrations and afterwards subjected to mechanical stimulation of either compression or tension for additional 24 h. Experimental force levels applied in this study were as following: 2.0 g/cm2 for static compressive force and 10 % and 15 % stretching for static tension force, both for the duration of 24 h. These force magnitudes are described in the literature as the “optimal” forces for application in experiments (Janjic et al. 2018; Sun et al. 2021). An “optimal” force is known as the force level not effecting cell viability and proliferation but leading to a modified expression of genes and metabolites. Cell viability and proliferation were assessed. OS/force-related genes and metabolites expression regulation was analysed in genes/metabolites related to inflammation, bone remodelling, autophagy and apoptosis. Gene expression at the transcriptional level was assessed using RT-qPCR and included the following genetic loci: (a) inflammation: CXCL8/IL8, IL6, PTRGS2/COX2; (b) bone remodelling: RUNX2, P2RX7, BGLAP, TNFRSF11B/OPG; (c) autophagy: MAP1LC3A/LC3, BECN1; (d) apoptosis: CASP3, CASP8. Additionally, IL6 and PGE2 secretion were determined by ELISA (Hosseini et al. 2024a; Hosseini et al. 2024b). Taken together, the main goal of this project was to examine the effect of increased oxidative stress environment on molecular events during OTM. Herein and to our knowledge, we established for the first time a dual model to study the effects of OS on mechanically stimulated hPDLCs and hOBs. The fundamental operating techniques of these two experimental setups can be simply defined by two approaches: tensile forces are applied through the deformation of the substrate (Sun et al. 2021), while compressive forces are applied through the application of weight (Janjic et al. 2018). Compressive force stimulation using WAB model: To mimic the orthodontic compressive force, the weight approach-based (WAB) in vitro loading model was utilized (Figure 2) (Janjic et al. 2018). The WAB model has been used since its introduction (Kanai 1992) to investigate the effects of static, compressive, unidirectional force on cellular responses during OTM (Janjic et al. 2018). This model makes it possible to study the cellular and molecular pathways, especially bone resorption and osteoclastogenesis induction, on the compression side during orthodontic tooth movement. In this model, cells are precultured in culture dishes and subjected to mechanical loading by placing a glass slide over the cell monolayer, with a glass cylinder filled with lead granules positioned on top to apply controlled weight. The proper force distribution is ensured by the glass slide, and the amount of the compressive force can be modulated by adjusting the number of lead granules in the cylinder (Janjic et al. 2018; Kanai 1992; Kanzaki et al. 2002). In the first study (Hosseini et al. 2024a), static mechanical compression was applied to hPDLCs. Periodontal ligament cells, in general, play a crucial role in mechanosensing by responding to mechanical loads applied to teeth, particularly in response to orthodontic forces (Sokos et al. 2015; Yang et al. 2015). When subjected to static compression, these cells trigger a cascade of biochemical reactions that drive bone resorption and tooth movement (Yang et al. 2015). Accordingly, our first study was designed to better understand how hPDLCs respond to compressive stimulation combined with OS pre-stimulation. Tension force stimulation using a custom-made tension apparatus: Different in vitro models have been developed to study cellular responses to tensile mechanical force (Sun et al. 2021). These models focused on two key parameters: temporal variation (continuous "static" or intermittent "dynamic" force) and direction of the force application (uniaxial tension along a single principal axis or equibiaxial tension across all directions) (Sun et al. 2021). In the current project, a custom-made tension apparatus was used to apply static equibiaxial tensile force (Figure 3) (Sun et al. 2022). In the second study (Hosseini et al. 2024b), static tensile force was applied to hOBs to investigate the role of tensile forces in promoting bone formation by triggering osteogenic regulation (Sun et al. 2021). As known, bone formation consists of matrix formation and mineralization and osteoblasts are key players in this process (Šromová et al. 2023). These cells contribute to matrix protein synthesis and mineralization in response to mechanical loading (Šromová et