Probiotics enhance alveolar bone microarchitecture, intestinal morphology and estradiol levels in osteoporotic animals.

BACKGROUND AND OBJECTIVE: Osteoporosis is associated with bone microarchitecture alterations, and the depletion of estrogen during menopause is a major contributing factor to its development. The literature highlights the noteworthy role of gut microbiota in bone metabolism, particularly in the progression of osteoporosis. Periodontal disease leads to alveolar bone loss, which may be influenced by estrogen deficiency, and this mechanism is intricately associated with an imbalance in systemic microbiota. The aim of this study was to evaluate the effects of Bifidobacterium animalis subsp. lactis HN019 (B. lactis HN019) and Lacticaseibacillus casei 01 (L. casei 01) administrations on an osteoporosis animal model. MATERIALS AND METHODS: Thirty-three female rats were randomly divided into three groups: control (C-OVX), C-OVX-HN019 and C-OVX-LC01. All animals were ovariectomized. In groups C-OVX-HN019 and C-OVX-LC01, the probiotics were administered for 4 months. All animals were euthanized after 16 weeks from ovariectomy. Microtomographic, histopathological and immunohistochemical examinations were conducted on periodontal tissues, whereas histomorphometry, histopathological and immunohistochemical analyses were carried out on the intestine. The levels of estradiol were assessed in blood using an immunoenzymatic assay. The data were subjected to statistical analyses (p < .05). RESULTS: The C-OVX-LC01 group exhibited a significant reduction in alveolar bone porosity and an increase in connective tissue density compared to C-OVX (p < .05). The C-OVX-HN019 and C-OVX-LC01 groups presented reduced expression of TRAP and RANKL compared to the C-OVX (p < .05). The C-OVX group presented villi defects, mild neutrophil infiltration, decrease in both villous height and intestinal crypts and reduced expression of intestinal junctional epithelium markers e-cadherin and claudin 01 compared to C-OVX-HN019 and C-OVX-LC01 (p < .05). The C-OVX group had lower estradiol levels than C-OVX-HN019 and C-OVX-LC01 (p < .05). CONCLUSION: The probiotic therapy promoted a reduction in alveolar bone destruction and intestinal permeability as well as an increase in estradiol levels in ovariectomized rats. Specifically, the probiotic strain Lacticaseibacillus casei 01 exhibited greater effectiveness compared to Bifidobacterium animalis subsp. lactis HN019, indicating strain-dependent outcomes.

Inflammatory profile of apical periodontitis associated with liver fibrosis in rats: histological and immunohistochemical analysis.

AIM: To investigate the effects of liver fibrosis (LF) on the pro-inflammatory mediators and periapical bone resorption of apical periodontitis (AP) in rats. METHODOLOGY: Forty male Wistar rats were distributed into four groups: C - control, AP - rats with AP, LF - rats with LF, AP + LF - rats with AP and LF. LF was induced by carbon tetrachloride administration for 8 weeks and surgical bile duct ligation for 4 weeks; AP was induced in the teeth of rats by dental pulp exposure to the oral environment for 30 days. Jaws and livers were removed after euthanasia. Haematoxylin and Eosin (H&E) and Picrosirius Red (PSR) staining were used to confirm fibrosis in the livers. The jaws were analysed using H&E staining, immunohistochemical assays of interleukin (IL)-1ß, IL-6 and tumour necrosis factor-alpha (TNF-α). Student's t-test and Mann-Whitney's U-test were used for statistical analysis (P < 0.05). RESULTS: Inflammatory infiltrate was moderate in the AP group and severe in the AP + LF group (P < 0.05). Periapical bone resorption was significantly larger in the AP + LF group compared with the AP group (P < 0.05). IL-1ß, IL-6 and TNF-α levels were significantly higher in AP + LF group when compared to the AP group (P < 0.05). CONCLUSION: More intense inflammatory infiltrate, greater amounts of pro-inflammatory cytokines and increased periapical bone resorption were observed in the presence of liver fibrosis in rats with exposed pulps.

Biocompatibility, induction of mineralization and antimicrobial activity of experimental intracanal pastes based on glass and glass-ceramic materials.

