In vitro evaluation of human dental pulp cells response to NeoMTA®2, ProRoot® MTA and TotalFill® BC RRM™ putty: biocompatibility and gene expression analysis

In vitro evaluation of human dental pulp cells response to NeoMTA®2, ProRoot® MTA and TotalFill® BC RRM™ putty: biocompatibility and gene expression analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In vitro evaluation of human dental pulp cells response to NeoMTA ® 2, ProRoot ® MTA and TotalFill ® BC RRM™ putty: biocompatibility and gene expression analysis Eirini Fragkouli, Kyriaki Kyriakidou, Maria Georgopoulou, Giorgos N. Tzanetakis This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8838821/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aim To comparatively evaluate the effects of NeoMTA®2, ProRoot® MTA, and TotalFill® BC RRM™ Putty on human dental pulp cells (hDPCs) in terms of cell viability, attachment, odontogenic differentiation and biomineralization potential. Materials and Methods Dental pulp tissue was obtained from seven intact third molars extracted from six patients aged 18–23 years. Isolated hDPCs were cultured in direct contact with the tested materials. Cell viability was assessed using an MTT assay at 24, 48, and 72h. Gene expression of mineralization- and odontogenic differentiation–related markers was analyzed by real-time PCR on days 3, 5, and 7. Cell morphology and attachment were evaluated using scanning electron microscopy (SEM). Quantile regression models were used to investigate possible differences by material and time of measurement. Results All materials supported cell viability, with significantly higher MTT values at 72h compared with earlier time points. NeoMTA®2 induced significantly higher expression of alkaline phosphatase (ALP) and osteocalcin (OCN) at day 7 compared with ProRoot® MTA and TotalFill® BC RRM™ Putty. SEM analysis revealed abundant, well-spread cells firmly attached to all material surfaces. Conclusions NeoMTA®2 demonstrated cellular responses comparable to or greater than those of ProRoot® MTA and TotalFill® BC RRM™ Putty, favoring cell viability, attachment, and gene expression associated with mineralization and odontogenic differentiation. Clinical relevance: NeoMTA®2 shows promising biological properties that may support its use in vital pulp treatment; however, further laboratory and clinical studies are required. Pulp capping materials Calcium silicate NeoMTA®2 Biocomatibility dental pulp cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Preservation of the dental pulp following injury caused by caries or trauma is essential for maintaining tissue vitality and long-term tooth function [ 1 ]. When pulp exposure occurs, undifferentiated mesenchymal cells within the pulp may differentiate into odontoblast-like cells under appropriate biological conditions, contributing to the formation of tertiary reparative dentin [ 2 ]. All therapeutic procedures aimed at preserving pulp vitality are collectively referred to as vital pulp treatment (VPT) [ 3 ]. These procedures rely on the use of biocompatible and bioactive materials that create a favorable microenvironment for cell migration, attachment, proliferation, and subsequent tissue regeneration [ 3 , 4 ]. Calcium hydroxide was historically considered the gold standard material for VPT. However, over the past two decades, calcium silicate–based materials have gradually replaced calcium hydroxide, providing satisfactory histological [ 5 ] and clinical outcomes [ 6 ]. Mineral trioxide aggregate (MTA) remains one of the most extensively studied, widely used, and clinically effective pulp capping materials [ 7 ]. Among its commercial formulations, ProRoot® MTA (Dentsply Tulsa Dental Specialties, Johnson City, TN, USA) has been thoroughly investigated and has demonstrated high biocompatibility, bioactivity, and the ability to promote odontogenic differentiation and mineralization of dental pulp cells [ 8 , 9 ]. To overcome certain limitations of MTA-like cements, such as handling difficulties and potential tooth discoloration, newer bioceramic materials have been introduced [ 4 ]. These materials typically consist of calcium silicates combined with calcium phosphates and radiopacifying agents. TotalFill® BC RRM™ Putty (FKG Dentaire SA, La Chaux-de-Fonds, Switzerland) and NeoMTA®2 (Avalon Biomed Inc, Houston, TX, USA) are representative examples of this category. TotalFill® BC RRM™ Putty has been shown to promote cell proliferation, migration, and attachment adhesion as well as mineralization, and dentinal bridge formation [ 10 , 11 ]. In addition, the material has shown a satisfactory success rate when used as pulp capping agent in partial pulpotomy cases with symptoms of irreversible pulpitis [ 7 ]. In contrast, limited information is available regarding the biological properties of NeoMTA®2. To date, only a small number of studies have evaluated this material, suggesting favorable biocompatibility and biomineralization potential without the need for osteogenic supplements [ 12 ]. Despite the growing clinical use of these newer calcium silicate cements, comparative data on their biological performance remain limited. In particular, further investigation is needed to clarify the biocompatibility and bioactive potential of NeoMTA®2 relative to well-established materials. Therefore, the aim of the present study was to evaluate the effects of NeoMTA®2 on human dental pulp cells (hDPCs) in terms of cell viability, attachment, odontogenic differentiation and biomineralization-related gene expression, and to compare these responses with those elicited by ProRoot® MTA and TotalFill® BC RRM™ Putty. The null hypothesis tested was that no significant differences would be observed among NeoMTA®2, ProRoot® MTA, and TotalFill® BC RRM™ Putty regarding hDPC viability, attachment, odontogenic differentiation, or mineralization-related gene expression over time. Materials and Methods The manuscript has been written according to Preferred Reporting Items for Laboratory Studies in Endodontology (PRILE) 2021 guidelines [13]. The flowchart of the study is shown in Figure S1 . The study was conducted using a blinded experimental design to minimize experimental bias. The tested materials were coded by an independent researcher. The operator who was responsible for cell culture and experimental procedures was unaware of the material group coding during the assays. Similarly, the examiner performing the quantitative analyses (MTT absorbance readings and qPCR data analysis) was blinded to the experimental groups until the statistical evaluation had been completed. Isolation and culture of hDPCs Dental pulp tissue was obtained from seven intact third molars with complete root formation, which, according to the patients' treatment plans (impaction, orthodontic reasons) had been scheduled for extraction. The research protocol was approved by the Research Ethics Committee of the School of Dentistry, National and Kapodistrian University of Athens (protocol ID 466/02.06.2021), and written consent was obtained from all the participants. Immediately after teeth extraction, periodontal tissue remnants were removed, and the external surfaces of the teeth were cleaned by using a sterile gauze. The teeth were placed in 50 ml centrifuge tubes containing 10 ml of DMEM 1g/L D-Glucose (Biowest, USA) transport medium, enriched in triplicate with a penicillin/streptomycin solution (Penicillin-Streptomycin Solution 100X, Biowest, USA) and an antifungal agent, Amphotericin B (Ampotericine B, Biowest, USA). Under aseptic conditions, in a vertical laminar flow chamber (Aura mini, BIOAIR EuroClone), each tooth was mechanically split, the pulp was removed with a sterile spatula, and the pulp was minced into very small tissue pieces with a sterile scalpel. The tissue pieces were then placed in a 100mm Petri dish with DMEM 1g/L D-Glucose enriched with fetal bovine serum (FBS) at 10% concentration (Fetal Bovine Serum, Biowest, USA), antibiotic solution (1% Pen-Strep ), and antifungal solution of amphotericin B. Cells were isolated from tissue fragments using a trypsin–EDTA solution (1x Trypsin-EDTA 1X in PBS w/o Calcium w/o Magnesium w/o Phenol Red, Biowest, USA) (passage 0). The same trypsinization and cell dilution procedure was repeated each time the cells covered 70–80% of the surface of the Petri dish. The cells were cultured in 100mm Petri dishes in a cell culture incubator (Thermo Scientific™ Heracell™ 150i CO2 Incubator) with DMEM 1g/L D-Glucose, 10% FBS (Fetal Bovine Serum, Biowest, USA), and antibiotic solution (1% Pen-Strept) under controlled conditions (37°C, 5% CO2, and 95% humidity). In this study, cells from the 4th passage were used. Preparation of the materials ProRoot® MTA and NeoMTA® 2 were prepared according to the manufacturers' instructions under aseptic conditions, while TotalFill® BC RRM™ putty is provided ready for use. The chemical composition of each tested material is summarized in Table 1. Discs were formed and standardized using single-use carriers (green disposable MTA carriers 1.6mm, Vista Apex) with 1.6 mm internal diameter, producing discs of 1.6 mm diameter × ~2 mm height. Setting time followed manufacturers’ instructions; discs were allowed to set for at least 24 hours under sterile humid conditions and then were transferred under aseptic conditions to the bottom of a 24-well cell culture plate and sterilized for 1 hour with UV radiation in the vertical laminar flow chamber [14]. Then, the disks were rinsed with PBS and transferred to new culture plates for cell culture, with a cell density of 5000 cells/cm². Cells were seeded directly onto the surface of fully set cement discs, without the use of Transwell inserts or membrane barriers. Cell counts were performed using a Neubauer hemocytometer. Cells from all donors were pooled to reduce inter-donor variability; therefore, results reflect a population-level cellular response rather than donor-specific effects. The control group consisted of cells from the same culture, which were