Biological evaluation of novel phosphorylated pullulan-based calcium hydroxide formulations as direct pulp capping materials: An in vivo study on a rat model.

AIM: Calcium hydroxide (CH) has been considered as a direct pulp capping materials (DPC) for the last decades despite having some limitations. Phosphorylate pullulan (PPL) incorporated with CH (CHPPL) is a novel biomaterial that was introduced as a promising DPC material. Thus, the aim of the study was to evaluate the inflammatory response and mineralized tissue formation (MTF) ability of PPL-based CH formulations on rat molars after DPC. METHODOLOGY: This study consisted of six groups: CH with 1% PPL (CHPPL-1); 3% PPL (CHPPL-3); 5% PPL (CHPPL-5); Dycal and NEX MTA Cement (N-MTA) as the positive control, and no capping materials (NC). One hundred twenty maxillary first molar cavities were prepared on Wistar rats. After capping, all the cavities were restored with 4-META/MMA-TBB resin and pulpal responses were evaluated at days 1, 7, and 28. Kruskal-Wallis followed by Mann-Whitney U-test was performed with a significance level of 0.05. Immunohistochemical expression of IL-6, Nestin, and DMP-1 was observed. RESULTS: At day 1, CHPPL-1, N-MTA, and Dycal exhibited no to mild inflammation, whilst CHPPL-3, CHPPL-5, and NC showed mild to moderate inflammation, and the results were significantly different (p < .05). At day 7, mild to moderate inflammation was observed in CHPPL-1, N-MTA, and Dycal, whereas CHPPL-3, CHPPL-5, and NC exhibited moderate to severe inflammation. Significant differences were observed between CHPPL-1 and N-MTA with NC (p < .05), CHPPL-1 and CHPPL-3 with CHPPL-5 and Dycal (p < .05), and CHPPL-3 with N-MTA (p < .05). A thin layer of mineralized tissue formation (MTF) was observed in all groups. At day 28, CHPPL-1, Dycal, and N-MTA showed no to mild inflammation, whilst CHPPL-3, CHPPL-5, and NC exhibited mild to severe inflammation, and statistically significant difference was detected (p < .05). CHPPL-1, Dycal, and N-MTA exhibited continuous MTF, whilst CHPPL-3, CHPPL-5, and NC had thicker and interrupted MTF. Significant differences were observed between CHPPL-1, CHPPL-3, and N-MTA with NC group (p < .05). Variable expressions of IL-6, Nestin, and DMP-1 indicated differences in the materials' impact on odontoblast-like cell formation and tissue mineralization. CONCLUSIONS: These findings suggest that CHPPL-1 has the potential to minimize pulpal inflammation and promote MTF and had similar efficacy as MTA cement.

Biocompatibility and bioactive potential of NeoPUTTY calcium silicate-based cement: An in vivo study in rats.

AIM: To evaluate the inflammatory reaction and the ability to induce mineralization activity of a new repair material, NeoPUTTY (NPutty; NuSmile, USA), in comparison with Bio-C Repair (BC; Angelus, Brazil) and MTA Repair HP (MTA HP; Angelus, Brazil). METHODOLOGY: Polyethylene tubes were filled with materials or kept empty (control group, CG) and implanted in subcutaneous tissue of rats for 7, 15, 30, and 60 days (n = 6/group). Capsule thickness, number of inflammatory cells (ICs), fibroblasts, collagen content, and von Kossa analysis were performed. Unstained sections were evaluated under polarized light and by immunohistochemistry for osteocalcin (OCN). Data were submitted to two-way anova followed by Tukey's test (p ≤ .05), except for OCN. OCN data were submitted to Kruskal-Wallis and Dunn and Friedman post hoc tests followed by the Nemenyi test at a significance level of 5%. RESULTS: At 7, 15, and 30 days, thick capsules containing numerous ICs were seen around the materials. At 60 days, a moderate inflammatory reaction was observed for NPutty, BC while MTA HP presented thin capsules with moderate inflammatory cells. In all periods, NPutty specimens contained the highest values of ICs (p < .05). From 7 to 60 days, the number of ICs reduced significantly while an increase in the number of fibroblasts and birefringent collagen content was observed. At 7 and 15 days, no significant difference was observed in the immunoexpression of OCN (p > .05). At 30 and 60 days, NPutty showed the lowest values of OCN (p < .05). At 60 days, a similar immunoexpression was observed for BC and MTA HP (p > .05). In all time intervals, capsules around NPutty, BC, and MTA HP showed von Kossa-positive and birefringent structures. CONCLUSIONS: Despite the greater inflammatory reaction promoted by NeoPutty than BC and MTA HP, the reduction in the thickness of capsules, the increase in the number of fibroblasts, and the reduction in the number of ICs indicate that this bioceramic material is biocompatible Furthermore, NeoPutty presents the ability to induce mineralization activity.

Osteoinductive and regenerative potential of premixed calcium-silicate bioceramic sealers on vascular wall mesenchymal stem cells.

AIM: The osteogenic potential of new premixed calcium-silicate-containing bioceramic sealers (Ca-Si sealers) was tested with porcine vascular wall-mesenchymal stem cells (pVW-MSCs). METHODOLOGY: Two Ca-Si-containing sealers: Ceraseal (MetaBiomed, Cheong-si, South Korea) and AH Plus Bioceramic (Maruchi, Wonju-si, South Korea), and an epoxy resin sealer (AH Plus; Dentsply, Konstanz, Germany) as a control, were prepared according to the manufacturers' indications. All samples were allowed to set for 100% of their setting time in a sterile humid cabinet at 37°C and 95% relative humidity. pVW-MSC seeding efficiency and osteogenic differentiation were analysed as marker of gene/protein expression for up to 12 days. Mineralization assay and immunofluorescence staining were performed and evaluated over a period of 21 days. Statistical analyses were conducted using one-way analysis of variance (p < .05). Additional samples were prepared and stored under the same conditions and inspected using an environmental scanning electron microscope equipped with an energy dispersive X-ray spectroscopy system. RESULTS: Significantly higher cell seeding efficiency (p < .05) was observed for both Ca-Si sealers from day 8. pVW-MSCs showed a significant shift towards the osteogenic lineage only when seeded in contact with Ca-Si sealers. Gene expression of osteopontin was upregulated significantly. Collagen I and osteocalcin were clearly expressed by cells in contact with Ca-Si sealers. Mineralization granules were observed in Alizarin red assays and confocal laser scanning microscopy analysis of both Ca-Si sealers. No gene expression or granule mineralization were observed on the epoxy resin sealer. CONCLUSIONS: Premixed Ca-Si sealers displayed a higher potential for osteogenic activity on pVW-MSCs. Epoxy resin sealer was unable to induce any osteogenic activity. The properties of both Ca-Si sealers suggest their potential as osteoinductive platforms for vascular MSCs in periapical bone.

In vitro and in vivo evaluation of iRoot BP Plus as a coronal sealing material for regenerative endodontic procedures.

OBJECTIVES: To investigate in vitro effects of a nanoparticle bioceramic material, iRoot BP Plus, on stem cells from apical papilla (SCAP) and in vivo capacity to induce pulp-dentin complex formation. MATERIALS AND METHODS: The sealing ability of iRoot BP Plus was measured via scanning electron microscopy (SEM). SCAP were isolated and treated in vitro by iRoot BP Plus conditioned medium, with mineral trioxide aggregate (MTA) conditioned medium and regular medium used as controls, respectively. Cell proliferation was assessed by BrdU labeling and MTT assay and cell migration was evaluated with wound healing and transwell assays. Osteo/odontogenic potential was evaluated by Alizarin red S staining and qPCR. Pulp-dentin complex formation in vivo was assessed by a tooth slice subcutaneous implantation model. RESULTS: iRoot BP Plus was more tightly bonded with the dentin. There was no difference in SCAP proliferation between iRoot BP Plus and control groups (P > 0.05). iRoot BP Plus had a greater capacity to elevated cell migration (P < 0.05) and osteo/odontogenic marker expression and mineralization nodule formation of SCAP compared with MTA groups (P < 0.05). Furthermore, the new continuous dentine layer and pulp-like tissue was observed in the iRoot BP Plus group in vivo. CONCLUSIONS: iRoot BP Plus showed excellent sealing ability, promoted the migration and osteo/odontogenesis of SCAP and induced pulp-dentin complex formation without affecting the cell proliferation, which indicated iRoot BP Plus was a promising coronal sealing material in REPs. CLINICAL RELEVANCE: The coronal sealing materials play crucial roles for the outcomes of REPs. This study showed that iRoot BP Plus has good coronal sealing and promote pulp-dentin complex formation compared with MTA, providing experimental evidences for the clinical application of iRoot BP Plus as a promising coronal seal material in REPs.

A three-dimensional cell culture approach to investigate cytotoxic effects and production of inflammatory mediators by epoxy resin-based and calcium silicate-based endodontic sealer.

