Biocompatibility And Apical Sealing Ability Of IRoot SP Root Canal Sealer

The ideal root canal filling material must possess certain characteristics, including biocompatibility, adequate marginal sealing quality, dimensional stability, the ability to allow or induce bone repair and antimicrobial activity. Biocompatibility is one of the factors that influence the clinician’s choice of filling materials in root canal treatment as they might be placed in contact with periapical tissues. Further, the cells and tissues response to these materials might influence the outcome of the root canal treatment. Many endodontic sealers are now used in clinical practice, including zinc-oxide-eugenol cements, glass ionomer cements, epoxy resins, and portland-based cements, but none meets all the appropriate requirements.iRoot SP (Innovative BioCreamix Inc, Vancouver, Canada), a new root canal sealer, is a convenient, premixed, ready-to-use injectable white hydraulic cement paste developed for permanent root canal filling and sealing applications. According to the manufacturer, iRoot SP is an aluminum-free, hydrophilic, calcium silicate based material which requires the presence of water to set and harden. iRoot SP requires no additional curing agent, no mixing, and delivers a consistent, homogeneous product for filling root canals with or without gutta-percha point. iRoot SP includes a similar composition to white mineral trioxide aggregate (MTA) material and has both excellent physical properties and antimicrobial activity.Superior qualities and handling abilities make iRoot SP an innovative and novel endodontic material for root canal sealer or root-end filling material. Although antimicrobial activity of iRoot SP have been investigated, the biocompatibility, sealing ability and effects of iRoot SP on mRNA expression of several mineralization-related genes are still not clear to date. AH-Plus (Dentsply DeTrey, Konstanz, Germany) is a popular resin-based sealer used for permanent root sealing. This sealer provides outstanding long-term dimensional stability and presents optimal handling and working time.The purpose of the present study was to evaluate iRoot SP’s biocompatibility, sealing ability and effects on the expression of mineralization-related genes (type I collagen, osteocalcin, bone sialoprotein and osteopontin) during hard-tissue formation in ostcoblast-like MG63 cells, while also comparing the results with those of AH Plus root canal sealer.Part One: Biocompatibility evaluation of iRoot SP in subcutaneous and intraosseous tissue of ratsObjective:The aim of this study was to evaluate the biocompatibility of iRoot SP in subcutaneous and intraosseous tissue of rats.Materials and Methods: Specimens of iRoot SP, AH Plus and mineral trioxide aggregate (MTA) were placed in the dorsal subcutaneous connective tissue and the tibialis of 28 Wistar rats. In the subcutaneous implant test, tissue specimens were collected after the rats were sacrificed after 7,15,30, and 60 days. In intraosseous implanting test, the specimens were collected after the rats were sacrificed after 15,30, 60 and 90 days. The specimens were fixed, stained, processed, and histologically evaluated under a light microscope. Inflammatory reactions were classified as grade 0, 1,2 and 3. Data were analyzed with the Kruskal-Wallis test.Results:In the subcutaneous implant test, on 7 and 15 days, all groups showed moderate chronic inflammatory cell infiltration (p> 0.05). On 30 days, iRoot SP, MTA and control group showed mild inflammatory cell infiltration and no differences (p>0.05). AH Plus group showed moderate inflammatory and thick fibrous capsules (P<0.05). On 60 days, iRoot SP, MTA, AH Plus and the control group showed mild reaction. There was no differences among the groups(P>0.05). AH Plus group had thick fibrous capsules. In intraosseous implanting test, the control group, with the time growing, new bone was formed and growed into the bone defect resion. The iRoot SP group, on the 15th day of observation, showed mild inflammatory infiltration. Few new bone was formed on the 30th day. Lots of osteoblast were seen along the edge of the material and the amount of new bone formed in direct contact with iRoot SP on the 60th day. New bone on surface of iRoot SP was observed and growed into the materials on the 90th day. The AH Plus group, on the 15th and 30th day, showed moderate inflammatory infiltration. On the 60th day, mild inflammatory was observed. Little new bone formed in direct contact with AH Plus. On the 90th day, the amount of new bone formed in direct contact