grown on plastic culture surfaces (TCP) for the same time periods. All tests were performed in triplicate in three independent runs. Cell viability/ MTT assay Cell viability was evaluated using the MTT spectrophotometric method. The culture medium from each well was replaced with 200 µl of MTT solution and 1,800 µl of DMEM. The plates were incubated for 4 hours at 37°C. After incubation, the solution was removed, and the formazan crystals were dissolved with DMSO. The absorption of formazan was measured using an ELISA reader at 570 nm (VersaMax ELISA Microplate Reader, Biocompare, USA). Finally, the results were quantified in relation to the controls. The cell cultures on the bioceramic materials were performed for 24, 48, and 72 hours. Gene expression analysis/ Real-Time PCR The gene expression of alkaline phosphatase (ALP), osteocalcin (OCN), collagen alpha-1 type I (COL1A1), dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein-1 (DMP1), nestin, and the transcription factor core-binding factor alpha-1 (Cbfa-1 or Runx2) was examined at mRNA level using real-time polymerase chain reaction (Real-time PCR) on days 3, 5, and 7. Total RNA isolation was performed using the monophasic reagent TRItidy G (AppliChem, USA). The quantity of isolated RNA was determined by measuring optical absorption in ultraviolet (UV) light at a wavelength of 260 nm (Biospec-nano, Shimadzu Biotech, Japan), while the purity was determined by measuring the solution at 280 nm and then calculating the OD260/OD280 ratio. Equal amounts of RNA from each sample were converted into cDNA [ProtoScript II, First Strand cDNA Synthesis Kit (New England Biolabs Inc)]. For gene detection in the cDNA, a 96-well PCR plate (4titude® PCR Plates, Azenta, USA) was used, where each well contained 15µl of the mixture containing forward and reverse primers, the master mix (Luna® Universal qPCR Master Mix, New England Biolabs, USA), RNAse-free H2O, and 5µl of the cDNA from each sample. Each sample was loaded in duplicate. Two wells in each plate were used as negative controls (NTC), to which the above mixture was added, but instead of cDNA, RNAse-free H2O was used. The 2-ΔΔCt method was used to calculate the relative expression values of the genes obtained from RT-PCR analysis for each gene, compared to the expression of the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Scanning electron microscopy For the observation of cell morphology and attachment under Scanning Electron Microscopy (SEM) (Quanta 200 FEI Co, Hillsboro, Oregon, USA), the cell cultures on bioceramic materials were performed for 3, 5, and 7 days. Initially, the samples were fixed with a mixture of 2% glutaraldehyde in 0.1M sodium cacodylate buffer at pH 7.4 (at 4°C for 60 minutes) and osmium tetroxide (OsO4) in a 1% aqueous solution in sodium cacodylate buffer (at 4°C for 60 minutes). They then underwent dehydration treatments with various concentrations of ethanol at 40°C, drying for 12 hours at room temperature, and subsequently gold coating. Fixation in 2% glutaraldehyde for 60 min at 4°C was selected to preserve fine cellular morphology and adherence structures. Post-fixation in osmium tetroxide for 60 min at 4°C stabilizes membrane lipids and enhances contrast. Graded ethanol dehydration was used to minimize cell collapse and preserve surface structures. Overnight drying at room temperature ensured complete dehydration before gold coating. Observation was performed under SEM in high vacuum, and representative SEI and BEI images were captured at magnifications of 500x, 1000x, and 1500x with a low electron beam intensity at a voltage of 15kV. Statistical Analysis Descriptive statistics for each variable are presented in relevant tables, by material and time of measurement. Three independent biological replicates were performed (cells derived from six separate donors within the total pool of seven teeth). For each biological replicate, three technical replicates were used per material and time point. Quantile regression models were used to investigate possible differences by material and time of measurement, due to skewed distributions and existence of outliers in the outcome variables. In these models, the outcome variable was each parameter while the explanatory variables were the material, the time of measurement and their interaction. Bonferroni correction for multiple comparisons was applied in the post-hoc tests. All tests were two-sided at α = 5% level of statistical significance. Stata v13 was used for the statistical analysis (StataCorp LP. College Station, Texas). Results Cell viability/ MTT assay An increasing trend in MTT values was observed in all materials. MTT median value in TotalFill® BC RRM™ putty was statistically significantly higher compared to control and ProRoot® MTA on 72h. MTT median values were significantly higher at 72h than at 24 and 48h for all materials ( Table 1 A, B, Fig. 1 A ) . Gene expression analysis/ Real-Time PCR ALP median value in NeoMTA®2 was statistically significantly higher compared to ProRoot® MTA on day 3, while on day 7 was statistically significantly higher compared to both ProRoot® MTA and TotalFill® BC RRM™ putty. On day 5, ALP median value in TotalFill® BC RRM™ putty was higher (statistically significant) compared to NeoMTA®2 ( Table 2 A, B, Fig. 1 B ) . OCN median value in NeoMTA®2 was lower on day 3 while on days 5 and 7 it was higher compared to ProRoot® MTA and TotalFill® BC RRM™ putty. The differences were statistically significant on day 7 ( Table 3 A, B, Fig. 1 C ) . On days 3 and 7, similar COL1A1 median values were recorded for all materials. On day 5 the median value in ProRoot® MTA was 0.60 units higher compared to TotalFill® BC RRM™ putty and 1.16 units higher compared to NeoMTA®2, but these differences were not statistically significant ( Table 4 A, B, Fig. 1 D ) . DMP1 median value was higher in TotalFill® BC RRM™ putty and NeoMTA®2 on day 3, on days 5 and 7 higher values were found only for NeoMTA®2. Lower values were found in ProRoot® MTA on all days. All these differences though were not statistically significant ( Table 5 A, B, Fig. 2 A ) . Although NESTIN median value in NeoMTA®2 was higher compared to other materials in all days, these differences were not statistically significant ( Table 6 A, B, Fig. 2 B ) . Finally, no significant differences in DSPP ( Table 7 A, B, Fig. 2 C ) and RUNX2 ( Table 8 A, B, Fig. 2 D ) median value were found between materials, on all days. Scanning electron microscopy All materials showed abundant, well-attached, and morphologically normal cells, with no significant differences between the materials, suggesting the absence of cytotoxic effects of these materials. Microphotographs were taken at random points, and some representative images are presented ( Figs. 3 , 4 , 5 ) . Discussion The present study evaluated the biological effects of three calcium silicate–based materials on human dental pulp cells (hDPCs) and represents one of the few in vitro investigations assessing the biocompatibility and bioactivity of NeoMTA®2. Human dental pulp cells were selected to simulate the clinical scenario of direct material–pulp interaction. Owing to their high differentiation potential and documented favorable response to calcium silicate materials, hDPCs constitute a suitable model for investigating cellular viability, odontogenic differentiation and mineralization-related behavior in vitro [ 15 , 16 ]. Real-time PCR was employed because of its high sensitivity and ability to detect early changes in odontogenic gene expression prior to mineralized tissue formation. In addition, this technique requires minimal RNA quantities and allows simultaneous evaluation of multiple genes across different time points, which was advantageous for comparing the biological performance of the tested materials over time. All materials supported cell viability throughout the observation period, with an overall increase in mitochondrial activity. TotalFill® BC RRM™ Putty demonstrated the highest MTT values, suggesting the lowest early cytotoxicity, whereas ProRoot® MTA showed comparatively lower viability values. These findings are consistent with previous reports demonstrating the favorable biocompatibility of TotalFill® BC RRM™ Putty materials in human cell cultures [ 17 ]. The response of hDPCs to NeoMTA®2 was also in agreement with earlier findings by Rodriguez-Lozano et al. [ 12 ], who reported a positive cellular response to this material. An initial delay in mitochondrial activity was observed for all materials, particularly for NeoMTA®2 during the first 48 hours. Rodriguez-Lozano et al. in their study showed that after 24 hours undiluted NeoMTA®2 and the 1:2 dilution of the material slightly affected the mitochondrial metabolism of human dental pulp stem cells. According to the authors, this might be associated with the significant release of Ca2 + ions observed during the first 24 hours, as excessive intracellular Ca2 + accumulation can lead to mitochondrial dysfunction, alteration of cytoskeletal organization, and activation of catabolic enzymes [ 18 ]. Regarding differentiation and mineralization-related markers, NeoMTA®2 induced significantly higher expression of ALP and OCN at day 7 compared with ProRoot® MTA and TotalFill® BC RRM™ Putty. ALP is an early marker of osteogenic and odontogenic differentiation and plays a key role in the mineralization of reparative dentin [ 19 ], whereas OCN is a late marker associated with matrix maturation and regulation of mineral deposition and calcium ion homeostasis [ 20 ]. ALP and OCN expression in ProRoot® MTA and TotalFill® BC RRM™ Putty groups peaked at day 5 and declined thereafter. The sustained expression of these markers in the NeoMTA®2 group perhaps suggests a prolonged stimulatory effect on mineralization-related pathways. Similar trends in ALP expression have been reported previously for NeoMTA®2 [ 12 ], supporting the consistency of the present finding. In contrast to the above differences, COL1A1 expression did not differ significantly among materials at any timepoint. COL1A1 is highly associated with the differentiation of mesenchymal cells into odontoblastic cells and early extracellular matrix formation [ 21 ]. In the study of Rodriguez-Lozano et al., NeoMTA®2 was found to exhibit significantly higher potential for COL1A1 cell expression, compared to NeoMTA Plus and Bio-C Repair [ 12 ]. In the present study, all three materials showed a significant decrease in COL1A1 levels upon cell exposure on day 7 compared to day 3. This pattern may reflect a temporal shift from matrix production toward later stages of mineralization. Markers associated with odontogenic differentiation and dentin matrix formation, including dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein-1 (DMP1), Nestin, and RUNX2, were also investigated. Although NeoMTA®2 tended to maintain numerically higher DSPP and DMP1 levels over time, these differences were not statistically significant. DSPP plays a critical role for dentin development, hydroxyapatite formation and remineralization of reparative dentin [ 22 ]. DMP1 gene is essential for the formation and mineralization of dentin, cementum, and bone tissue [ 23 ] participating also in the regulation of DSPP gene expression [ 24 ]. Its overexpression leads to the differentiation of mesenchymal stem cells into fully differentiated odontoblast-like cells [ 25 ]. Their sustained expression may indicate continued odontoblastic activity. This observation is consistent with the previous findings of Rodriguez-Lozano et al., who found that DSPP values for NeoMTA®2 were significantly higher compared to NeoMTA Plus and Bio-C Repair [ 12 ]. Comparable results were observed among the tested materials regarding Nestin and RUNX2 expression at all time points. Although NESTIN expression in cells exposed to NeoMTA®2 was numerically higher throughout the observation period, these differences did not reach statistical significance. In addition, RUNX2 values in the ProRoot® MTA and TotalFill® BC RRM™ Putty groups were significantly lower at day 7 compared with days 3 and 5. Rodriguez-Lozano et al. reported significantly higher RUNX2 expression in cell cultures exposed to NeoMTA®2 compared with Bio-C Repair [ 12 ]. Nestin is a filamentous cytoskeletal protein expressed in differentiating odontoblast-like cells and is upregulated under pathological conditions such as caries or following restorative procedures [ 26 , 27 ]. RUNX2 is a key transcription factor involved in early odontogenic differentiation; however, its expression decreases as cells reach a fully differentiated odontoblastic phenotype [ 21 , 28 ]. It may be re-expressed by odontoblast-like cells during reparative dentin formation, such as after pulp capping or pulpotomy procedures [ 29 ]. Taken together, these findings suggest that all materials were capable of initiating early odontogenic differentiation of hDPCs, whereas differences among materials became more evident during the mineralization phase of differentiation. Scanning electron microscopy further supported the biocompatibility of all materials by demonstrating cell attachment and spreading on their surfaces. Although each material exhibited distinct surface morphology, abundant, well-spread cells firmly attached to all materials were observed, indicating active cell–material interactions. SEM images demonstrated that all three materials exhibited progressive surface changes over time, characterized by increased cell spreading, features suggestive of extracellular matrix deposition, and possible hydroxyapatite crystal formation, without evidence of adverse effects on cell morphology. Several limitations should be considered when interpreting these findings. Physicochemical properties such as ion release and pH changes were not evaluated. These factors are known to influence early cellular responses and may partly explain the differences observed among the tested materials. Future studies incorporating ion release analysis and pH monitoring would help clarify the mechanisms underlying the biological behavior of these cements. Another limitation of the present study is that hDPCs were cultured in direct contact with the tested materials without the use of Transwell inserts or membrane barriers. While this model simulates clinical scenarios such as direct pulp capping, it primarily reflects the biological response of cells located at the immediate material–tissue interface. In vivo, however, deeper pulpal cells are more likely to be exposed to soluble factors and ionic eluates diffusing through dentinal tubules and pulp tissue rather than to the bulk material itself. Future studies employing indirect-contact models or material eluates could provide complementary information regarding the effects of diffusible components on cells located at greater distances from the exposure site. Finally, as an in vitro investigation, this study cannot fully reproduce the complexity of the in vivo pulp environment, where inflammatory mediators, vascular, immunological, and mechanical factors influence healing. Therefore, further in vivo and clinical studies are necessary to confirm the translational relevance of these findings. Overall, the present results indicate that all tested calcium silicate materials supported hDPC viability and attachment. Within this context, NeoMTA®2 demonstrated a biological performance comparable to or, for selected differentiation markers, greater than that of ProRoot® MTA and TotalFill® BC RRM™ Putty, suggesting a sustained stimulatory effect on mineralization-related cellular pathways. Conclusions Within the limitations of this in vitro study, NeoMTA®2 favored cellular responses comparable to or greater than those observed with ProRoot® MTA and TotalFill® BC RRM™ Putty, supporting cell viability, attachment, and gene expression associated with mineralization and odontogenic differentiation. Declarations Funding: No funding was associated with the present study Ethical clearance: The protocol of the study has been approved by the Research Ethics Committee of the School of Dentistry, National and Kapodistrian University of Athens (protocol ID 466/02.06.2021), Conflict of Interest: The authors have stated explicitly that there are no conflicts of interest in connection with this article. Authorship Declaration : All authors have contributed significantly, and all authors are in agreement with the manuscript. Author contribution: EF, GNT conceptualization; EF, KK, data procurement; EF, KK, data analysis; EF, KK, GNT, data interpretation; EF, KK drafted the manuscript; GNT, MG review and editing. All authors contributed to reviewing and editing the manuscript. All authors have read and approved the final manuscript. Data availability statement : The data that support the findings of this study are available from the corresponding author upon reasonable request. ORCID ID Giorgos N. Tzanetakis 0000-0002-1826-2688 Kyriaki Kyriakidou 0000-0002-7842-6058 References European Society of Endodontology (ESE) developed by:, Duncan HF, Galler KM, Tomson PL, Simon S, El-Karim I, Kundzina R, Krastl G, Dammaschke T, Fransson H, Markvart M, Zehnder M, Bjørndal L (2019) European Society of Endodontology position statement: Management of deep caries and the exposed pulp. Int Endod J 52:923–934 Lin ML, Riccuci D, Saoud TM et al (2019) Vital pulp therapy of mature permanent teeth with irreversible pulpitis from the perspective of pulp biology. Aust Endod J 46:154–166 Duncan HF (2022) Present status and future directions-Vital pulp treatment and pulp preservation strategies. Int Endod J 55(Suppl 3):497–511 Prati C, Gandolfi MG (2015) Calcium silicate bioactive cements: Biological perspectives and clinical applications. Dent Mater 31:351–370 Nair PN, Duncan HF, Pitt Ford TR, Luder HU (2008) Histological, ultrastructural and quantitative investigations on the response of healthy human pulps to experimental capping with mineral trioxide aggregate: a randomized controlled trial. Int Endod J 41:128–150 Mente J, Hufnagel S, Leo M, Michel A, Gehrig H, Panagidis D, Saure D, Pfefferle T (2014) Treatment outcome of mineral trioxide aggregate or calcium hydroxide direct pulp capping: long-term results. J Endod 40:1746–1751 Tzanetakis GN, Koletsi D, Georgopoulou M (2023) Treatment outcome of partial pulpotomy using two different calcium silicate materials in mature permanent teeth with symptoms of irreversible pulpitis: A randomized clinical trial. Int Endod J 56:1178–1196 Paranjpe A, Smoot T, Zhang H, Johnson JD (2011) Direct contact with mineral trioxide aggregate activates and differentiates human dental pulp cells. J Endod 37:1691–1695 Seo MS, Hwang GK, Lee J et al (2013) The effect of mineral trioxide aggregate on odontogenic differentiation in dental pulp stem cells. J Endod 39:242–248 Zhang S, Yang X, Fan M (2013) BioAggregate and iRoot BP Plus optimize the proliferation and mineralization ability of human dental pulp cells. Int Endod J 46:923–929 Öncel Torun Z, Torun D, Demirkaya K et al (2015) Effects of iRoot BP and white mineral trioxide aggregate on cell viability and the expression of genes associated with mineralization. Int Endod J 48:986–993 Rodriguez-Lozano FJ, Lozano A, López-Garcia S et al (2022) Biomineralization potential and biological properties of a new tantalum oxide (Ta 2 O 5 )-containing calcium silicate cement. Clin Oral Invest 26:1427–1441 Nagendrababu V, Murray PE, Ordinola-Zapata R et al (2021) PRILE 2021 Guidelines for Reporting Laboratory Studies in Endodontics: Explanation and Elaboration. Int Endod J 54:1491–1515 Rodrigues EM, Cornelio ALG, Mestieri L et al (2017) Human dental pulp cells response to mineral trioxide aggregate (MTA) and MTA Plus: cytotoxicity and gene expression analysis. Int Endod J 50:780–789 Chen CC, Shie MY, Ding SJ (2011) Human dental pulp cell responses to new calcium silicate-based endodontic materials. Int Endod J 44:836–842 Chang SW, Lee SY, Ann HJ, Kum KY, Kim EC (2014) Effects of calcium silicate endodontic cements on biocompatibility and