ABSTRACT
The effects of compressive strain during orthodontic treatment on gene expression profiles of periodontal ligament fibroblasts (PDLFs) have mostly been studied in 2D cell culture. However, cells behave differently in many aspects in 3D culture. Therefore, the effect of pressure application on PDLFs in different 3D structures was investigated. PDLFs were either conventionally seeded or embedded into different 3D structures (spheroids, Mebiol® gel, 3D scaffolds) and exposed to compressive force or incubated without pressure. For one 3D scaffold (POR), we also tested the effect of different compressive forces and application times. Expression of an angiogenic gene (VEGF), a gene involved in extracellular matrix synthesis (COL1A2), inflammatory genes (IL6, PTGS2), and genes involved in bone remodelling (OPG, RANKL) were investigated by RT-qPCR. Depending on the used 3D cell culture model, we detected different effects of compressive strain on expression profiles of PDLFs. COL1A2 was downregulated in all investigated 3D culture models. Angiogenetic and proinflammatory genes were regulated differentially between models. In 3D scaffolds, regulation of bone-remodelling genes upon compressive force was contrary to that observed in 3D gels. 3D cell culture models provide better approximations to in vivo physiology, compared with conventional 2D models. However, it is crucial which 3D structures are used, as these showed diverse effects on the expression profiles of PDLFs during mechanical strain.
Subject(s)
Fibroblasts/metabolism , Periodontal Ligament/cytology , Tissue Engineering/methods , Cells, Cultured , Collagen Type I/genetics , Collagen Type I/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Fibroblasts/cytology , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Osteoprotegerin/genetics , Osteoprotegerin/metabolism , Pressure , Primary Cell Culture/methods , RANK Ligand/genetics , RANK Ligand/metabolism , Tissue Scaffolds/chemistry , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
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.
Subject(s)
Fibroblasts/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mechanotransduction, Cellular , Periodontal Ligament/metabolism , Adolescent , Adult , Cells, Cultured , Female , Fibroblasts/drug effects , Focal Adhesion Kinase 1/antagonists & inhibitors , Genistein/pharmacology , Glycine/analogs & derivatives , Glycine/pharmacology , Glycosaminoglycans/antagonists & inhibitors , Humans , Indazoles/pharmacology , Integrins/antagonists & inhibitors , Male , Mechanotransduction, Cellular/drug effects , Mechanotransduction, Cellular/genetics , Periodontal Ligament/cytology , Periodontal Ligament/drug effects , Phosphorylation , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/metabolism , Prostaglandins/biosynthesis , Prostaglandins/metabolism , Protein Stability/drug effects , Signal Transduction/drug effects , Stress, Mechanical , Tooth Movement Techniques , Urea/analogs & derivatives , Urea/pharmacology , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
BACKGROUND: The viability of periodontal ligament fibroblasts (PDLF) can affect the long-term prognosis of replanted avulsed teeth. When immediate replantation of an avulsed tooth is not possible, the cells should be incubated in a physiological storage medium instantly to maintain their biological activity. The ability of different storage media to preserve PDLF viability has been previously evaluated. However, few studies have showed the effect of temperature on the viability of PDLF cultured with various storage media in vitro. MATERIAL AND METHODS: This study was designed to measure PDLF activity by CCK-8 assay to compare the effectiveness at 4, 22 (room temperature), and 37°C under various storage media. RESULTS: Statistical analysis demonstrated that tap water, saline, and saliva decreased cell viability as the storage temperature increased. But the temperature played only a minor role on cell viability when cells were incubated in Hank's balanced salt solution (HBSS), Dubelco's modified Eagle's medium (DMEM), or milk. CONCLUSIONS: Within the parameters of this study, it seems that room temperature is adequate for storing the avulsed teeth in HBSS, DMEM, or milk in the extra-alveolar period.
Subject(s)
Fibroblasts/physiology , Organ Preservation Solutions/pharmacology , Periodontal Ligament/cytology , Tissue Preservation/methods , Animals , Cell Survival , Cells, Cultured , Humans , In Vitro Techniques , Isotonic Solutions/pharmacology , Milk , Saliva , Sodium Chloride/pharmacology , Temperature , Water/pharmacologyABSTRACT
This work focuses on the improvement of the R-PDLF heteronuclear recoupling scheme, a method that allows quantification of molecular dynamics up to the microsecond timescale in heterogeneous materials. We show how the stability of the sequence towards rf-imperfections, one of the main sources of error of this technique, can be improved by the insertion of windows without irradiation into the basic elements of the symmetry-based recoupling sequence. The impact of this modification on the overall performance of the sequence in terms of scaling factor and homonuclear decoupling efficiency is evaluated. This study indicates the experimental conditions for which precise and reliable measurement of dipolar couplings can be obtained using the popular R18(1)(7) recoupling sequence, as well as alternative symmetry-based R sequences suited for fast MAS conditions. An analytical expression for the recoupled dipolar modulation has been derived that applies to a whole class of sequences with similar recoupling properties as R18(1)(7). This analytical expression provides an efficient and precise way to extract dipolar couplings from the experimental dipolar modulation curves. We hereby provide helpful tools and information for tailoring R-PDLF recoupling schemes to specific sample properties and hardware capabilities. This approach is particularly well suited for the study of materials with strong and heterogeneous molecular dynamics where a precise measurement of dipolar couplings is crucial.