with AH Plus. The MTA group, on the 15th and 30th day, showed mild inflammatory infiltration. On the 60th day, the amount of new bone formed in direct contact with MTA. On the 90th day, the mature osseous tissue was formed but not observed inside of MTA.Conclusion:iRoot SP and MTA had biocompatibility in subcutaneous and intraosseous tissue of rats.Part Two:Ex vivo cytotoxicity of a new calcium silicate-based canal filling materialObjective:The purpose of this study was to evaluate the biocompatibility of iRoot SP root canal filling material to L929 mouse fibroblasts.Materials and Methods: Specimens of iRoot SP, AH Plus and ProRoot mineral trioxide aggregate (MTA) were prepared for direct contact using the Millipore filter diffusion test, as well as extracts in MTT assay. Mouse fibroblasts (L929) were used as toxicity targets. Differences in cytotoxicity between fresh and set specimens and between root canal material extracts were determined by t-test. A P-value<0.05 was considered statistically significant.Results:In the filter diffusion test, iRoot SP was slightly cytotoxic when tested as a fresh mixture and not cytotoxic in a set condition tested after 24 h. AH Plus was rated moderately cytotoxic when tested as a fresh mixture and slightly cytotoxic in a set (24 h) condition. MTA was noncytotoxic in both a fresh or set state. AH Plus was significantly more toxic than iRoot SP and MTA (P<0.05). In addition, iRoot SP was significantly more toxic than MTA (P<0.05). In the MTT assay with set specimens, extracts of iRoot SP and MTA were noncytotoxic, whilst extracts of AH Plus were rated as slightly cytotoxic. AH Plus was significantly more toxic than iRoot SP and MTA (P<0.05).Conclusion:AH Plus root canal sealer was significantly more toxic to L-929 cells than MTA and iRoot SP. iRoot SP had intermediate toxicity.Part Three:Effects of iRoot SP on Mineralization-related Genes Expression in MG63 CellsObjective: The purpose of this study was to assess iRoot SP root canal sealer’s effects on the expression of mineralization-related genes in human MG63 osteoblast-like cells.Materials and Methods:Specimens (diameter 5 mm and height 2 mm) of iRoot SP and AH Plus were extracted in culture medium. MG63 cells were exposed to various dilutions (1/1,1/2 and 1/4) of the extracts. The MTT assay was used for assessing dental materials’nonspecific cytotoxicity. Expression of mineralization- related genes including collagen typeⅠ(COLⅠ), osteocalcin (OCN), bone sialoprotein (BSP) and osteopontin (OPN) were detected on day 1,3, and 6 by real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay experiment was used for evaluating COL I and BSP protein changes. The data were analyzed by one-way ANOVA and Turkey tests.Results:In the MTT assay, undiluted extracts of iRoot SP were noncytotoxic, while undiluted extracts of AH Plus were rated as slightly cytotoxic. iRoot SP upregulated COLⅠ, OCN and BSP messenger RNA expression after 3 and 6 days. In ELISA experiment, iRoot SP increased COL I and BSP protein levels compare(?)to AH Plus and control group on day 6.Conclusion:In the presence of iRoot SP, MG63 cells can produce more mineralized matrix gene and protein expression. Based on these results, iRoot SP can be considered as a favorable material regarding cell-material interaction.Part four: Assessment of a new root canal sealer’s apical sealing abilityObjective:The aim of this study was to investigate the apical sealing ability of a newly introduced root canal sealer: iRoot SP Root Canal Sealer.Materials and Methods:Sixty-eight extracted human anterior single-root teeth were used. The coronal part of each tooth was removed and the root canals were prepared with ProTaper files. The specimens were divided into 3 groups of 20 teeth each. Group A specimens were filled with iRoot SP using the continuous wave condensation technique; Group B specimens were obturated with iRoot SP using a single cone technique; Group C specimens were filled with AH plus by means of the continuous wave condensation technique. Evaluation of the apical leakage was performed with a fluid filtration method at 24 hours and 1,4, and 8 weeks. Scanning electron microscopy (SEM) was used to qualitatively assess what mechanisms might be responsible for leakage of the different groups.Results:There was no significant difference in fluid leakage among the groups, as well as no time effect on leakage (P<0.05). SEM revealed both gap-free regions and gap-containing regions in canals filled with both materials.Conclusion:iRoot SP was equivalent to AH Plus sealer in apical sealing ability.