mineralization-inducing potentials in human dental pulp cells. J Endod 40:1194–1200 Sheela S, Nassar M, AlGhalban FM, Gorduysus MO (2023) In Vitro Cytotoxicity and Mineralization Potential of an Endodontic Bioceramic Material. Eur J Dent 17:548–555 Nicotera P, Bellomo G, Orrenius S (1992) Calcium-mediated mechanisms in chemically induced cell death. Annu Rev Pharmacol Toxicol 32:449–470 Gandolfi MG, Spagnuolo G, Siboni F et al (2015) Calcium silicate/calcium phosphate biphasic cements for vital pulp therapy: chemical-physical properties and human pulp cells response. Clin Oral Invest 19:2075–2089 Wu BC, Youn SC, Kao CT et al (2015) The effects of calcium silicate cement/fibroblast growth factor-2 composite on osteogenesis accelerator in human dental pulp cells. J Dent Sci 10:145–153 D’Souza RN, Bachman T, Baumgardner KR et al (1995) Characterization of cellular responses involved in reparative dentinogenesis in rat molars. J Dent Res 74:702–709 Min KS, Lee HJ, Kim SH, Lee SK, Kim HR, Pae HO, Chung HT, Shin HI, Lee SK, Kim EC (2008) Hydrogen peroxide induces heme oxygenase-1 and dentin sialophosphoprotein mRNA in human pulp cells. J Endod 34:983–989 Sun Y, Lu Y, Chen L et al (2011) DMP1 Processing is Essential to Dentin and Jaw Formation. J Dent Res 90:619–624 Ye L, MacDougall M, Zhang S et al (2004) Deletion of dentin matrix protein-1 leads to a partial failure of maturation of predentin into dentin, hypomineralization and expanded cavities of pulp and root canal during postnatal tooth development. J Biol Chem 279:19141–19148 Narayanan K, Srinivas R, Ramachandran A et al (2001) A differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1. Proc. Natl. Acad. Sci. U. S. A. ; 98: 4516–21 Quispe-Salcedo A, Ida-Yonemochi H, Nakatomi M et al (2012) Expression patterns of nestin and dentin sialoprotein during dentinogenesis in mice. Biomed Res 33:119–132 Kuratate M, Yoshiba K, Shigetani Y et al (2008) Immunohistochemical analysis of nestin, osteopontin, and proliferating cells in the reparative process of exposed dental pulp capped with mineral trioxide aggregate. J Endod 34:970–974 Chen S, Gluhak-Heinrich J, Wang YH et al (2009) Runx2, Osx , and Dspp in tooth development. J Dent Res 88:904–909 Takeuchi R, Ohkura N, Yoshiba K et al (2020) Immunohistochemistry and gene expression of GLUT1, RUNX2 and MTOR in reparative dentinogenesis. Oral Dis 26:341–349 Tables Tables are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files FigureS1.pdf Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8838821","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":599726413,"identity":"d4b52c14-e850-4115-97b6-099d4f55ae58","order_by":0,"name":"Eirini Fragkouli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBAC9gYwZcEDIiUYKoAkM3MDXi08B8CUBFTLGZAWRuK0QEjGNhBFSIv04ccvPrZJyDCw9x688XFebTR/O1DLj4ptuLXwpZlZzmwDOoznXLLlzG3Hc2ccZmxg7DlzG6cWex4GM2NekBaJHDNp3m3HchuAWpgZ23Br4eFh/wbRIv8GqGXOsdz5hLXwGD+G2MID1NJQk7uBCC1ljDPOSfCw8eQYW844diB3I1DLQXx+ATps84cPZTb2/OxnDG98qKnLnXf+8MEHPypwawECNnCksEE4h8HkAXzqgYD5AxKnjoDiUTAKRsEoGIkAAF3nT1kYwb4NAAAAAElFTkSuQmCC","orcid":"","institution":"National and Kapodistrian University of Athens","correspondingAuthor":true,"prefix":"","firstName":"Eirini","middleName":"","lastName":"Fragkouli","suffix":""},{"id":599726414,"identity":"1e7e0e6a-3bc9-4631-b05b-4f655e91a0f9","order_by":1,"name":"Kyriaki Kyriakidou","email":"","orcid":"","institution":"National and Kapodistrian University of Athens","correspondingAuthor":false,"prefix":"","firstName":"Kyriaki","middleName":"","lastName":"Kyriakidou","suffix":""},{"id":599726415,"identity":"83077422-3847-468d-b0a3-2abf2e8fdb63","order_by":2,"name":"Maria Georgopoulou","email":"","orcid":"","institution":"National and Kapodistrian University of Athens","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Georgopoulou","suffix":""},{"id":599726416,"identity":"b91ebfc1-f297-45de-9452-fca6b2df15ff","order_by":3,"name":"Giorgos N. Tzanetakis","email":"","orcid":"","institution":"National and Kapodistrian University of Athens","correspondingAuthor":false,"prefix":"","firstName":"Giorgos","middleName":"N.","lastName":"Tzanetakis","suffix":""}],"badges":[],"createdAt":"2026-02-10 09:09:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8838821/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8838821/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104179166,"identity":"d958ea93-1f32-4a5b-9a4b-82d83645c53d","added_by":"auto","created_at":"2026-03-08 17:02:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":289677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Estimated median values and 95% confidence intervals (95% CI) for MTT by material and time of measurement, \u003cstrong\u003eB.\u003c/strong\u003e Estimated median values and 95% confidence intervals (95% CI) for ALP by material and time of measurement, \u003cstrong\u003eC.\u003c/strong\u003e Estimated median values and 95% confidence intervals (95% CI) for OCN by material and time of measurement, \u003cstrong\u003eD.\u003c/strong\u003eEstimated median values and 95% confidence intervals (95% CI) for COL1A1 by material and time of measurement.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8838821/v1/13ef75defa84c6e8828e926f.png"},{"id":104179165,"identity":"54f193b1-4e44-45a5-b84f-8810c799f99e","added_by":"auto","created_at":"2026-03-08 17:02:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":282637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Estimated median values and 95% confidence intervals (95% CI) for DMP1 by material and time of measurement, \u003cstrong\u003eB.\u003c/strong\u003e Estimated median values and 95% confidence intervals (95% CI) for NESTIN by material and time of measurement, \u003cstrong\u003eC.\u003c/strong\u003e Estimated median values and 95% confidence intervals (95% CI) for DSPP by material and time of measurement, \u003cstrong\u003eD.\u003c/strong\u003e Estimated median values and 95% confidence intervals (95% CI) for RUNX2 by material and time of measurement.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8838821/v1/bfe78e30b45e1333110e710c.png"},{"id":104779568,"identity":"ebe98dcb-97d6-4254-af3a-8fd5383d1a24","added_by":"auto","created_at":"2026-03-17 07:42:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1435503,"visible":true,"origin":"","legend":"\u003cp\u003eThe initial attachment of dental pulp cells to the surfaces of all three bioceramic materials is evident. The presence of cells is relatively sparse. The cells are distinguished as small, elongated structures, exhibiting thin filamentous extensions, as if the cells are attempting to cover the pores of the materials, extending from one end of the pore to the other. All materials show rough, porous surfaces that favor the initial attachment of cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrographs α,δ,η:\u003c/strong\u003e cells cultured on NeoMTA®2 for 3 days at different magnifications of 500x, 1000x, and 1500x, \u003cstrong\u003eMicrographs β,ε,θ:\u003c/strong\u003e cells cultured on TotalFill® BC RRM™ Putty for 3 days at different magnifications of 500x, 1000x, and 1500x, \u003cstrong\u003eMicrographs γ,στ,ι:\u003c/strong\u003e cells cultured on ProRoot® MTA for 3 days at different magnifications of 500x, 1000x, and 1500x.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8838821/v1/0da19c97b208e2892356fd52.png"},{"id":104179170,"identity":"c5d577ba-7244-4c15-ab1c-a2a08d955385","added_by":"auto","created_at":"2026-03-08 17:02:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1388746,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM images from day 5 show significant progress in cellular activity. On the surface of each material, more cells are observed with numerous filamentous extensions, more extensive cell spreading on the surface, and more pronounced bridging of the pores compared to day 3 images, \u003cstrong\u003eMicrographs α,δ,η:\u003c/strong\u003e Cells cultured on NeoMTA®2 for 5 days at different magnifications of 500x, 1000x, and 1500x, \u003cstrong\u003eMicrographs β,ε,θ:\u003c/strong\u003e Cells cultured on TotalFill® BC RRM™ Putty for 5 days at different magnifications of 500x, 1000x, and 1500x, \u003cstrong\u003eMicrographs γ,στ,ι:\u003c/strong\u003e Cells cultured on ProRoot® MTA for 5 days at different magnifications of 500x, 1000x, and 1500x.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8838821/v1/153fc87ef522d037d59ee02b.png"},{"id":104179168,"identity":"f3a29663-bb33-44f5-adad-4d8c08fa138f","added_by":"auto","created_at":"2026-03-08 17:02:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1482118,"visible":true,"origin":"","legend":"\u003cp\u003eAt 7 days of cultivation, abundant, flattened, well-adhered dental pulp cells are shown, which appear to cover a larger portion of the surface of all three bioceramic materials compared to days 3 and 5. The surfaces of the materials appear more uniformly covered with multiple layers, \u003cstrong\u003eMicrographs α,δ,η:\u003c/strong\u003e Cells cultured on NeoMTA®2 for 7 days at different magnifications of 500x, 1000x, and 1500x, \u003cstrong\u003eMicrographs β,ε,θ:\u003c/strong\u003e Cells cultured on TotalFill® BC RRM™ Putty for 7 days at different magnifications of 500x, 1000x, and 1500x, \u003cstrong\u003eMicrographs γ,στ,ι:\u003c/strong\u003e Cells cultured on ProRoot® MTA for 7 days at different magnifications of 500x, 1000x, and 1500x.