Citric Acid Conditioning as an Alternative to EDTA for Growth Factors Release and Stem Cell Response in Regenerative Endodontics: A Systematic Review of In Vitro Studies.

INTRODUCTION: Citric acid (CA) conditioning may be a promising alternative to ethylenediaminetetraacetic acid (EDTA) in regenerative endodontic procedures, as reported to improve growth factors' release from dentin. This review systematically investigated the effect of CA conditioning on the growth factors release from dentin and cell behavior compared to EDTA conditioning. METHODS: Searches were conducted (PubMed/MEDLINE, Scopus, Web of Science, Embase, SciELO, Cochrane Library, and grey literature) until May-2023. Only in vitro studies that evaluated the effects of CA on growth factors' release from dentin and cell behavior outcomes compared to EDTA were included. The studies were critically appraised using a modified Joanna Briggs Institute's checklist. Meta-analysis was unfeasible. RESULTS: Out of the 335 articles screened, nine were included. Among these, three studies used dentin discs/roots from permanent human teeth; the rest combined them with stem cells. 10% CA for 5 or 10 minute was the most used protocol. Meanwhile, EDTA concentrations ranged from 10% to 17%. In eight studies examining the release of growth factors, five reported a significant release of transforming growth factor-ß after dentin conditioning with 10% CA compared to 17% EDTA. Regarding cell behavior (6 studies), three studies assessed cell viability. The findings revealed that 10% CA conditioning showed cell viability similar to those of 17% EDTA. Additionally, in two out of three studies, it was observed that 10% CA conditioning did not affect cell morphology. The studies had a low risk of bias. CONCLUSIONS: The use of 10% CA to condition dentin for 5-10 minutes resulted in a notable transforming growth factor -ß1 release, but its cell responses were similar to those of EDTA.

Reduced bone resorption and inflammation in apical periodontitis evoked by dietary supplementation with probiotics in rats.

AIM: To evaluate the relationship between systemic administration of probiotics and inflammation/resorption processes associated with apical periodontitis (AP) in a rat model. METHODOLOGY: Twenty-four male Wistar rats were used. AP was induced in the mandibular left/right first molars. The animals were arranged into three groups: Control, Lactobacillus rhamnosus and L. acidophilus. Probiotics were orally administered via gavage (109 colony-forming units (CFU) diluted in 5 mL of water) for 30 days during the development of AP. On the 30th day, blood was collected to analyse the calcium, phosphorus and alkaline phosphatase concentrations in plasma. Then, the animals were euthanized and the jaws removed for micro-computed tomography and immune-histopathological analysis for receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phosphatase (TRAP). After the Shapiro-Wilk test of normality, the Kruskal-Wallis followed by Dunn's test was performed for nonparametric data, and analysis of variance followed by the Tukey test was performed for parametric data (P < 0.05). RESULTS: There was no significant difference in the calcium and phosphorus levels in plasma amongst the groups (P > 0.05). The level of alkaline phosphatase was significantly higher in the groups that consumed probiotics (P < 0.05). A significantly lower volume of bone resorption was observed in groups that consumed probiotics (P < 0.05). The inflammatory infiltrates and the immunolabelling for RANKL and TRAP were significantly lower in probiotic groups when compared to the control (P < 0.05). Also, the OPG was significantly more immunolabelled in the L. acidophilus group than in the L. rhamnosus and control groups (P < 0.05). CONCLUSION: Probiotic supplementation through gavage (L. rhamnosus and L. acidophilus) had a significant effect on the reduction of inflammation and bone resorption in apical periodontitis development in rats.

Systemic administration of probiotics reduces the severity of apical periodontitis.

AIM: To evaluate the effect of systemic administration of probiotics on the severity of apical periodontitis (AP). METHODOLOGY: Twenty-four male Wistar rats were used. AP was induced in the maxillary left/right first molars. The animals were arranged into groups: Control, Lactobacillus rhamnosus, and Lactobacillus acidophilus. Probiotics were administered orally for gavage (109 colony-forming units diluted in 5 mL of water for 30 days) during the development of AP. After 30 days, cardiac puncture was performed to analyse the complete blood count. Moreover, microbiological analysis of the root canal contents and saliva was performed. Then, the animals were euthanized and the jaw removed for histopathological and IL-10, IL-1ß and IL-6 immunolabeling analyses. After the Shapiro-Wilk test of normality, the Kruskal-Wallis followed by Dunn's test was performed for nonparametric data, and analysis of variance followed by the Tukey test was performed for parametric data (P < 0.05). RESULTS: No significance difference was observed in the blood profiles and in the counts of microorganisms from the saliva samples among the groups (P > 0.05). Total microorganism counts in the root canal, the inflammatory infiltrate and the immunostaining for IL-1ß and IL-6 in AP were significantly lower in the probiotic groups when compared with the control group (P < 0.05). IL-10 was significantly more immunolabled in the probiotic groups than in the control group (P < 0.05). CONCLUSION: Supplementation with probiotics (Lactobacillus rhamnosus and Lactobacillus acidophilus) had a significant effect on the severity of apical periodontitis in rats, demonstrating the anti-inflammatory effect of probiotics on the development of apical periodontitis.

