Effects Of IRoot FS On Biological Behavior Of Osteoblasts And Dental Pulp Cells

Bioceramic material is commonly used in endodontic procedures involving pulpal regeneration and hard tissue repair,such as pulp capping,perforation repair,and root-end filling.iRoot FS(Innovative BioCeramix Inc.,Vancouver,BC,Canada)is a nanoparticle bioceramic material with a short setting time and ready-to-use characteristics.According to the manufacturer’s instructions,iRoot FS can set within 20 min.Pulp capping and root repair material should possess great biocompatibility and bioactivity,especially the capacity to promote hard tissue regeneration in dental pulp or periapical zone.Osteoblast is the main component of bone tissue.In root perforation repair or apical surgery,iRoot FS is applied to contact with bone tissue directly or indirectly through microcirculation.So the effect of iRoot FS on osteoblast is the main criteria to evaluate the root filling and repair material.During direct pulp capping,the capping material directly contact with the exposed dental pulp to maintain its vitality and promote reparative dentine formation.Dental pulp cells are the main components of pulp tissue.Therefore,the effect of capping material on dental pulp cells can be used to evaluate the biocompatibility and bioactivity of pulp capping material.However,little information is avaliable on the biological roles of iRoot FS in osteoblast and dental pulp cells.The present study investigates the effect of bioceramic material iRoot FS on biological behavior of osteoblasts and dental pulp cells.The performance of iRoot BP Plus and MTA were also compared with that of iRoot FS,aiming to provide information for the clinical application of this new fast-setting bioceramic material.Part Ⅰ:Effect of iRoot FS on Biological Behavior of Osteoblasts in vitroObjective:To investigate the in vitro effect of iRoot FS(Innovative BioCeramix Inc.,Vancouver,BC,Canada)on biological behavior of MC3T3-E1 cells and to compare the performance of iRoot FS with those of iRoot BP Plus(Innovative BioCeramix Inc.)and ProRoot mineral trioxide aggregate(MTA;Dentsply Tulsa Dental,Tulsa,OK,USA).Methods:Various dilutions of iRoot FS,iRoot BP Plus and MTA extracts were obtained after 7 days of setting.MC3T3-E1 cells were cultured in material extract for 1,2 and 3 days.Then cell viability was assessed by using a cell counting kit-8.The cell apoptosis induced by the set material extracts was evaluated through annexin V-propidium iodide flow cytometry.Changes in the cytoskeletal organization and stress fibres were observed through immunofluorescence.Cell attachment was observed under a scanning electron microscope after the MC3T3-E1 cells were seeded on the surface iRoot FS.Alkaline phosphate activity and staining Kit was used to detect the expression of ALP.Alizarine staining was used to detect the calcium deposition.The osteogenic mRNA and protein expression of osteoblast was detected by real-time PCR and Western Blot.Results:iRoot FS extracts induced higher cell viability than the control extracts without evident cytotoxicity.Stretched stress fibres and cytoskeletons were detected in the cells treated with iRoot FS extracts.Scanning electron microscopy revealed that the MC3T3-E1 cells attached to iRoot FS appeared flatter and exhibited better stretch than those attached to the other extracts.iRoot FS increased ALP activity and the formation of mineralized nodules and up-regulated the expression of osteoblastic marker mRNA and protein.Conclusions:iRoot FS promoted in vitro viability,attachment,cytoskeletal organization and osteogenesis of MC3T3-E1 cells without causing cell apoptosis.Part II:Effect of iRoot FS on Biological Behavior of Dental Pulp Cells in vitro and in vivoExperiment one:Effect of iRoot FS on Biological Behavior of Dental Pulp Cells in vitroObjective:To investigate the effects on biological behavior of human dental pulp cells by iRoot FS in vitro,and to explore the underlying molecular mechanism of the effect on dental pulp cell odontogenesis by iRoot FS.Methods:Primary cultured human dental pulp cells were treated with various dilutions of iRoot FS and MTA extracts to assess cell viability using a cell counting kit-8.Cell adhesion assay was performed using type I collagen-coated plates.In vitro scratch wound healing model and Transwell assay were used to determine dental pulp cells migration.ALP activity assessment was performed to evaluate the cell differentiation potential on days 3,7 and 10.Alizarin Red staining were used to detect the calcium deposition after treating dental pulp cells with iRoot FS extracts for 21 days.Also,we performed western blot assessment to detect the expression of odontogenic and migration molecules in material-treated dental pulp cells,as well as the molecules in underlying molecular mechanism.Results:The viability of cells treated with iRoot FS extracts was comparable to that of control extracts.The effect was similar with MTA and iRoot BP Plus.Treatment with iRoot FS extracts promoted dental pulp cells adhension as well as horizontal and vertical migration.iRoot FS enhanced the odontogenic capacity of dental pulp cells,including ALP activity and calcium deposition.iRoot FS treatment resulted in the accumulation of odontogenic and migration molecules.In addition,AMPK/ERK signaling pathway was activated to promote dental pulp cells odontogenesis by iRoot FS.Conclusion:iRoot FS,as a new direct pulp-capping material,showed cytocompatibility on dental pulp cells and induced adhesion,migration,and odontogenic differentiation in vitro.Experiment two:Effect of iRoot FS on Biological Behavior of Dental Pulp Cells in vivoObjective:To investigate the in vivo effects of iRoot FS on biological behavior of dental pulp cells,and to explore the underlying molecular mechanism of dentine bridge formation.Methods:After direct pulp capping with MTA,iRoot BP Plus and iRoot FS,the maxillary first molar segments dissected from male Wistar rats were used to observe the formation of dentine bridge and expression of odontogenic and migration molecules at the injury site of pulp repair model.Related molecular signaling pathways were detected by histologic analysis.Results:iRoot FS promoted dentine bridge formation and the expression of odontogenic and migration molecules in pulp capping therapy model in vivo.AMPK/ERK signaling pathway was activated by iRoot FS in the formation of dentine bridge.Conclusion:iRoot FS facilitated dentin bridge formation in a pulp capping model in vivo.