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8838821/v1/5af189432453e856482f8631.png"},{"id":105877231,"identity":"9f7a43b2-c74d-455f-b014-6c0d76ba2751","added_by":"auto","created_at":"2026-04-01 05:56:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5600229,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8838821/v1/f9e8d044-af2b-43d1-97bd-86fc5633462d.pdf"},{"id":104179164,"identity":"8e33ed96-32b3-43d5-a2f6-83815a31450a","added_by":"auto","created_at":"2026-03-08 17:02:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":39489,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8838821/v1/a98b1e32e3e5b1299de6c3d7.pdf"},{"id":104404763,"identity":"e964b354-d22b-4c53-9b4d-0c13191c2222","added_by":"auto","created_at":"2026-03-11 12:21:02","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":46424,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8838821/v1/0f639dc434a10138711b8cb6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eIn vitro evaluation of human dental pulp cells response to NeoMTA\u003csup\u003e®\u003c/sup\u003e2, ProRoot\u003csup\u003e\u003csub\u003e®\u003c/sub\u003e\u003c/sup\u003e MTA and TotalFill\u003csup\u003e®\u003c/sup\u003e BC RRM™ putty: biocompatibility and gene expression analysis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreservation of the dental pulp following injury caused by caries or trauma is essential for maintaining tissue vitality and long-term tooth function [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. When pulp exposure occurs, undifferentiated mesenchymal cells within the pulp may differentiate into odontoblast-like cells under appropriate biological conditions, contributing to the formation of tertiary reparative dentin [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. All therapeutic procedures aimed at preserving pulp vitality are collectively referred to as vital pulp treatment (VPT) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These procedures rely on the use of biocompatible and bioactive materials that create a favorable microenvironment for cell migration, attachment, proliferation, and subsequent tissue regeneration [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCalcium hydroxide was historically considered the gold standard material for VPT. However, over the past two decades, calcium silicate\u0026ndash;based materials have gradually replaced calcium hydroxide, providing satisfactory histological [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and clinical outcomes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Mineral trioxide aggregate (MTA) remains one of the most extensively studied, widely used, and clinically effective pulp capping materials [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among its commercial formulations, ProRoot\u0026reg; MTA (Dentsply Tulsa Dental Specialties, Johnson City, TN, USA) has been thoroughly investigated and has demonstrated high biocompatibility, bioactivity, and the ability to promote odontogenic differentiation and mineralization of dental pulp cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo overcome certain limitations of MTA-like cements, such as handling difficulties and potential tooth discoloration, newer bioceramic materials have been introduced [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These materials typically consist of calcium silicates combined with calcium phosphates and radiopacifying agents. TotalFill\u0026reg; BC RRM\u0026trade; Putty (FKG Dentaire SA, La Chaux-de-Fonds, Switzerland) and NeoMTA\u0026reg;2 (Avalon Biomed Inc, Houston, TX, USA) are representative examples of this category. TotalFill\u0026reg; BC RRM\u0026trade; Putty has been shown to promote cell proliferation, migration, and attachment adhesion as well as mineralization, and dentinal bridge formation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, the material has shown a satisfactory success rate when used as pulp capping agent in partial pulpotomy cases with symptoms of irreversible pulpitis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In contrast, limited information is available regarding the biological properties of NeoMTA\u0026reg;2. To date, only a small number of studies have evaluated this material, suggesting favorable biocompatibility and biomineralization potential without the need for osteogenic supplements [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the growing clinical use of these newer calcium silicate cements, comparative data on their biological performance remain limited. In particular, further investigation is needed to clarify the biocompatibility and bioactive potential of NeoMTA\u0026reg;2 relative to well-established materials. Therefore, the aim of the present study was to evaluate the effects of NeoMTA\u0026reg;2 on human dental pulp cells (hDPCs) in terms of cell viability, attachment, odontogenic differentiation and biomineralization-related gene expression, and to compare these responses with those elicited by ProRoot\u0026reg; MTA and TotalFill\u0026reg; BC RRM\u0026trade; Putty.\u003c/p\u003e \u003cp\u003eThe null hypothesis tested was that no significant differences would be observed among NeoMTA\u0026reg;2, ProRoot\u0026reg; MTA, and TotalFill\u0026reg; BC RRM\u0026trade; Putty regarding hDPC viability, attachment, odontogenic differentiation, or mineralization-related gene expression over time.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe manuscript has been written according to Preferred Reporting Items for Laboratory Studies in Endodontology (PRILE) 2021 guidelines [13]. The flowchart of the study is shown in \u003cstrong\u003eFigure S1\u003c/strong\u003e. The study was conducted using a blinded experimental design to minimize experimental bias. The tested materials were coded by an independent researcher. The operator who was responsible for cell culture and experimental procedures was unaware of the material group coding during the assays. Similarly, the examiner performing the quantitative analyses (MTT absorbance readings and qPCR data analysis) was blinded to the experimental groups until the statistical evaluation had been completed.\u003c/p\u003e\n\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eIsolation and culture of hDPCs\u003c/h2\u003e\n \u003cp\u003eDental pulp tissue was obtained from seven intact third molars with complete root formation, which, according to the patients\u0026apos; treatment plans (impaction, orthodontic reasons) had been scheduled for extraction. The research protocol was approved by the Research Ethics Committee of the School of Dentistry, National and Kapodistrian University of Athens (protocol ID 466/02.06.2021), and written consent was obtained from all the participants.\u003c/p\u003e\n \u003cp\u003eImmediately after teeth extraction, periodontal tissue remnants were removed, and the external surfaces of the teeth were cleaned by using a sterile gauze. The teeth were placed in 50 ml centrifuge tubes containing 10 ml of DMEM 1g/L D-Glucose (Biowest, USA) transport medium, enriched in triplicate with a penicillin/streptomycin solution (Penicillin-Streptomycin Solution 100X, Biowest, USA) and an antifungal agent, Amphotericin B (Ampotericine B, Biowest, USA). Under aseptic conditions, in a vertical laminar flow chamber (Aura mini, BIOAIR EuroClone), each tooth was mechanically split, the pulp was removed with a sterile spatula, and the pulp was minced into very small tissue pieces with a sterile scalpel. The tissue pieces were then placed in a 100mm Petri dish with DMEM 1g/L D-Glucose enriched with fetal bovine serum (FBS) at 10% concentration (Fetal Bovine Serum, Biowest, USA), antibiotic solution (1% Pen-Strep ), and antifungal solution of amphotericin B. Cells were isolated from tissue fragments using a trypsin\u0026ndash;EDTA solution (1x Trypsin-EDTA 1X in PBS w/o Calcium w/o Magnesium w/o Phenol Red, Biowest, USA) (passage 0). The same trypsinization and cell dilution procedure was repeated each time the cells covered 70\u0026ndash;80% of the surface of the Petri dish. The cells were cultured in 100mm Petri dishes in a cell culture incubator (Thermo Scientific\u0026trade; Heracell\u0026trade; 150i CO2 Incubator) with DMEM 1g/L D-Glucose, 10% FBS (Fetal Bovine Serum, Biowest, USA), and antibiotic solution (1% Pen-Strept) under controlled conditions (37\u0026deg;C, 5% CO2, and 95% humidity). In this study, cells from the 4th passage were used.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePreparation of the materials\u003c/h3\u003e\n\u003cp\u003eProRoot\u0026reg; MTA and NeoMTA\u0026reg; 2 were prepared according to the manufacturers\u0026apos; instructions under aseptic conditions, while TotalFill\u0026reg; BC RRM\u0026trade; putty is provided ready for use. The chemical composition of each tested material is summarized in Table 1. Discs were formed and standardized using single-use carriers (green disposable MTA carriers 1.6mm, Vista Apex) with 1.6 mm internal diameter, producing discs of 1.6 mm diameter \u0026times; ~2 mm height. Setting time followed manufacturers\u0026rsquo; instructions; discs were allowed to set for at least 24 hours under sterile humid conditions and then were transferred under aseptic conditions to the bottom of a 24-well cell culture plate and sterilized for 1 hour with UV radiation in the vertical laminar flow chamber [14].\u003c/p\u003e\n\u003cp\u003eThen, the disks were rinsed with PBS and transferred to new culture plates for cell culture, with a cell density of 5000 cells/cm\u0026sup2;. Cells were seeded directly onto the surface of fully set cement discs, without the use of Transwell inserts or membrane barriers. Cell counts were performed using a Neubauer hemocytometer. Cells from all donors were pooled to reduce inter-donor variability; therefore, results reflect a population-level cellular response rather than donor-specific effects. The control group consisted of cells from the same culture, which were grown on plastic culture surfaces (TCP) for the same time periods. All tests were performed in triplicate in three independent runs.\u003c/p\u003e\n\u003ch3\u003eCell viability/ MTT assay\u003c/h3\u003e\n\u003cp\u003eCell viability was evaluated using the MTT spectrophotometric method. The culture medium from each well was replaced with 200 \u0026micro;l of MTT solution and 1,800 \u0026micro;l of DMEM. The plates were incubated for 4 hours at 37\u0026deg;C. After incubation, the solution was removed, and the formazan crystals were dissolved with DMSO. The absorption of formazan was measured using an ELISA reader at 570 nm (VersaMax ELISA Microplate Reader, Biocompare, USA). Finally, the results were quantified in relation to the controls. The cell cultures on the bioceramic materials were performed for 24, 48, and 72 hours.