al. 2023; Xiao et al. 2023). Moreover, they play a significant role in tension-induced osteogenesis, further highlighting their involvement in bone remodelling (Xiao et al. 2023). In this regard, our second study was designed to better understand how hOBs respond to tension stimulation in combination with OS pre-stimulation. Clinical relevance of the project: Better understanding of the cellular and biological responses could enhance both the effectiveness and duration of orthodontic tooth movement (OTM). This understanding will particularly help to improve treatment strategies for adult patients, who often require more efficient interdisciplinary treatment approaches with lower risks. From a therapeutic point, improved knowledge of biology and mechanobiology of the underlying mechanisms in regulation of OTM, will guide to the development of more targeted orthodontic materials, leading to a “biologization” of orthodontic therapy. Further research is required to bridge the gap between biological concepts and clinical practice. This concept could implement personalized treatment strategies based on each patient's individual biological response and would also consider age-related variations and/or coexistence of other oral pathologies such as periodontitis. Given that OS is a common factor in numerous oral diseases, the findings from this research could be used as an initial step for future clinical studies. Summary of the papers and key findings: Both studies employed an in vitro model using H2O2 to induce OS and examined its impact on hPDLCs and hOBs under mechanical forces relevant to OTM. In both cell types, pre-exposure to H2O2 altered mechanosensing under mechanical force, and affected cell viability and proliferation. In hPDLCs, changes in gene expression related to inflammation (IL6, CXCL8/IL8, PTGS2/COX2), bone remodelling (RUNX2, TNFRSF11B/OPG, BGLAP), apoptosis (CASP3, CASP8), and autophagy (MAP1LC3A/LC3, BECN1) were examined (Hosseini et al. 2024a). Static compression upregulated PTGS2/COX2 expression, particularly in H2O2-prestimulated groups, which was accompanied by increased PGE2 levels. Similarly, static compression enhanced CXCL8/IL8 expression, especially when pretreatment with H2O2 was done. IL6 expression was downregulated. H2O2 pre-exposure reduced osteogenesis-related gene expression after compression. While autophagy-related genes showed a slight upregulation, apoptosis-related genes were generally downregulated (Hosseini et al. 2024a). In hOBs, changes in gene expression related to inflammation (IL6, CXCL8/IL8, PTGS2/COX2), bone remodelling (RUNX2, TNFRSF11B/OPG, P2RX7), apoptosis (CASP3, CASP8), and autophagy (MAP1LC3A/LC3, BECN1) were examined (Hosseini et al. 2024b). OS influenced tension-induced mechanotransduction. In general, 15 % of tension force upregulated the expression of examined genes. While tension alone upregulated genes related to osteogenic differentiation, OS pre-stimulation did not significantly enhance this effect. Key inflammatory markers (CXCL8/IL8, IL6, PTGS2/COX2) and apoptosis/autophagy-related genes (CASP3, CASP8, MAP1LC3A/LC3, BECN1) showed varied responses. Additionally, increased PTGS2/COX2 expression with higher concentrations of H2O2 correlated with elevated PGE2 levels (Hosseini et al. 2024b). The findings of these two in vitro projects confirm the altering effects of OS on both cell types. The results also showed the potential prolonged influence of OS on important cellular processes related to inflammation, bone remodelling, autophagy and apoptosis during OTM. These findings and modulations of gene expressions show the complexity of OS in relation to mechanical stimulation and underscore the need for more studies in more physiologically relevant environments, in vivo. Such studies will lead to the betterment of clinical orthodontic treatments by having more personalized treatment plans specially in adult patients with presented periodontal diseases. Such studies will improve clinical orthodontic treatment by enabling more personalized treatment planning, particularly for adult patients with coexisting chronic inflammatory oral conditions, such as periodontal disease.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: oxidative stress, human periodontal ligament cells, human osteoblasts, tensile strain, static compressive force, bone remodeling, orthodontic tooth movement
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 26. Januar 2026
• 1. Bericht­erstatter:in: Baumert, Uwe