Role and Regulation of Mechanotransductive HIF-1α Stabilisation in Periodontal Ligament Fibroblasts

Orthodontic tooth movement (OTM) creates compressive and tensile strain in the periodontal ligament, causing circulation disorders. Hypoxia-inducible factor 1α (HIF-1α) has been shown to be primarily stabilised by compression, but not hypoxia in periodontal ligament fibroblasts (PDLF) during mechanical strain, which are key regulators of OTM. This study aimed to elucidate the role of heparan sulfate integrin interaction and downstream kinase phosphorylation for HIF-1α stabilisation under compressive and tensile strain and to which extent downstream synthesis of VEGF and prostaglandins is HIF-1α-dependent in a model of simulated OTM in PDLF. PDLF were subjected to compressive or tensile strain for 48 h. In various setups HIF-1α was experimentally stabilised (DMOG) or destabilised (YC-1) and mechanotransduction was inhibited by surfen and genistein. We found that HIF-1α was not stabilised by tensile, but rather by compressive strain. HIF-1α stabilisation had an inductive effect on prostaglandin and VEGF synthesis. As expected, HIF-1α destabilisation reduced VEGF expression, whereas prostaglandin synthesis was increased. Inhibition of integrin mechanotransduction via surfen or genistein prevented stabilisation of HIF-1α. A decrease in VEGF expression was observed, but not in prostaglandin synthesis. Stabilisation of HIF-1α via integrin mechanotransduction and downstream phosphorylation of kinases seems to be essential for the induction of VEGF, but not prostaglandin synthesis by PDLF during compressive (but not tensile) orthodontic strain.

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Myeloid HIF1α Is Involved in the Extent of Orthodontically Induced Tooth Movement

Biomedicines ◽

10.3390/biomedicines9070796 ◽

2021 ◽

Vol 9 (7) ◽

pp. 796

Author(s):

Christian Kirschneck ◽

Nadine Straßmair ◽

Fabian Cieplik ◽

Eva Paddenberg ◽

Jonathan Jantsch ◽

...

Keyword(s):

Bone Density ◽

Periodontal Ligament ◽

Tooth Movement ◽

Hypoxia Inducible Factor ◽

Myeloid Cells ◽

Orthodontic Tooth Movement ◽

Periodontal Ligament Fibroblasts ◽

Expression Of Genes ◽

Periodontal Bone Loss

During orthodontic tooth movement, transcription factor hypoxia-inducible factor 1α (HIF1α) is stabilised in the periodontal ligament. While HIF1α in periodontal ligament fibroblasts can be stabilised by mechanical compression, in macrophages pressure application alone is not sufficient to stabilise HIF1α. The present study was conducted to investigate the role of myeloid HIF1α during orthodontic tooth movement. Orthodontic tooth movement was performed in wildtype and Hif1αΔmyel mice lacking HIF1α expression in myeloid cells. Subsequently, µCT images were obtained to determine periodontal bone loss, extent of orthodontic tooth movement and bone density. RNA was isolated from the periodontal ligament of the control side and the orthodontically treated side, and the expression of genes involved in bone remodelling was investigated. The extent of tooth movement was increased in Hif1αΔmyel mice. This may be due to the lower bone density of the Hif1αΔmyel mice. Deletion of myeloid Hif1α was associated with increased expression of Ctsk and Acp5, while both Rankl and its decoy receptor Opg were increased. HIF1α from myeloid cells thus appears to play a regulatory role in orthodontic tooth movement.

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Role of Oxygen Supply in Macrophages in a Model of Simulated Orthodontic Tooth Movement

Mediators of Inflammation ◽

10.1155/2020/5802435 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

Agnes Schröder ◽

Leonie Barschkies ◽

Jonathan Jantsch ◽

Peter Proff ◽

Lina Gölz ◽

...

Keyword(s):