\u003c/p\u003e\n\u003ch3\u003eGene expression analysis/ Real-Time PCR\u003c/h3\u003e\n\u003cp\u003eThe gene expression of alkaline phosphatase (ALP), osteocalcin (OCN), collagen alpha-1 type I (COL1A1), dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein-1 (DMP1), nestin, and the transcription factor core-binding factor alpha-1 (Cbfa-1 or Runx2) was examined at mRNA level using real-time polymerase chain reaction (Real-time PCR) on days 3, 5, and 7.\u003c/p\u003e\n\u003cp\u003eTotal RNA isolation was performed using the monophasic reagent TRItidy G (AppliChem, USA). The quantity of isolated RNA was determined by measuring optical absorption in ultraviolet (UV) light at a wavelength of 260 nm (Biospec-nano, Shimadzu Biotech, Japan), while the purity was determined by measuring the solution at 280 nm and then calculating the OD260/OD280 ratio. Equal amounts of RNA from each sample were converted into cDNA [ProtoScript II, First Strand cDNA Synthesis Kit (New England Biolabs Inc)]. For gene detection in the cDNA, a 96-well PCR plate (4titude\u0026reg; PCR Plates, Azenta, USA) was used, where each well contained 15\u0026micro;l of the mixture containing forward and reverse primers, the master mix (Luna\u0026reg; Universal qPCR Master Mix, New England Biolabs, USA), RNAse-free H2O, and 5\u0026micro;l of the cDNA from each sample. Each sample was loaded in duplicate. Two wells in each plate were used as negative controls (NTC), to which the above mixture was added, but instead of cDNA, RNAse-free H2O was used. The 2-\u0026Delta;\u0026Delta;Ct method was used to calculate the relative expression values of the genes obtained from RT-PCR analysis for each gene, compared to the expression of the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene.\u003c/p\u003e\n\u003ch3\u003eScanning electron microscopy\u003c/h3\u003e\n\u003cp\u003eFor the observation of cell morphology and attachment under Scanning Electron Microscopy (SEM) (Quanta 200 FEI Co, Hillsboro, Oregon, USA), the cell cultures on bioceramic materials were performed for 3, 5, and 7 days. Initially, the samples were fixed with a mixture of 2% glutaraldehyde in 0.1M sodium cacodylate buffer at pH 7.4 (at 4\u0026deg;C for 60 minutes) and osmium tetroxide (OsO4) in a 1% aqueous solution in sodium cacodylate buffer (at 4\u0026deg;C for 60 minutes). They then underwent dehydration treatments with various concentrations of ethanol at 40\u0026deg;C, drying for 12 hours at room temperature, and subsequently gold coating. Fixation in 2% glutaraldehyde for 60 min at 4\u0026deg;C was selected to preserve fine cellular morphology and adherence structures. Post-fixation in osmium tetroxide for 60 min at 4\u0026deg;C stabilizes membrane lipids and enhances contrast. Graded ethanol dehydration was used to minimize cell collapse and preserve surface structures. Overnight drying at room temperature ensured complete dehydration before gold coating. Observation was performed under SEM in high vacuum, and representative SEI and BEI images were captured at magnifications of 500x, 1000x, and 1500x with a low electron beam intensity at a voltage of 15kV.\u003c/p\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cp\u003eDescriptive statistics for each variable are presented in relevant tables, by material and time of measurement. Three independent biological replicates were performed (cells derived from six separate donors within the total pool of seven teeth). For each biological replicate, three technical replicates were used per material and time point.\u003c/p\u003e\n \u003cp\u003eQuantile regression models were used to investigate possible differences by material and time of measurement, due to skewed distributions and existence of outliers in the outcome variables. In these models, the outcome variable was each parameter while the explanatory variables were the material, the time of measurement and their interaction. Bonferroni correction for multiple comparisons was applied in the post-hoc tests.\u003c/p\u003e\n \u003cp\u003eAll tests were two-sided at \u0026alpha;\u0026thinsp;=\u0026thinsp;5% level of statistical significance. Stata v13 was used for the statistical analysis (StataCorp LP. College Station, Texas).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell viability/ MTT assay\u003c/h2\u003e \u003cp\u003eAn increasing trend in MTT values was observed in all materials. MTT median value in TotalFill\u0026reg; BC RRM\u0026trade; putty was statistically significantly higher compared to control and ProRoot\u0026reg; MTA on 72h. MTT median values were significantly higher at 72h than at 24 and 48h for all materials \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGene expression analysis/ Real-Time PCR\u003c/h2\u003e \u003cp\u003eALP median value in NeoMTA\u0026reg;2 was statistically significantly higher compared to ProRoot\u0026reg; MTA on day 3, while on day 7 was statistically significantly higher compared to both ProRoot\u0026reg; MTA and TotalFill\u0026reg; BC RRM\u0026trade; putty. On day 5, ALP median value in TotalFill\u0026reg; BC RRM\u0026trade; putty was higher (statistically significant) compared to NeoMTA\u0026reg;2 \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab10\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. OCN median value in NeoMTA\u0026reg;2 was lower on day 3 while on days 5 and 7 it was higher compared to ProRoot\u0026reg; MTA and TotalFill\u0026reg; BC RRM\u0026trade; putty. The differences were statistically significant on day 7 \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab11\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eOn days 3 and 7, similar COL1A1 median values were recorded for all materials. On day 5 the median value in ProRoot\u0026reg; MTA was 0.60 units higher compared to TotalFill\u0026reg; BC RRM\u0026trade; putty and 1.16 units higher compared to NeoMTA\u0026reg;2, but these differences were not statistically significant \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab12\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. DMP1 median value was higher in TotalFill\u0026reg; BC RRM\u0026trade; putty and NeoMTA\u0026reg;2 on day 3, on days 5 and 7 higher values were found only for NeoMTA\u0026reg;2. Lower values were found in ProRoot\u0026reg; MTA on all days. All these differences though were not statistically significant \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab13\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Although NESTIN median value in NeoMTA\u0026reg;2 was higher compared to other materials in all days, these differences were not statistically significant \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab14\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, no significant differences in DSPP \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab15\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e and RUNX2 \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab17\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e median value were found between materials, on all days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy\u003c/h2\u003e \u003cp\u003eAll materials showed abundant, well-attached, and morphologically normal cells, with no significant differences between the materials, suggesting the absence of cytotoxic effects of these materials. Microphotographs were taken at random points, and some representative images are presented \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003eThe present study evaluated the biological effects of three calcium silicate\u0026ndash;based materials on human dental pulp cells (hDPCs) and represents one of the few in vitro investigations assessing the biocompatibility and bioactivity of NeoMTA\u0026reg;2. Human dental pulp cells were selected to simulate the clinical scenario of direct material\u0026ndash;pulp interaction. Owing to their high differentiation potential and documented favorable response to calcium silicate materials, hDPCs constitute a suitable model for investigating cellular viability, odontogenic differentiation and mineralization-related behavior in vitro [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Real-time PCR was employed because of its high sensitivity and ability to detect early changes in odontogenic gene expression prior to mineralized tissue formation. In addition, this technique requires minimal RNA quantities and allows simultaneous evaluation of multiple genes across different time points, which was advantageous for comparing the biological performance of the tested materials over time.