OBJECTIVES: The aim of the present study was to assess the cytocompatibility of epoxy resin-based AH Plus Jet (Dentsply De Trey, Konstanz, Germany), Sealer Plus (MK Life, Porto Alegre, Brazil), calcium silicate-based Bio-C Sealer (Angelus, Londrina, PR, Brazil), Sealer Plus BC (MK Life) and AH Plus BC (Dentsply) through a tridimensional (3D) culture model of human osteoblast-like cells. METHODS: Spheroids of MG-63 cells were produced and exposed to fresh root canal sealers extracts by 24 h, and the cytotoxicity was assessed by the Lactate Dehydrogenase assay (LDH). The distribution of dead cells within the microtissue was assessed by fluorescence microscopy, and morphological effects were investigated by histological analysis. The secreted inflammatory mediators were detected in cell supernatants through flow luminometry (XMap Luminex). RESULTS: Cells incubated with AH Plus Jet, AH Plus BC, Sealer Plus BC and Bio-C Sealer extracts showed high rates of cell viability, while the Sealer Plus induced a significant reduction of cell viability, causing reduction on the spheroid structure. Sealer Plus and Seaker Plus BC caused alterations on 3D microtissue morphology. The AH Plus BC extract was associated with the downregulation of secretion of pro-inflammatory cytokines IL-5, IL-7, IP-10 and RANTES. CONCLUSIONS: The new AH Plus BC calcium silicate-based endodontic sealer did not reduce cell viability in vitro, while led to the downregulation of pro-inflammatory cytokines. CLINICAL SIGNIFICANCE: Choosing the appropriate endodontic sealer is a crucial step. AH Plus BC demonstrated high cell viability and downregulation of pro-inflammatory cytokines, appearing reliable for clinical use, while Sealer Plus presented lower cytocompatibility.

Biocompatibility, bioactivity and immunomodulatory properties of three calcium silicate-based sealers: an in vitro study on hPDLSCs.

OBJECTIVES: To assess the biocompatibility, bioactivity, and immunomodulatory properties of three new calcium silicate cement-based sealers: Ceraseal (CS), Totalfill BC Sealer (TFbc) and WellRoot ST (WR-ST) on human periodontal ligament stem cells (hPDLSCs). MATERIALS AND METHODS: HPDLSCs were isolated from extracted third molars from healthy patients. Eluates (1:1, 1:2, and 1:4 ratio) and sample discs of CS, TFbc and WR-ST after setting were prepared. A series of assays were performed: cell characterization, cell metabolic activity (MTT assay) cell attachment and morphology (SEM assay), cell migration (wound-healing assay), cytoskeleton organization (phaloidin-based assay); IL-6 and IL-8 release (ELISA); differentiation marker expression (RT-qPCR assay), and cell mineralization (Alizarin Red S staining). HPDLSCs cultured in unconditioned (negative control) or osteogenic (positive control) culture media were used as a comparison. Statistical significance was established at p < 0.05. RESULTS: All the tested sealers exhibited similar results in the cytocompatibility assays (cell metabolic activity, migration, attachment, morphology, and cytoskeleton organization) compared with a negative control group. CS and TFbc exhibited an upregulation of at least one osteo/cementogenic marker compared to the negative and positive control groups. CS and TFbc also showed a significantly higher calcified nodule formation than the negative and positive control groups. Both the marker expression and calcified nodule formation were significantly higher in CS-treated cells than TFbc treated cells. WR-ST exhibited similar results to the control group. CS and TFbc-treated cells exhibited a significant downregulation of IL-6 after 72 h of culture compared to the negative control group (p < 0.05). CONCLUSION: All the tested sealers exhibited an adequate cytocompatibility. CS significantly enhances cell differentiation by upregulating the expression of key genes associated with bone and cementum formation. Additionally, CS was observed to facilitate the mineralization of the extracellular matrix effectively. In contrast, the effects of TFbc and WR-ST on these processes were less pronounced compared to CS. Furthermore, both CS and TFbc exhibited an anti-inflammatory potential, contributing to their potential therapeutic benefits in regenerative endodontics. CLINICAL RELEVANCE: This is the first study to compare the biological properties and immunomodulatory potential of Ceraseal, Totalfill BC Sealer, and WellRoot ST. The results act as supporting evidence for their use in root canal treatment.

Effects of mineral trioxide aggregate and methyl sulfonyl methane on pulp exposure via RUNX2 and RANKL pathways.

The aim of this study was to determine the therapeutic effects of mineral trioxide aggregate (MTA) and methyl sulfonyl methane (MSM) on pulp damage due to pulp exposure through the RUNX2 and RANKL pathways. Seventy-two male Sprague-Dawley rats aged 4-6 months and weighing 250-300 g were divided into healthy, control, MTA, and MSM groups. After experimental applications, all rats at 2, 4, and 8 weeks were killed anesthetically with xylazine hydrochloride (Rompun, Bayer) 30 mg/kg and ketamine hydrochloride (Ketalar, Pfizer) 50 mg/kg injections (i.p.). We observed that necrotic odontoblasts, edema, inflammation, and vascular congestion findings were reduced from week 2 to week 8 in the MSM treatment group after pulp capping compared to the control group and MTA group. Similarly, we found a decrease in RUNX2 and RANKL levels in the MSM application group compared to the control and MTA groups (p < 0.05). MSM material has shown therapeutic effects on pulp capping treatment-induced pulp injury via increased RUNX2 ve RANKL expression.