Chronic alcohol consumption increases inflammation and osteoclastogenesis in apical periodontitis.

AIM: To investigate the effect of chronic alcohol consumption on apical periodontitis in rats. METHODOLOGY: Thirty-two male Wistar rats were arranged into four groups: Control (C): without apical periodontitis and nonalcoholic diet; (AL): without apical periodontitis and alcoholic diet; (AP): with apical periodontitis and nonalcoholic diet; and (AP + AL): with apical periodontitis and alcoholic diet. The alcoholic solution at 20% was given to the AL and AP + AL groups as the sole source of hydration throughout the experiment. AP was induced in the mandibular left first molars at the end of the 4th week. Weight changes and the amount of solid and liquid foods were recorded for 8 weeks. At the end, the animals were euthanized and the jaws removed followed by histological processing for histopathological and RANKL, OPG, TRAP and HIF-1α analyses. The Mann-Whitney test was used for nonparametric data, and anova followed by the Tukey test was performed for parametric data, with P < 0.05. RESULTS: Animals that received the alcoholic diet had a lower weight gain than the other groups (P < 0.05). Control and AL groups did not have an inflammatory response in the periapical tissues. The median score of inflammatory infiltrate was significantly higher in the AP + AL group (2.5) compared to the AP group (1.5; P < 0.05). In the same comparison, AP + AL was associated with score 3 for RANKL and HIF-1α versus score 2 for AP group (P < 0.05). Moreover, the values for TRAP were 3.88 ± 0.70 cells mm-1 for the AP + AL group and 2.43 ± 0.94 cells mm-1 for the AP group (P < 0.05). CONCLUSION: In rats, an alcoholic diet had a significant effect on the severity of apical periodontitis, exacerbating the inflammatory response and osteoclastogenesis.

Biocompatibility and immunohistochemical evaluation of a new calcium silicate-based cement, Bio-C Pulpo.

AIM: To evaluate the inflammatory response and ability to induce mineral deposition through histological and immunohistochemical analysis for osteocalcin (OCN), osteopontin (OPN) and bone sialoprotein (BSP) of a new calcium silicate-based cement, Bio-C Pulpo (Angelus), compared to white mineral trioxide aggregate (White MTA-Ang) (Angelus). METHODOLOGY: Polyethylene tubes containing Bio-C Pulpo and White MTA-Ang as well as empty tubes were implanted into the dorsal connective tissue of 30 Wistar rats, which were arranged in five groups according to the period of analysis: 7, 15, 30, 60 and 90 days. After each experimental period, the tubes with surrounding tissue were removed and histologically processed to be analysed using haematoxylin-eosin and immunohistochemistry for the detection of OCN, OPN and BSP. The data were statistically analysed (Friedman's test) at a 5% significance level. RESULTS: The inflammatory response observed with Bio-C Pulpo and White MTA-Ang was greater after 7 and 15 days and decreased from 30 days onwards. No significant difference was found between the control, Bio-C Pulpo and White MTA-Ang at the different periods of analysis (P > 0.05). The immunolabelling for OCN, OPN and BSP was more intense for Bio-C Pulpo and White MTA-Ang after 60 and 90 days, but there was no difference between Bio-C Pulpo and White MTA-Ang at the different periods of analysis (P > 0.05). CONCLUSION: Bio-C Pulpo is biocompatible and induces immunolabelling of osteogenic markers such as OCN, OPN and BSP similar to White MTA-Ang.

Systemic bone marker expression induced by grey and white mineral trioxide aggregate in normal and diabetic conditions.

AIM: To investigate the relationship between diabetes mellitus and local/systemic effects of both grey and white mineral trioxide aggregate (MTA) Angelus on bone marker expression. METHODOLOGY: Wistar rats were divided into two groups: healthy and diabetic (Alloxan induced), which were further divided into three subgroups (control, GMTA Angelus and WMTA Angelus). Polyethylene tubes filled with MTA materials or empty tubes were implanted in dorsal connective tissue. On days 7 and 30, blood samples were collected for calcium, phosphorus and ALP measurement. The animals were euthanized; implanted tubes were removed and processed for immunohistochemical analysis of osteocalcin (OCN) and osteopontin (OPN). Kruskal-Wallis followed by Dunn's multiple comparison test was performed for nonparametric data, and anova followed by Tukey's test for parametric data. RESULTS: No difference in systemic serum calcium levels between both groups was observed. On day 7, serum phosphorus levels within the WMTA healthy group were higher than that of the diabetic group. On day 30, healthy rats exhibited lower phosphorus levels than diabetic ones. At both time points, the diabetic group was associated with more ALP activity than the healthy group. Immunohistochemical analyses of the healthy group revealed OCN- and OPN-positive cells in the presence of both MTA materials. However, under diabetic conditions, both OCN and OPN were absent. CONCLUSION: Both MTA materials were associated with an increase in serum calcium, phosphorus and ALP, suggesting a potential systemic effect, along with triggered differentiation of OCN- and OPN-positive cells. Moreover, in diabetic conditions, an inhibitory effect on MTA-induced differentiation of OCN- and OPN-positive cells was detected.