\u003c/p\u003e \u003cp\u003eAll materials supported cell viability throughout the observation period, with an overall increase in mitochondrial activity. TotalFill\u0026reg; BC RRM\u0026trade; Putty demonstrated the highest MTT values, suggesting the lowest early cytotoxicity, whereas ProRoot\u0026reg; MTA showed comparatively lower viability values. These findings are consistent with previous reports demonstrating the favorable biocompatibility of TotalFill\u0026reg; BC RRM\u0026trade; Putty materials in human cell cultures [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The response of hDPCs to NeoMTA\u0026reg;2 was also in agreement with earlier findings by Rodriguez-Lozano et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], who reported a positive cellular response to this material. An initial delay in mitochondrial activity was observed for all materials, particularly for NeoMTA\u0026reg;2 during the first 48 hours. Rodriguez-Lozano et al. in their study showed that after 24 hours undiluted NeoMTA\u0026reg;2 and the 1:2 dilution of the material slightly affected the mitochondrial metabolism of human dental pulp stem cells. According to the authors, this might be associated with the significant release of Ca2\u0026thinsp;+\u0026thinsp;ions observed during the first 24 hours, as excessive intracellular Ca2\u0026thinsp;+\u0026thinsp;accumulation can lead to mitochondrial dysfunction, alteration of cytoskeletal organization, and activation of catabolic enzymes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding differentiation and mineralization-related markers, NeoMTA\u0026reg;2 induced significantly higher expression of ALP and OCN at day 7 compared with ProRoot\u0026reg; MTA and TotalFill\u0026reg; BC RRM\u0026trade; Putty. ALP is an early marker of osteogenic and odontogenic differentiation and plays a key role in the mineralization of reparative dentin [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], whereas OCN is a late marker associated with matrix maturation and regulation of mineral deposition and calcium ion homeostasis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. ALP and OCN expression in ProRoot\u0026reg; MTA and TotalFill\u0026reg; BC RRM\u0026trade; Putty groups peaked at day 5 and declined thereafter. The sustained expression of these markers in the NeoMTA\u0026reg;2 group perhaps suggests a prolonged stimulatory effect on mineralization-related pathways. Similar trends in ALP expression have been reported previously for NeoMTA\u0026reg;2 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], supporting the consistency of the present finding.\u003c/p\u003e \u003cp\u003eIn contrast to the above differences, COL1A1 expression did not differ significantly among materials at any timepoint. COL1A1 is highly associated with the differentiation of mesenchymal cells into odontoblastic cells and early extracellular matrix formation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the study of Rodriguez-Lozano et al., NeoMTA\u0026reg;2 was found to exhibit significantly higher potential for COL1A1 cell expression, compared to NeoMTA Plus and Bio-C Repair [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the present study, all three materials showed a significant decrease in COL1A1 levels upon cell exposure on day 7 compared to day 3. This pattern may reflect a temporal shift from matrix production toward later stages of mineralization.\u003c/p\u003e \u003cp\u003eMarkers associated with odontogenic differentiation and dentin matrix formation, including dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein-1 (DMP1), Nestin, and RUNX2, were also investigated. Although NeoMTA\u0026reg;2 tended to maintain numerically higher DSPP and DMP1 levels over time, these differences were not statistically significant. DSPP plays a critical role for dentin development, hydroxyapatite formation and remineralization of reparative dentin [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. DMP1 gene is essential for the formation and mineralization of dentin, cementum, and bone tissue [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] participating also in the regulation of DSPP gene expression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Its overexpression leads to the differentiation of mesenchymal stem cells into fully differentiated odontoblast-like cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Their sustained expression may indicate continued odontoblastic activity. This observation is consistent with the previous findings of Rodriguez-Lozano et al., who found that DSPP values for NeoMTA\u0026reg;2 were significantly higher compared to NeoMTA Plus and Bio-C Repair [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eComparable results were observed among the tested materials regarding Nestin and RUNX2 expression at all time points. Although NESTIN expression in cells exposed to NeoMTA\u0026reg;2 was numerically higher throughout the observation period, these differences did not reach statistical significance. In addition, RUNX2 values in the ProRoot\u0026reg; MTA and TotalFill\u0026reg; BC RRM\u0026trade; Putty groups were significantly lower at day 7 compared with days 3 and 5. Rodriguez-Lozano et al. reported significantly higher RUNX2 expression in cell cultures exposed to NeoMTA\u0026reg;2 compared with Bio-C Repair [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNestin is a filamentous cytoskeletal protein expressed in differentiating odontoblast-like cells and is upregulated under pathological conditions such as caries or following restorative procedures [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. RUNX2 is a key transcription factor involved in early odontogenic differentiation; however, its expression decreases as cells reach a fully differentiated odontoblastic phenotype [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It may be re-expressed by odontoblast-like cells during reparative dentin formation, such as after pulp capping or pulpotomy procedures [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Taken together, these findings suggest that all materials were capable of initiating early odontogenic differentiation of hDPCs, whereas differences among materials became more evident during the mineralization phase of differentiation.\u003c/p\u003e \u003cp\u003eScanning electron microscopy further supported the biocompatibility of all materials by demonstrating cell attachment and spreading on their surfaces. Although each material exhibited distinct surface morphology, abundant, well-spread cells firmly attached to all materials were observed, indicating active cell\u0026ndash;material interactions. SEM images demonstrated that all three materials exhibited progressive surface changes over time, characterized by increased cell spreading, features suggestive of extracellular matrix deposition, and possible hydroxyapatite crystal formation, without evidence of adverse effects on cell morphology.\u003c/p\u003e \u003cp\u003eSeveral limitations should be considered when interpreting these findings. Physicochemical properties such as ion release and pH changes were not evaluated. These factors are known to influence early cellular responses and may partly explain the differences observed among the tested materials. Future studies incorporating ion release analysis and pH monitoring would help clarify the mechanisms underlying the biological behavior of these cements.\u003c/p\u003e \u003cp\u003eAnother limitation of the present study is that hDPCs were cultured in direct contact with the tested materials without the use of Transwell inserts or membrane barriers. While this model simulates clinical scenarios such as direct pulp capping, it primarily reflects the biological response of cells located at the immediate material\u0026ndash;tissue interface. In vivo, however, deeper pulpal cells are more likely to be exposed to soluble factors and ionic eluates diffusing through dentinal tubules and pulp tissue rather than to the bulk material itself. Future studies employing indirect-contact models or material eluates could provide complementary information regarding the effects of diffusible components on cells located at greater distances from the exposure site.\u003c/p\u003e \u003cp\u003eFinally, as an in vitro investigation, this study cannot fully reproduce the complexity of the in vivo pulp environment, where inflammatory mediators, vascular, immunological, and mechanical factors influence healing. Therefore, further in vivo and clinical studies are necessary to confirm the translational relevance of these findings.\u003c/p\u003e \u003cp\u003eOverall, the present results indicate that all tested calcium silicate materials supported hDPC viability and attachment. Within this context, NeoMTA\u0026reg;2 demonstrated a biological performance comparable to or, for selected differentiation markers, greater than that of ProRoot\u0026reg; MTA and TotalFill\u0026reg; BC RRM\u0026trade; Putty, suggesting a sustained stimulatory effect on mineralization-related cellular pathways.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWithin the limitations of this in vitro study, NeoMTA\u0026reg;2 favored cellular responses comparable to or greater than those observed with ProRoot\u0026reg; MTA and TotalFill\u0026reg; BC RRM\u0026trade; Putty, supporting cell viability, attachment, and gene expression associated with mineralization and odontogenic differentiation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eNo funding was associated with the present study\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical clearance:\u0026nbsp;\u003c/strong\u003eThe protocol of the study has been approved\u0026nbsp;by the Research Ethics Committee of the School of Dentistry, National and Kapodistrian University of Athens (protocol ID 466/02.06.2021),\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eThe authors have stated explicitly that there are no conflicts of interest in connection with this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Declaration\u003c/strong\u003e: All authors have contributed significantly, and all authors are in agreement with the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u0026nbsp;\u003c/strong\u003eEF,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eGNT conceptualization; EF, KK, data procurement; EF, KK, data analysis; EF, KK, GNT, data interpretation; EF, KK drafted the manuscript; GNT, MG review and editing. All authors contributed to reviewing and editing the manuscript. All authors have read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e: The data that support the findings of this study are available from the corresponding author upon reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID ID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiorgos N. Tzanetakis 0000-0002-1826-2688\u003c/p\u003e\n\u003cp\u003eKyriaki Kyriakidou \u0026nbsp; \u0026nbsp;0000-0002-7842-6058\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEuropean Society of Endodontology (ESE) developed by:, Duncan HF, Galler KM, Tomson PL, Simon S, El-Karim I, Kundzina R, Krastl G, Dammaschke T, Fransson H, Markvart M, Zehnder M, Bj\u0026oslash;rndal L (2019) European Society of Endodontology position statement: Management of deep caries and the exposed pulp. Int