Laponite/lactoferrin hydrogel loaded with eugenol for methicillin-resistant Staphylococcus aureus-infected chronic skin wound healing.

Severe bacterial infections can give rise to protracted wound healing processes, thereby posing a significant risk to a patient's well-being. Consequently, the development of a versatile hydrogel dressing possessing robust bioactivity becomes imperative, as it holds the potential to expedite wound healing and yield enhanced clinical therapeutic outcomes. In this context, the present study involves the formulation of an injectable multifunctional hydrogel utilizing laponite (LAP) and lactoferrin (LF) as foundational components and loaded with eugenol (EG). This hydrogel is fabricated employing a straightforward one-pot mixing approach that leverages the principle of electrostatic interaction. The resulting LAP/LF/EG2% composite hydrogel can be conveniently injected to address irregular wound geometries effectively. Once administered, the hydrogel continually releases lactoferrin and eugenol, mitigating unwarranted oxidative stress and eradicating bacterial infections. This orchestrated action culminates in the acceleration of wound healing specifically in the context of MRSA-infected wounds. Importantly, the LAP/LF/EG2% hydrogel exhibits commendable qualities including exceptional injectability, potent antioxidant attributes, and proficient hemostatic functionality. Furthermore, the hydrogel composition notably encourages cellular migration while maintaining favorable cytocompatibility. Additionally, the hydrogel manifests noteworthy bactericidal efficacy against the formidable multidrug-resistant MRSA bacterium. Most significantly, this hydrogel formulation distinctly expedites the healing of MRSA-infected wounds by promptly inducing hemostasis, curbing bacterial proliferation, and fostering angiogenesis, collagen deposition, and re-epithelialization processes. As such, the innovative hydrogel material introduced in this investigation emerges as a promising dressing for the facilitation of bacterial-infected wound healing and consequent tissue regeneration.

Comparison of remineralization ability of tricalcium silicate and of glass ionomer cement on residual dentin: an in vitro study.

OBJECTIVE: This study aimed to compare the remineralization effects of a calcium silicate-based cement (Biodentine) and of a glass ionomer cement (GIC: Fuji IX) on artificially demineralized dentin. METHODS: Four standard cavities were prepared in dentin discs prepared from 34 extracted sound human third molars. In each disc, one cavity was covered with an acid-resistant varnish before demineralization (Group 1). The specimens were soaked in a chemical demineralization solution for 96 h to induce artificial carious lesions. Thereafter, one cavity each was filled with Biodentine (Group 2) and GIC (Group 3), respectively, and one carious lesion was left unrestored as a negative control (Group 4). Next, specimens were immersed in simulated body fluid (SBF) for 21 days. After cross-sectioning the specimens, the Ca/P ratio was calculated in each specimen by using scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDX). Finally, data were analyzed using repeated-measures ANOVA with post-hoc Bonferroni correction. RESULTS: Both cement types induced dentin remineralization as compared to Group 4. The Ca/P ratio was significantly higher in Group 2 than in Group 3 (p < 0.05). CONCLUSION: The dentin lesion remineralization capability of Biodentine is higher than that of GIC, suggesting the usefulness of the former as a bioactive dentin replacement material. CLINICAL RELEVANCE: Biodentine has a higher remineralization ability than that of GIC for carious dentin, and its interfacial properties make it a promising bioactive dentin restorative material.

The effects of mineral trioxide aggregate and second-generation autologous growth factor on pulpotomy via TNF-α and NF-kß/p65 pathways.

This study aims to investigate the effect of Mineral Trioxide Aggregate (MTA), a bioactive endodontic cement, and Concentrated Growth Factor (CGF), a second-generation autologous growth factor, on pulpotomy-induced pulp inflammation. The study utilized the maxillary anterior central teeth of thirty-six young male Sprague Dawley rats. Forty-eight teeth were randomly assigned to two groups (12 rats/group; 24 teeth/group) based on the capping material (MTA or CGF). Subsequently, two subgroups (MTAG and CGFG) were formed per group (12 teeth/group) based on the time following pulpotomy (2-weeks and 4-weeks). The central teeth of the 12 animals assigned to the control group (CG) were not manipulated in any way, both in the 2-week group and in the 4-week group. Tissue samples extracted from rats at the end of the experiment were stained with H&E for histopathological analysis. For immunohistochemical analysis, primary antibodies for TNF-α and NF-kß/65 were incubated. Data obtained from semi-quantitative analysis were assessed for normal distribution using Skewness-Kurtosis values, Q-Q plot, Levene's test, and the Shapiro-Wilk test on statistical software. A P value < 0.05 was considered significant. When compared with the control group, both MTAG and CGFG showed increased edematous and inflammatory areas. In MTAG, edematous and inflammatory areas decreased significantly from the 2nd week (2(2-2), 2(1-2)) to the 4th week (1(1-1), 1(0-1)), while in CGFG, edematous areas decreased (2(2-3), 1.5(1-2)), and inflammatory areas increased significantly (2(2-3), 3(2-2.5)). When compared with the control group, TNF-α and NF-kß/p65 positivity were higher in both MTAG and CGFG. In MTAG, TNF-α [2(1.5-2)] and NF-kß/p65 [1.5(1-2)] positivity decreased significantly from the 2nd week to the 4th week [TNF-α: 1(1-1), NF-kß/p65: 1(1-2)], while no significant change was observed in CGFG. In conclusion, this study revealed a reduction in cells showing TNF-α and NF-kß/p65 positivity in the MTA treatment group compared to the CGF group. Although MTA demonstrated more favorable results than CGF in mitigating pulpal inflammation within the scope of this study, further experimental and clinical investigations are warranted to obtain comprehensive data regarding CGF.