Cell Culture ◽

Periodontal Ligament ◽

Oxygen Supply ◽

Tooth Movement ◽

Mechanical Strain ◽

Orthodontic Tooth Movement ◽

Hypoxic Cell ◽

Periodontal Ligament Fibroblasts ◽

Hypoxic Conditions ◽

Expression Of Genes

Apart from periodontal ligament fibroblasts, immune cells like macrophages also play an important mediating role in orthodontic tooth movement (OTM). Upon orthodontic force application to malpositioned teeth, macrophages in the periodontal ligament get exposed to both mechanical strain and hypoxic conditions (via a compression of blood vessels). In this study, we assessed the relative impact of orthodontically induced mechanical strain and hypoxic conditions on macrophages for the mediation and regulation of OTM. Macrophages were stimulated with physiological orthodontic compressive forces of 2 g/cm2 for 4 h and 24 h on gas-impermeable or gas-permeable cell culture plates under normoxic or hypoxic cell culture conditions. We quantified expression of genes involved in inflammation (Tnf, Il-6, and Cox-2), extracellular remodelling (Mmp-9), and angiogenesis (Vegf) by RT-qPCR. Furthermore, we analysed HIF-1α, prostaglandin-E2, and VEGF protein expression via immunoblotting or ELISA. Mechanical strain and oxygen supply both differentially affected expression of genes and proteins involved in inflammation and angiogenesis. In this context, we found that HIF-1α protein levels were elevated by combined mechanical strain and hypoxic conditions, whereas gas-permeable plates providing sufficient oxygen supply prevented HIF-1α stabilization at the protein level after pressure application on macrophages. Our results thus indicate that macrophages involved in the mediation of OTM are affected by and respond differently to hypoxic conditions and mechanical compressive strain, which occur concomitantly during OTM, than periodontal ligament fibroblasts (PDLF), thus indicating different roles of these cells in the regulation of OTM at the cellular-molecular level. We further observed that contrary to PDLF HIF-1α stabilization in macrophages is rather induced via the decreased oxygen supply associated with OTM than via mechanotransduction by mechanical strain.

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Effects of the Highly COX-2-Selective Analgesic NSAID Etoricoxib on Human Periodontal Ligament Fibroblasts during Compressive Orthodontic Mechanical Strain

Mediators of Inflammation ◽

10.1155/2019/2514956 ◽

2019 ◽

Vol 2019 ◽

pp. 1-14 ◽

Cited By ~ 13

Author(s):

Christian Kirschneck ◽

Erika Calvano Küchler ◽

Michael Wolf ◽

Gerrit Spanier ◽

Peter Proff ◽

...

Keyword(s):

Extracellular Matrix ◽

Periodontal Ligament ◽

Tooth Movement ◽

Compressive Strain ◽

Mechanical Strain ◽

Orthodontic Tooth Movement ◽

Extracellular Matrix Remodeling ◽

Matrix Remodeling ◽

Periodontal Inflammation ◽

Cox 2

Human periodontal ligament (hPDL) fibroblasts play a major role during periodontitis and orthodontic tooth movement, mediating periodontal inflammation, osteoclastogenesis, and collagen synthesis. The highly COX-2-selective NSAID etoricoxib has a favorable systemic side effect profile and high analgesic efficacy, particularly for orthodontic pain. In this in vitro study, we investigated possible side effects of two clinically relevant etoricoxib concentrations on the expression pattern of mechanically strained hPDL fibroblasts and associated osteoclastogenesis in a model of simulated orthodontic compressive strain occurring during orthodontic tooth movement. hPDL fibroblasts were incubated for 72 h under physiological conditions with etoricoxib at 0 μM, 3.29 μM, and 5.49 μM, corresponding to clinically normal and subtoxic dosages, with and without mechanical strain by compression (2 g/cm2) for the final 48 h, simulating conditions during orthodontic tooth movement in compressive areas of the periodontal ligament. We then determined gene and/or protein expression of COX-2, IL-6, PG-E2, RANK-L, OPG, ALPL, VEGF-A, P4HA1, COL1A2, and FN1 via RT-qPCR, ELISA, and Western blot analyses as well as apoptosis, necrosis, cell viability, and cytotoxicity via FACS, MTT, and LDH assays. In addition, hPDL fibroblast-mediated osteoclastogenesis was assessed by TRAP staining in coculture with RAW267.4 cells for another 72 h. Gene and protein expression of all evaluated factors was significantly induced by the mechanical compressive strain applied. Etoricoxib at 3.29 μM and 5.49 μM significantly inhibited PG-E2 synthesis, but not COX-2 and IL-6 gene expression nor RANK-L-/OPG-mediated osteoclastogenesis or angiogenesis (VEGF-A). Extracellular matrix remodeling (COL1A2, FN1) and bone anabolism (ALPL), by contrast, were significantly stimulated particularly at 5.49 μM. In general, no adverse etoricoxib effects on hPDL fibroblasts regarding apoptosis, necrosis, cell viability, or cytotoxicity were detected. Clinically dosed etoricoxib, that is, a highly selective COX-2 inhibition, did not have substantial effects on hPDL fibroblast-mediated periodontal inflammation, extracellular matrix remodeling, RANK-L/OPG expression, and osteoclastogenesis during simulated orthodontic compressive strain.