Endod J 52:923\u0026ndash;934\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin ML, Riccuci D, Saoud TM et al (2019) Vital pulp therapy of mature permanent teeth with irreversible pulpitis from the perspective of pulp biology. Aust Endod J 46:154\u0026ndash;166\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuncan HF (2022) Present status and future directions-Vital pulp treatment and pulp preservation strategies. Int Endod J 55(Suppl 3):497\u0026ndash;511\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrati C, Gandolfi MG (2015) Calcium silicate bioactive cements: Biological perspectives and clinical applications. Dent Mater 31:351\u0026ndash;370\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNair PN, Duncan HF, Pitt Ford TR, Luder HU (2008) Histological, ultrastructural and quantitative investigations on the response of healthy human pulps to experimental capping with mineral trioxide aggregate: a randomized controlled trial. Int Endod J 41:128\u0026ndash;150\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMente J, Hufnagel S, Leo M, Michel A, Gehrig H, Panagidis D, Saure D, Pfefferle T (2014) Treatment outcome of mineral trioxide aggregate or calcium hydroxide direct pulp capping: long-term results. J Endod 40:1746\u0026ndash;1751\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTzanetakis GN, Koletsi D, Georgopoulou M (2023) Treatment outcome of partial pulpotomy using two different calcium silicate materials in mature permanent teeth with symptoms of irreversible pulpitis: A randomized clinical trial. Int Endod J 56:1178\u0026ndash;1196\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParanjpe A, Smoot T, Zhang H, Johnson JD (2011) Direct contact with mineral trioxide aggregate activates and differentiates human dental pulp cells. J Endod 37:1691\u0026ndash;1695\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeo MS, Hwang GK, Lee J et al (2013) The effect of mineral trioxide aggregate on odontogenic differentiation in dental pulp stem cells. J Endod 39:242\u0026ndash;248\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Yang X, Fan M (2013) BioAggregate and iRoot BP Plus optimize the proliferation and mineralization ability of human dental pulp cells. Int Endod J 46:923\u0026ndash;929\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Ouml;ncel Torun Z, Torun D, Demirkaya K et al (2015) Effects of iRoot BP and white mineral trioxide aggregate on cell viability and the expression of genes associated with mineralization. Int Endod J 48:986\u0026ndash;993\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodriguez-Lozano FJ, Lozano A, L\u0026oacute;pez-Garcia S et al (2022) Biomineralization potential and biological properties of a new tantalum oxide (Ta\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e)-containing calcium silicate cement. Clin Oral Invest 26:1427\u0026ndash;1441\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagendrababu V, Murray PE, Ordinola-Zapata R et al (2021) PRILE 2021 Guidelines for Reporting Laboratory Studies in Endodontics: Explanation and Elaboration. Int Endod J 54:1491\u0026ndash;1515\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodrigues EM, Cornelio ALG, Mestieri L et al (2017) Human dental pulp cells response to mineral trioxide aggregate (MTA) and MTA Plus: cytotoxicity and gene expression analysis. Int Endod J 50:780\u0026ndash;789\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen CC, Shie MY, Ding SJ (2011) Human dental pulp cell responses to new calcium silicate-based endodontic materials. Int Endod J 44:836\u0026ndash;842\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang SW, Lee SY, Ann HJ, Kum KY, Kim EC (2014) Effects of calcium silicate endodontic cements on biocompatibility and mineralization-inducing potentials in human dental pulp cells. J Endod 40:1194\u0026ndash;1200\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSheela S, Nassar M, AlGhalban FM, Gorduysus MO (2023) In Vitro Cytotoxicity and Mineralization Potential of an Endodontic Bioceramic Material. Eur J Dent 17:548\u0026ndash;555\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicotera P, Bellomo G, Orrenius S (1992) Calcium-mediated mechanisms in chemically induced cell death. Annu Rev Pharmacol Toxicol 32:449\u0026ndash;470\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGandolfi MG, Spagnuolo G, Siboni F et al (2015) Calcium silicate/calcium phosphate biphasic cements for vital pulp therapy: chemical-physical properties and human pulp cells response. Clin Oral Invest 19:2075\u0026ndash;2089\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu BC, Youn SC, Kao CT et al (2015) The effects of calcium silicate cement/fibroblast growth factor-2 composite on osteogenesis accelerator in human dental pulp cells. J Dent Sci 10:145\u0026ndash;153\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026rsquo;Souza RN, Bachman T, Baumgardner KR et al (1995) Characterization of cellular responses involved in reparative dentinogenesis in rat molars. J Dent Res 74:702\u0026ndash;709\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMin KS, Lee HJ, Kim SH, Lee SK, Kim HR, Pae HO, Chung HT, Shin HI, Lee SK, Kim EC (2008) Hydrogen peroxide induces heme oxygenase-1 and dentin sialophosphoprotein mRNA in human pulp cells. J Endod 34:983\u0026ndash;989\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Lu Y, Chen L et al (2011) DMP1 Processing is Essential to Dentin and Jaw Formation. J Dent Res 90:619\u0026ndash;624\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe L, MacDougall M, Zhang S et al (2004) Deletion of dentin matrix protein-1 leads to a partial failure of maturation of predentin into dentin, hypomineralization and expanded cavities of pulp and root canal during postnatal tooth development. J Biol Chem 279:19141\u0026ndash;19148\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNarayanan K, Srinivas R, Ramachandran A et al (2001) A differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1. Proc. Natl. Acad. Sci. U. S. A. ; 98: 4516\u0026ndash;21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuispe-Salcedo A, Ida-Yonemochi H, Nakatomi M et al (2012) Expression patterns of nestin and dentin sialoprotein during dentinogenesis in mice. Biomed Res 33:119\u0026ndash;132\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuratate M, Yoshiba K, Shigetani Y et al (2008) Immunohistochemical analysis of nestin, osteopontin, and proliferating cells in the reparative process of exposed dental pulp capped with mineral trioxide aggregate. J Endod 34:970\u0026ndash;974\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Gluhak-Heinrich J, Wang YH et al (2009) \u003cem\u003eRunx2, Osx\u003c/em\u003e, and \u003cem\u003eDspp\u003c/em\u003e in tooth development. J Dent Res 88:904\u0026ndash;909\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeuchi R, Ohkura N, Yoshiba K et al (2020) Immunohistochemistry and gene expression of GLUT1, RUNX2 and MTOR in reparative dentinogenesis. Oral Dis 26:341\u0026ndash;349\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pulp capping materials, Calcium silicate, NeoMTA®2, Biocomatibility, dental pulp cells","lastPublishedDoi":"10.21203/rs.3.rs-8838821/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8838821/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAim\u003c/h2\u003e \u003cp\u003eTo comparatively evaluate the effects of NeoMTA\u0026reg;2, ProRoot\u0026reg; MTA, and TotalFill\u0026reg; BC RRM\u0026trade; Putty on human dental pulp cells (hDPCs) in terms of cell viability, attachment, odontogenic differentiation and biomineralization potential.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eDental pulp tissue was obtained from seven intact third molars extracted from six patients aged 18\u0026ndash;23 years. Isolated hDPCs were cultured in direct contact with the tested materials. Cell viability was assessed using an MTT assay at 24, 48, and 72h. Gene expression of mineralization- and odontogenic differentiation\u0026ndash;related markers was analyzed by real-time PCR on days 3, 5, and 7. Cell morphology and attachment were evaluated using scanning electron microscopy (SEM). Quantile regression models were used to investigate possible differences by material and time of measurement.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAll materials supported cell viability, with significantly higher MTT values at 72h compared with earlier time points. NeoMTA\u0026reg;2 induced significantly higher expression of alkaline phosphatase (ALP) and osteocalcin (OCN) at day 7 compared with ProRoot\u0026reg; MTA and TotalFill\u0026reg; BC RRM\u0026trade; Putty. SEM analysis revealed abundant, well-spread cells firmly attached to all material surfaces.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eNeoMTA\u0026reg;2 demonstrated cellular responses comparable to or greater than those of ProRoot\u0026reg; MTA and TotalFill\u0026reg; BC RRM\u0026trade; Putty, favoring cell viability, attachment, and gene expression associated with mineralization and odontogenic differentiation.\u003c/p\u003e\u003ch2\u003eClinical relevance:\u003c/h2\u003e \u003cp\u003eNeoMTA\u0026reg;2 shows promising biological properties that may support its use in vital pulp treatment; however, further laboratory and clinical studies are required.\u003c/p\u003e","manuscriptTitle":"In vitro evaluation of human dental pulp cells response to NeoMTA®2, ProRoot® MTA and TotalFill® BC RRM™ putty: biocompatibility and gene expression analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-08 17:02:46","doi":"10.21203/rs.3.rs-8838821/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7c93f43f-6726-42d6-b37c-e56f30755cea","owner":[],"postedDate":"March 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T05:55:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-08 17:02:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8838821","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8838821","identity":"rs-8838821","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}