Effect of exposure conditions on chemical properties of materials for surgical endodontic procedures.

This study investigated the role of aging and changes in environmental conditions on selected properties of a prototype radiopacified calcium silicate-based cement (TZ-base) with or without incorporation of silver nanoparticles or bioactive glass, and two commercial materials, Biodentine and intermediate restorative material. Materials were immersed in ultrapure water or fetal bovine serum for 28 days and were characterized with scanning electron microscopy and energy dispersive x-ray analysis. Immersion media were either replaced weekly or not replenished at all and were assessed for alkalinity and calcium release after 1, 7, 14, 21, and 28 days; antibacterial effect against 2-day monospecies biofilms; and cytotoxicity by the 3-(4,5 dimethylthiazolyl-2-yl)-2,5-diphenyl tetrazolium bromide assay after 1, 7, or 28 days. Alkalinity, calcium release, antibacterial activity, and cell cytotoxicity increased over time when the medium was not changed but decreased with medium replenishment. Immersion in fetal bovine serum resulted in lower alkalinity, less bactericidal properties, and lower cytotoxicity of prototype cements and Biodentine than did water immersion. Biodentine and 20% bioactive glass-containing cement had overall lower alkalinity, calcium release, and antibacterial activity than TZ-base, and Biodentine was less cytotoxic than TZ-base. In conclusion, exposure conditions and cement modifications significantly affected materials' leaching properties. Exposure conditions warrant consideration when evaluating cements' clinical properties.

Effects of phosphate and silicate on stiffness and viscoelasticity of mature biofilms developed with simulated drinking water.

Biofilms, a porous matrix of cells aggregated with extracellular polymeric substances under the influence of chemical constituents in the feed water, can develop a viscoelastic response to mechanical stresses. In this study, the roles of phosphate and silicate, common additives in corrosion control and meat processing, on the stiffness, viscoelasticity, porous structure networks, and chemical properties of biofilm were investigated. Three-year biofilms on PVC coupons were grown from sand-filtered groundwater with or without one of the non-nutrient (silicate) or nutrient additives (phosphate or phosphate blends). Compared with non-nutrient additives, the phosphate and phosphate-blend additives led to a biofilm with the lowest stiffness, most viscoelastic, and more porous structure, including more connecting throats with greater equivalent radii. The phosphate-based additives also led to more organic species in the biofilm matrix than the silicate additive did. This work demonstrated that nutrient additives could promote biomass accumulation but also reduce mechanical stability.

Inflammatory and differentiation cellular response to calcium silicate cements: An in vitro study.

AIM: Inflammatory-regenerative cell interaction is believed to mediate hard tissue formation. This study aimed to investigate the interaction between human inflammatory monocytes with human regenerative fibroblasts after exposure to different calcium silicate materials. METHODOLOGY: Human monocytes were cultured on three materials, polystyrene (PS), mineral trioxide aggregates (MTA) and biodentine (BD), in the presence or absence of lipopolysaccharide (LPS). Half of the monocyte-conditioned media (MoCM) of each group was used to analyse inflammatory cytokine secretion, namely TNF-α, IL-1ß, IL-1RA and IL-6. The remaining MoCM was used to culture recipient fibroblasts, measuring the cell number (proliferation) and levels of alkaline phosphatase (differentiation) and lactic acid dehydrogenase (cytotoxicity). RESULTS: In absence of LPS, MTA was associated with higher secretion of TNF-α and lower secretion of IL-1ß, while BD triggered higher secretions of both cytokines when both materials were compared to control (PS). When LPS was added, higher levels of all analysed cytokines were observed in the PS and BD groups, whereas for the MTA group, only TNF-α and IL-6 were increased. Fibroblasts responded differently to the MoCM from the different groups, revealing significant increases in proliferation and differentiation capacities, particularly when cultured in CM from monocytes exposed to MTA. The morphological evaluation revealed different patterns of fibroblast shape and spread in the different MoCM groups. CONCLUSION: Calcium silicate materials modulate the monocyte inflammatory response, which subsequently induce differential effects on the recipient fibroblasts. MTA appears to promote the secretion of prodifferentiation signals from the monocytes, which are received by fibroblasts, promoting their proliferation and differentiation. The model represents a promising tool to evaluate the interaction of different cells in response to different materials.