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The role of mechanotransduction versus hypoxia during simulated orthodontic compressive strain—an in vitro study of human periodontal ligament fibroblasts

International Journal of Oral Science ◽

10.1038/s41368-019-0066-x ◽

2019 ◽

Vol 11 (4) ◽

Cited By ~ 9

Author(s):

Niklas Ullrich ◽

Agnes Schröder ◽

Jonathan Jantsch ◽

Gerrit Spanier ◽

Peter Proff ◽

...

Keyword(s):

Periodontal Ligament ◽

Tooth Movement ◽

Compressive Strain ◽

Orthodontic Tooth Movement ◽

In Vitro Study ◽

Minor Role ◽

Periodontal Ligament Fibroblasts ◽

Signalling Molecules ◽

Hypoxic Conditions

Abstract During orthodontic tooth movement (OTM) mechanical forces trigger pseudo-inflammatory, osteoclastogenic and remodelling processes in the periodontal ligament (PDL) that are mediated by PDL fibroblasts via the expression of various signalling molecules. Thus far, it is unknown whether these processes are mainly induced by mechanical cellular deformation (mechanotransduction) or by concomitant hypoxic conditions via the compression of periodontal blood vessels. Human primary PDL fibroblasts were randomly seeded in conventional six-well cell culture plates with O2-impermeable polystyrene membranes and in special plates with gas-permeable membranes (Lumox®, Sarstedt), enabling the experimental separation of mechanotransducive and hypoxic effects that occur concomitantly during OTM. To simulate physiological orthodontic compressive forces, PDL fibroblasts were stimulated mechanically at 2 g·cm−2 for 48 h after 24 h of pre-incubation. We quantified the cell viability by MTT assay, gene expression by quantitative real-time polymerase chain reaction (RT-qPCR) and protein expression by western blot/enzyme-linked immunosorbent assays (ELISA). In addition, PDL-fibroblast-mediated osteoclastogenesis (TRAP+ cells) was measured in a 72-h coculture with RAW264.7 cells. The expression of HIF-1α, COX-2, PGE2, VEGF, COL1A2, collagen and ALPL, and the RANKL/OPG ratios at the mRNA/protein levels during PDL-fibroblast-mediated osteoclastogenesis were significantly elevated by mechanical loading irrespective of the oxygen supply, whereas hypoxic conditions had no significant additional effects. The cellular–molecular mediation of OTM by PDL fibroblasts via the expression of various signalling molecules is expected to be predominantly controlled by the application of force (mechanotransduction), whereas hypoxic effects seem to play only a minor role. In the context of OTM, the hypoxic marker HIF-1α does not appear to be primarily stabilized by a reduced O2 supply but is rather stabilised mechanically.

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Effects of mechanical strain on periodontal ligament fibroblasts in presence of Aggregatibacter actinomycetemcomitans lysate

BMC Oral Health ◽

10.1186/s12903-021-01761-3 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Agnes Schröder ◽

Julia Stumpf ◽

Eva Paddenberg ◽

Patrick Neubert ◽

Valentin Schatz ◽

...

Keyword(s):

Periodontal Ligament ◽

Tooth Movement ◽

Bone Remodelling ◽

Mechanical Strain ◽

Orthodontic Tooth Movement ◽

Aggregatibacter Actinomycetemcomitans ◽

Periodontal Ligament Fibroblasts ◽

Reduced Pressure ◽

Expression Of Genes ◽

Ros Formation

Abstract Purpose Many adult orthodontic patients suffer from periodontitis, which is caused by oral pathogens such as the gram-negative Aggregatibacter actinomycetemcomitans (Agac). Like orthodontic tooth movement, periodontitis is associated with inflammation and alveolar bone remodelling thereby affecting orthodontic treatment. Interactions of both processes, however, are not sufficiently explored, particularly with regard to oxidative stress. Methods After preincubation with Agac lysate for 24 h periodontal ligament fibroblasts (PDLF) were either stretched or compressed for further 48 h simulating orthodontic forces in vitro. We analysed the expression of genes and proteins involved in the formation of reactive oxygen species (NOX-4, ROS) and nitric oxide (NOS-2), inflammation (TNF, IL-6, PTGS-2) and bone remodelling (OPG, RANKL). Results Agac lysate elevated the expression of NOX-4, NOS-2, inflammatory IL-6 and PTGS-2 and the bone-remodelling RANKL/OPG ratio during compressive, but not tensile mechanical strain. Agac lysate stimulated pressure-induced inflammatory signalling, whereas surprisingly ROS formation was reduced. Pressure-induced downregulation of OPG expression was inhibited by Agac lysate. Conclusions Agac lysate impact on the expression of genes and proteins involved in inflammation and bone remodelling as well as ROS formation, when PDLF were subjected to mechanical forces occurring during orthodontic tooth movement.

