Selection and validation of reference genes by RT-qPCR for murine cementoblasts in mechanical loading experiments simulating orthodontic forces in vitro

Niederau, Christian; Craveiro, Rogerio B.; Azraq, Irma; Brockhaus, Julia; Bastian, Asisa; Kirschneck, Christian; Wolf, Michael

doi:10.1038/s41598-020-67449-w

Cited by 13 publications

(15 citation statements)

References 45 publications

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“…[37,38] During mechanical loading and OTM, alteration of the ECM features occurs due to a release of soluble factors, that may exhibit either positive or negative effects on PDL remodeling and eventually trigger a local inflammatory response. [39][40][41][42] Periodontal ligament fibroblasts (PDLFs), PDLSCs, and cementoblasts have been investigated in in vitro and in vivo models focusing on the secretion of cytokines and growth factors turnover during remodeling and regeneration. [10,43] All of these modifications inevitably activate mechanotransduction signals that advance the internal signal propagation cascades and gene transcription.…”

Section: Molecular Mechanisms Of Periodontal Remodeling and Orthodontmentioning

confidence: 99%

Current Trends in In Vitro Modeling to Mimic Cellular Crosstalk in Periodontal Tissue

Aveic

Craveiro

Wolf

et al. 2020

Adv Healthcare Materials

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Clinical evidence indicates that in physiological and therapeutic conditions a continuous remodeling of the tooth root cementum and the periodontal apparatus is required to maintain tissue strength, to prevent damage, and to secure teeth anchorage. Within the tooth's surrounding tissues, tooth root cementum and the periodontal ligament are the key regulators of a functional tissue homeostasis. While the root cementum anchors the periodontal fibers to the tooth root, the periodontal ligament itself is the key regulator of tissue resorption, the remodeling process, and mechanical signal transduction. Thus, a balanced crosstalk of both tissues is mandatory for maintaining the homeostasis of this complex system. However, the mechanobiological mechanisms that shape the remodeling process and the interaction between the tissues are largely unknown. In recent years, numerous 2D and 3D in vitro models have sought to mimic the physiological and pathophysiological conditions of periodontal tissue. They have been proposed to unravel the underlying nature of the cell–cell and the cell–extracellular matrix interactions. The present review provides an overview of recent in vitro models and relevant biomaterials used to enhance the understanding of periodontal crosstalk and aims to provide a scientific basis for advanced regenerative strategies.

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Section: Molecular Mechanisms Of Periodontal Remodeling and Orthodontmentioning

confidence: 99%

Current Trends in In Vitro Modeling to Mimic Cellular Crosstalk in Periodontal Tissue

Aveic

Craveiro

Wolf

et al. 2020

Adv Healthcare Materials

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“…Together from previous studies [ 12 – 15 ], the ribosomal 60S protein L22 ( RPL22 ), Peptidylprolyl isomerase B ( PPIB ), polymerase RNA II polypeptide A ( POLR2A ), Ribosomal protein, large, P0 ( RPLP0 ), glucuronidase, beta ( GUSB ), Actin-beta ( β-actin ), TATA-binding protein ( TBP ), ubiquitin C ( UBC ), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Tyrosine 3-monooxygenase/tryptophan 5-mono-oxygenase activate protein, zeta ( YWHAZ ), eukaryotic translation elongation factor 1 alpha 1 ( EEF1A1 ), Ribosomal protein L18 ( RPL ) and beta-5 class I ( TUBB ) were identified as reliable reference genes.…”

Section: Introductionmentioning

confidence: 83%

“…Immortalized murine mouse cementoblast (OCCM-30) cell line [ 16 ] and immortalized mouse murine periodontal ligament (SV-PDL) cells [ 17 ] were provided by Prof. Martha J. Somerman (Laboratory of Oral Connective Tissue Biology, NIH, Bethesda, USA). The optimal density of OCCM-30 [ 18 ] and SV-PDL cells [ 17 ] were previously determined, thus 60—100% confluence status of cells were used for cell confluence experiments [ 15 ].…”

Section: Methodsmentioning

confidence: 99%

Selection and validation of reference gene for RT-qPCR studies in co-culture system of mouse cementoblasts and periodontal ligament cells

et al. 2022

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Objective RT-qPCR is a reliable method for gene expression analysis, but the accuracy of the quantitative data depends on the appropriate selection of reference genes. A Co-culture system consisting of periodontal ligament cells (SV-PDL) and cementoblasts (OCCM-30) to investigate the crosstalk between these two cell lines under orthodontic condition is essential for experimental orthodontic setups in-vitro. Therefore, we aimed to identify a set of reliable reference genes suitable for RT-qPCR studies for prospective co-culture systems of OCCM-30 and SV-PDL cells. Results The results demonstrated that PPIB, GUSB and RPLP0 turned out to be the three most stable reference genes for OCCM-30 in the co-culture system, while PPIB, POLR2A and RPLP0 have the three highest rankings for SV-PDL cells in the co-culture system. The most stable gene combination were PPIB and POLR2A in the co-culture system. In conclusion, PPIB is overall the most stably expressed reference gene for OCCM-30 or SV-PDL cell line in the system. The combination of PPIB and POLR2A as reference genes are indicated to be the potential and mandatory to obtain accurate quantification results for normalizing RT-qPCR data in genes of interest expression in these two cell lines co-culture systems.

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“…Therefore, cyclic tension induces a decreased RANKL/OPG ratio whereas the opposite is shown on the compression side 81 . Furthermore, static compression forces applied on OCCM-30 initiate an upregulation in sub-confluent conditions whereas it is downregulated in confluent monolayer 43 82 . In vitro experiments with human cementoblasts show consistent results with static compressive loadings after 6 hours of stimulation 61 .…”

Section: Main Signaling Pathways Induced By Mechanical Forcesmentioning

confidence: 96%

Mechanical Loading on Cementoblasts: A Mini Review

et al. 2022

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Orthodontic treatments are concomitant with mechanical forces and thereby cause teeth movements. The applied forces are transmitted to the tooth root and the periodontal ligaments which is compressed on one side and tensed up on the other side. Indeed, strong forces can lead to tooth root resorption and the crown-to-tooth ratio is reduced with the potential for significant clinical impact. The cementum, which covers the tooth root, is a thin mineralized tissue of the periodontium that connects the periodontal ligament with the tooth and is build up by cementoblasts. The impact of tension and compression on these cells is investigated in several in vivo and in vitro studies demonstrating differences in protein expression and signaling pathways. In summary, osteogenic marker changes indicate that cyclic tensile forces support whereas static tension inhibits cementogenesis. Furthermore, cementogenesis experiences the same protein expression changes in static conditions as static tension, but cyclic compression leads to the exact opposite of cyclic tension. Consistent with marker expression changes, the singaling pathways of Wnt/ß-catenin and RANKL/OPG show that tissue compression leads to cementum degradation and tension forces to cementogenesis. However, the cementum, and in particular its cementoblasts, remain a research area which should be explored in more detail to understand the underlying mechanism of bone resorption and remodeling after orthodontic treatments.

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Selection and validation of reference genes by RT-qPCR for murine cementoblasts in mechanical loading experiments simulating orthodontic forces in vitro

Cited by 13 publications

References 45 publications

Current Trends in In Vitro Modeling to Mimic Cellular Crosstalk in Periodontal Tissue

Current Trends in In Vitro Modeling to Mimic Cellular Crosstalk in Periodontal Tissue

Selection and validation of reference gene for RT-qPCR studies in co-culture system of mouse cementoblasts and periodontal ligament cells

Mechanical Loading on Cementoblasts: A Mini Review

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