In vitro inflammatory modulation of bioceramic endodontic sealer in macrophages stimulated by bacterial lipopolysaccharide.

AIM: To evaluate the effects of AH Plus (Dentsply), Sealer 26 (Dentsply), and Sealer Plus BC (Produtos Médicos e Odontológicos) on cytotoxicity and inflammation in macrophage cultures exposed to bacterial lipopolysaccharide (LPS). METHODOLOGY: After initial setting, the sealers were conditioned with serum-free culture medium for 24 h (1 ml/cm2 ). Macrophages from the RAW 264.7 strain were exposed to sealer extracts in a 1:16 ratio in a culture medium with or without LPS. Cell morphology, viability, mitochondrial activity, oxidative stress and gene expression of interleukin 1ß (IL-1ß) and tumour necrosis factor-alpha (TNF-α) were evaluated. Data on mitochondrial activity, oxidative stress and TNF-α were analysed using a two-way analysis of variance (anova) test, followed by the Student-Newman-Keuls post-test. IL-1ß data were analysed using one-way anova, followed by SNK, and the t-test was used for intragroup comparison. The significance level was set at 5%. RESULTS: In the absence of LPS, only AH Plus and Sealer 26 showed a reduction in cell density, while in the presence of LPS, Sealer 26 had the lowest density compared to the other groups. In terms of mitochondrial activity, at 24 and 48 h, Sealer Plus BC had significantly higher mean values than Sealer 26 and AH Plus (p < .05). Sealer 26 exhibited the lowest levels of oxidative stress and IL-1ß and TNF-α expression, regardless of the presence of LPS (p < .05). CONCLUSIONS: Although all sealers interfere with the response of macrophages to LPS, contact with epoxy resin-based sealers can impair cell activity in vitro, while bioceramic sealer seems to favour the inflammatory functions of these cells.

Participation of fibroblast growth factor-1 and interleukin-10 in connective tissue repair following subcutaneous implantation of bioceramic materials in rats.

AIM: To evaluate whether the bioceramic materials Bio-C Pulpo (Bio-C, Angelus) and mineral trioxide aggregate (MTA) Repair HP (MTA-HP, Angelus) induce fibroblast proliferation and release of interleukin-10 (IL-10), an anti-inflammatory cytokine, stimulating connective tissue remodelling. The tissue response of Bio-C and MTA-HP was compared with the White MTA (WMTA; Angelus) since studies have demonstrated that WMTA induces tissue repair. METHODOLOGY: Bio-C, MTA-HP and WMTA were inserted into polyethylene tubes and implanted in the subcutaneous tissue of Holtzman rats for 7, 15, 30 and 60 days. As a control group (CG), empty tubes were implanted subcutaneously. The number of fibroblasts (FB), Ki-67-, fibroblast growth factor-1- (FGF-1) and IL-10-immunolabelled cells and collagen content in the capsules was obtained. The data were subjected to two-way anova followed by Tukey's test (p ≤ .05). RESULTS: At 7 days, significant differences in the number of FB were not detected amongst Bio-C, MTA-HP and WMTA groups (p Ëƒ .05). The capsules of all groups exhibited a significant increase in the number of FB and content of collagen over time. From 7 to 60 days, a significant reduction in the number of FGF-1- and Ki-67-immunolabelled cells was seen in the capsules of all specimens. In all periods, no significant difference in the number of FGF-1-immunolabelled cells was detected between Bio-C and CG specimens. At 60 days, significant differences in the immunoexpression of FGF-1 were not observed amongst the groups. At 7 and 15 days, the highest immunoexpression for Ki-67 was present in Bio-C specimens whilst, after 30 and 60 days, no significant difference was observed amongst the bioceramic materials. At 7 days, few IL-10 immunolabelled cells were present in the capsules of all specimens whereas, at 60 days, a significant increase in the IL-10-immunostaining was present in all groups. At 60 days, the Bio-C, MTA-HP and WMTA groups showed a greater number of IL-10-immunolabelled cells than in the CG specimens (p < .0001). CONCLUSIONS: Bio-C, MTA-HP and WMTA stimulate fibroblast proliferation, leading to the formation of collagen-rich capsules. FGF-1 and IL-10 may mediate the remodelling of capsules around Bio-C, MTA-HP and WMTA bioceramic materials.

Biocompatibility and bioactive potential of an experimental tricalcium silicate-based cement in comparison with Bio-C repair and MTA Repair HP materials.