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Impact of Leptin on Periodontal Ligament Fibroblasts during Mechanical Strain

International Journal of Molecular Sciences ◽

10.3390/ijms22136847 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6847

Author(s):

Agnes Schröder ◽

Andrea Meyer ◽

Gerrit Spanier ◽

Anna Damanaki ◽

Eva Paddenberg ◽

...

Keyword(s):

Periodontal Ligament ◽

Orthodontic Treatment ◽

Tooth Movement ◽

Mechanical Strain ◽

Orthodontic Tooth Movement ◽

Tartrate Resistant Acid Phosphatase ◽

Obese Patients ◽

Periodontal Ligament Fibroblasts ◽

Gene And Protein Expression ◽

Proinflammatory Factors

Orthodontic treatment to correct dental malocclusions leads to the formation of pressure zones in the periodontal ligament resulting in a sterile inflammatory reaction, which is mediated by periodontal ligament fibroblasts (PDLF). Leptin levels are elevated in obesity and chronic inflammatory responses. In view of the increasing number of orthodontic patients with these conditions, insights into effects on orthodontic treatment are of distinct clinical relevance. A possible influence of leptin on the expression profile of PDLF during simulated orthodontic mechanical strain, however, has not yet been investigated. In this study, PDLF were exposed to mechanical strain with or without different leptin concentrations. The gene and protein expression of proinflammatory and bone-remodelling factors were analysed with RT-qPCR, Western-blot and ELISA. The functional analysis of PDLF-induced osteoclastogenesis was analysed by TRAP (tartrate-resistant acid phosphatase) staining in coculture with human macrophages. Pressure-induced increase of proinflammatory factors was additionally elevated with leptin treatment. PDLF significantly increased RANKL (receptor activator of NF-kB ligand) expression after compression, while osteoprotegerin was downregulated. An additional leptin effect was demonstrated for RANKL as well as for subsequent osteoclastogenesis in coculture after TRAP staining. Our results suggest that increased leptin concentrations, as present in obese patients, may influence orthodontic tooth movement. In particular, the increased expression of proinflammatory factors and RANKL as well as increased osteoclastogenesis can be assumed to accelerate bone resorption and thus the velocity of orthodontic tooth movement in the orthodontic treatment of obese patients.

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In Vitro Compression Model for Orthodontic Tooth Movement Modulates Human Periodontal Ligament Fibroblast Proliferation, Apoptosis and Cell Cycle

Biomolecules ◽

10.3390/biom11070932 ◽

2021 ◽

Vol 11 (7) ◽

pp. 932

Author(s):

Julia Brockhaus ◽

Rogerio B. Craveiro ◽

Irma Azraq ◽

Christian Niederau ◽

Sarah K. Schröder ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Periodontal Ligament ◽

Environmental Changes ◽

Tooth Movement ◽

Orthodontic Tooth Movement ◽

Compressive Force ◽

Periodontal Ligament Fibroblasts ◽

Human Periodontal Ligament Fibroblasts

Human Periodontal Ligament Fibroblasts (hPDLF), as part of the periodontal apparatus, modulate inflammation, regeneration and bone remodeling. Interferences are clinically manifested as attachment loss, tooth loosening and root resorption. During orthodontic tooth movement (OTM), remodeling and adaptation of the periodontium is required in order to enable tooth movement. hPDLF involvement in the early phase-OTM compression side was investigated for a 72-h period through a well-studied in vitro model. Changes in the morphology, cell proliferation and cell death were analyzed. Specific markers of the cell cycle were investigated by RT-qPCR and Western blot. The study showed that the morphology of hPDLF changes towards more unstructured, unsorted filaments under mechanical compression. The total cell numbers were significantly reduced with a higher cell death rate over the whole observation period. hPDLF started to recover to pretreatment conditions after 48 h. Furthermore, key molecules involved in the cell cycle were significantly reduced under compressive force at the gene expression and protein levels. These findings revealed important information for a better understanding of the preservation and remodeling processes within the periodontium through Periodontal Ligament Fibroblasts during orthodontic tooth movement. OTM initially decelerates the hPDLF cell cycle and proliferation. After adapting to environmental changes, human Periodontal Ligament Fibroblasts can regain homeostasis of the periodontium, affecting its reorganization.

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