AIM: To evaluate the tissue reaction of a tricalcium silicate-based repair material associated with 30% calcium tungstate (TCS + CaWO4 ) in comparison to Bio-C Repair (Bio-C; Angelus) and to MTA Repair HP (MTA HP; Angelus). METHODOLOGY: Polyethylene tubes filled with one of the materials or left empty (control group, CG) were implanted into the subcutaneous tissues of rats for 7, 15, 30 and 60 days (n = 32/group). The capsule thickness, number of inflammatory cells, collagen content, interleukin-6 (IL-6), osteocalcin (OCN), von Kossa reaction and analysis under polarized light were evaluated. The data were subjected to generalized linear models for repeated measures, except the OCN. OCN data were submitted to Kruskal-Wallis and Dunn's post hoc test and Friedman followed by Nemenyi's test at significance level of 5%. RESULTS: At all time points, significant differences in the number of inflammatory cells were not observed between TCS + CaWO4 and Bio-C, whereas, at 15, 30 and 60 days, no significant difference was detected between TCS + CaWO4 and MTA HP. At all periods, significant differences were not detected in the number of fibroblasts in TCS + CaWO4 versus MTA HP, and, at 60 days, no significant difference was demonstrated between these groups and CG. Significant differences in the immunoexpression of IL-6 were not detected amongst bioceramic materials at all periods. From 7 to 60 days, significant reduction in the number of inflammatory cells, number of IL-6-immunopositive cells and in the capsule thickness was accompanied by significant increase in the collagen in all groups. OCN-immunolabelled cells, von Kossa-positive structures and amorphous calcite deposits were observed around all materials, whereas, in the CG, these structures were not seen. CONCLUSIONS: These findings indicate that the experimental material (TCS + CaWO4 ) is biocompatible and has a bioactive potential, similar to the MTA HP and Bio-C Repair, and suggest its use as a root repair material.

Inflammatory response and odontogenic differentiation of inflamed dental pulp treated with different pulp capping materials: An in vivo study.

AIM: Previous studies have evaluated the pulpal responses to calcium silicate cements (CSCs) on normal dental pulp, but investigations on the effects of CSCs on inflamed pulp are limited. This study aimed to test the inflammatory response and odontogenic differentiation of inflamed rat dental pulp after direct pulp capping with CSCs. METHODOLOGY: Wistar rat molars pulps were exposed for 48 h to induce inflammation and then capped with ProRoot MTA (Dentsply), Biodentine (Septodont), RetroMTA (Bio MTA) and Dycal (Dentsply Caulk). The degree of pulpal inflammation and hard tissue formation was evaluated by histological analysis. Immunofluorescence staining for interleukin (IL)-6, osteocalcin (OCN) and runt-related transcription factor 2 (RUNX2) was also performed. RESULTS: After 4 weeks, complete recovery from inflammation was evident in 22%, 37.5%, 10% and none of the ProRoot MTA, Biodentine, RetroMTA and Dycal samples, respectively. Heavy hard tissue deposition as a continuous hard tissue bridge was observed in 77.8%, 75%, 70% and 60% of the ProRoot MTA, Biodentine, RetroMTA and Dycal samples, respectively. IL-6, OCN and RUNX2 were detected in all materials, mainly adjacent to areas of inflammation and reparative dentine formation. At one, two and 4 weeks, significant differences were not observed between the inflammation and hard tissue formation scores of the four material groups (p > .05). CONCLUSIONS: In this study, pulpal inflammation was still present in most specimens at 4 weeks after pulp capping and a significant number of samples showed incomplete and discontinuous dentine bridge formation. The results of this study suggest that initial inflammatory conditions of the pulp may risk the prognosis of teeth treated with CSCs.

Evaluation of the effectiveness of different treatment approaches in preventing coronal discoloration caused by regenerative endodontic treatment.

OBJECTIVES: This study aimed to evaluate the effectiveness of Teethmate desensitizer, a dentin bonding agent (DBA), Nd:YAG laser, and Er:YAG laser, which provides dentin tubule occlusion in the pulp chamber with different mechanisms, in preventing tooth discoloration due to regenerative endodontic treatment. MATERIALS AND METHODS: One hundred five extracted maxillary human incisors with single roots and single canals were included in the study. The apical third of each tooth was resected below the enamel-cementum junction (CEJ) to obtain a standard root length as 10 ± 1 mm. Root canal preparation was performed using the ProTaper Next files up to X5. Root canals were prepared with Gates Glidden (# 2-4) burs to simulate the immature root apex and an apical diameter of 1.1 ± 0.1 mm was obtained. The teeth were randomly divided into 7 groups (n = 15): DBA, Teethmate, Nd:YAG, Er:YAG, Biodentine, Blood, and Negative Control. Relevant dentin tubule occlusion methods were applied to DBA, Teethmate, Nd:YAG, and Er:YAG groups. Following dentin tubule occlusion procedures, Biodentine was placed on the blood clot after filling the root canals with blood up to 4 mm below the CEJ. No dentin tubule occlusion procedure was applied for Blood and Biodentine groups. Color measurement was performed with the spectrophotometer Vita Easyshade Advance before treatment, immediately after treatment, and at days 7, 30, and 90. Data were converted to L*a*b color values of Commission International de I'Eclairage (CIE L*a*b) and ΔE values were calculated. Two-way ANOVA and post hoc Tukey test (p = 0.05) were performed for statistical analysis. RESULTS: A clinically detectable color change was observed in all groups except for the negative control (ΔE ≥ 3,3). It was observed that Biodentine used alone has a potential for discoloration. It was determined that as the contact time with blood increased, tooth discoloration increased. However, no significant difference was found between dentin tubule occlusion methods in preventing color change (p > 0.05). CONCLUSIONS: It was determined that no dentin tubule occlusion method could 100% prevent discoloration caused by RET. CLINICAL RELEVANCE: DBA and Teethmate, which do not have a significant difference in terms of preventing color change, are considered to be suitable for dentin tubule occlusion due to their ease of application and low cost compared to Nd:YAG laser and Er:YAG laser.

Comparative biological properties of resin-free and resin-based calcium silicate-based endodontic repair materials on human periodontal ligament stem cells.

OBJECTIVES: To investigate the effect of three different calcium silicate-based materials (CSBM) on the biological behavior of human periodontal ligament stem cells (hPDLSCs). METHODS: Eluates of Biodentine, NeoPutty and TheraCal PT prepared at 1:1, 1:2, and 1:4 ratios were extracted under sterile conditions. The cytotoxicity of the extracts to the hPDLSCs was assessed using the MTT assay. Scratch wound healing assay was utilized for assessing cell migration. Scanning electron microscopy was used to detect cell attachment and morphology. Calcium ion release was measured using inductively coupled plasma-optical emission spectrometry; the pH-value was evaluated with a pH-meter. ANOVA with post hoc Tukey test was used for statistical analysis. RESULTS: Cell viability was significantly higher for Biodentine and NeoPutty at day 1 with all dilutions (p < 0.05), while at day 3 and day 7 with dilutions 1:2 and 1:4; all materials showed similar behavior (p > 0.05). Biodentine had the highest percentage of cell migration into the scratched area at day 1 for all dilutions (p < 0.05). Stem cells were attached favorably on Biodentine and NeoPutty with evident spreading, and intercellular communications; however, this was not shown for TheraCal PT. Biodentine showed the highest pH values and calcium ion release (p < 0.05). CONCLUSIONS: The resin-free CSBM showed better performance and favorable biological effects on hPDLSCs and were therefore considered promising for usage as endodontic repair materials. CLINICAL SIGNIFICANCE: Proper selection of materials with favorable impact on the host stem cells is crucial to ensure outcome in different clinical scenarios.

Biological properties of Ceraputty as a retrograde filling material: an in vitro study on hPDLSCs.

OBJECTIVES: To assess the cytocompatibility and bioactive potential of the new calcium silicate-based cement Ceraputty on human periodontal ligament stem cells (hPDLSCs) compared to Biodentine and Endosequence BC root repair material (ERRM). MATERIALS AND METHODS: hPDLSCs were isolated from extracted third molars from healthy donors. Standardized sample discs and 1:1, 1:2, and 1:4 eluates of the tested materials were prepared. The following assays were performed: surface element distribution via SEM-EDX, cell attachment and morphology via SEM, cell viability via a MTT assay, osteo/cemento/odontogenic marker expression via RT-qPCR, and cell calcified nodule formation via Alizarin Red S staining. hPDLSCs cultured in unconditioned or osteogenic media were used as negative and positive control groups, respectively. Statistical analysis was performed using one-way ANOVA or two-way ANOVA and Tukey's post hoc test. Statistical significance was established at p < 0.05. RESULTS: The highest Ca2+ peak was detected from Biodentine samples, followed by ERRM and Ceraputty. hPDLSC viability was significantly reduced in Ceraputty samples (p < 0.001), while 1:2 and 1:4 Biodentine and ERRM samples similar results to that of the negative control (p > 0.05). Biodentine and ERRM exhibited an upregulation of at least one cemento/odonto/osteogenic marker compared to the negative and positive control groups. Cells cultured with Biodentine produced a significantly higher calcified nodule formation than ERRM and Ceraputty (p < 0.001), which were also higher than the control groups (p < 0.001). CONCLUSION: Ceraputty evidenced a reduced cytocompatibility towards hPDLSCs on its lowest dilutions compared to the other tested cements and the control group. Biodentine and ERRM promoted a significantly higher mineralization and osteo/cementogenic marker expression on hPDLSCs compared with Ceraputty. Further studies are necessary to verify the biological properties of this new material and its adequacy as a retrograde filling material. CLINICAL RELEVANCE: This is the first study to elucidate the adequate biological properties of Ceraputty for its use as a retrograde filling material.