AIM: To evaluate the biocompatibility, induction of mineralization and antimicrobial activity of experimental intracanal pastes based on two glass and glass-ceramic materials. Calcium hydroxide (Ca(OH)2 ) paste was used as the positive control. METHODOLOGY: The glass-ceramic powder [two-phased Biosilicate (BS-2P)] and F18 bioactive glass were mixed with distilled water (ratio 2 : 1), inserted in polyethylene tubes and implanted in the subcutaneous tissues of 16 rats. Empty tubes were used as negative control. After 7 and 30 days (n = 8), the rats were euthanized for haematoxylin-eosin, von Kossa, polarized light and osteopontin (OPN) immunolabeling analysis. Direct contact tests using a suspension of each paste were performed with Enterococcus faecalis planktonic cells to evaluate antimicrobial activity (24 h of contact), in a pilot study. The number of CFU mL-1 was calculated for each group. The antimicrobial analysis data were submitted to one-way anova and Tukey tests, whilst biocompatibility and immunohistochemical data were submitted to the Kruskal-Wallis and Dunn tests (P < 0.05). RESULTS: Most specimens of the control, BS-2P and Ca(OH)2 groups were associated with moderate inflammation seven days following implantation, whilst F18 was associated with moderate to severe inflammation, without differences amongst the groups (P > 0.05). At 30 days, most specimens of control, F18 and BS-2P groups had mild inflammation, whilst Ca(OH)2 had mild to moderate inflammation; however, no differences were determined amongst the groups (P > 0.05). The fibrous capsule was thick at 7 days, becoming thin at 30 days. All pastes induced von Kossa-positive structures and were birefringent to polarized light. At seven days, the BS-2P group had significantly more OPN immunolabeling compared to the control and Ca(OH)2 groups (P < 0.05). At 30 days, the F18 group had significantly more OPN immunolabeling compared to the control and Ca(OH)2 groups (P < 0.05). All pastes reduced the total number of E. faecalis; however, the reduction was only significant when comparing BS-2P and Ca(OH)2 groups to the control (P < 0.05). Only calcium hydroxide eliminated E. faecalis. CONCLUSIONS: Experimental BS-2P and F18 pastes were biocompatible, stimulated biomineralization and induced significant OPN immunolabeling compared to Ca(OH)2 . Only the BS-2P paste demonstrated antimicrobial activity comparable to Ca(OH)2 .

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.

In vivo analysis of the presence of heme oxygenase-1, transcription factor Jun-D and CD90+/CD73+/CD105+/CD45- cells in the pulp of bleached teeth.

AIM: To investigate hydrogen peroxide (H2 O2 )-induced responsiveness in pulp cells using heme oxygenase-1 (HO-1) immunolabelling, Jun-D immunolabelling to study the effects of H2 O2 on odontoblastic differentiation and CD90+/CD73+/CD105+/CD45- cell counting for in vivo identification of mesenchymal stem cells in the pulp. METHODOLOGY: The maxillary molars of 50 rats were treated with a bleaching gel (35% H2 O2 , 1 × 30 min) or placebo gel (control groups). At 2, 3, 7, 15 and 30 days after the treatment (n = 10), inflammation in pulp tissue was analysed by haematoxylin-eosin staining, HO-1- and Jun-D-immunolabelled cells were counted in each third of the pulp chamber, and the number of CD90+/CD73+/CD105+/CD45- cells was quantified by immunofluorescence. The results were assessed using the Paired t-test or Wilcoxon signed-rank test (P < 0.05). RESULTS: Significant H2 O2 -induced inflammation was noted at 2 and 3 days (P < 0.05), with tertiary dentine formation occurring from 7 days. The bleached specimens had greater HO-1 immunolabelling in the middle and cervical thirds of the coronal pulp at 2 and 3 days, in all thirds at 7 days, and in the occlusal third at 15 days (P < 0.05), and significant nuclear Jun-D immunolabelling in the cervical third at 2 and 3 days and in the occlusal and middle thirds at 7 days (P < 0.05). Bleached and control groups had low numbers of CD90+/CD73+/CD105+/CD45- cells in the pulp at all periods (P > 0.05). CONCLUSIONS: Pulp cells responded to oxidative stress by expressing HO-1 during the post-bleaching inflammation phase until the beginning of the repair phase. Jun-D expression occurred during the reduction of inflammation and the beginning of tertiary dentine production. The presence of oxidative stress did not influence the number of CD90+/CD73+/CD105+/CD45- cells identified in vivo in the dental pulp.

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.

The presence of osteocalcin, osteopontin and reactive oxygen species-positive cells in pulp tissue after dental bleaching.

AIM: To analyse the influence of H2 O2 on pulp repair through osteocalcin and osteopontin immunolabelling and in cellular defence by using the antireactive oxygen species (ROS) antibody. METHODOLOGY: The maxillary molars of 50 rats were treated with 35% H2 O2 (Ble groups) or placebo gel (control groups). At 0 h and 2, 7, 15 and 30 days (n = 10 hemimaxillae), the rats were killed and pulp tissue was evaluated using inflammation and immunolabelling scores (osteocalcin/osteopontin); ROS-positive cells were counted. Paired t-test and Wilcoxon signed-rank test were used (P < 0.05). RESULTS: The Ble group had necrosis in the coronal pulp at 0 h and in the occlusal third of the coronal pulp at 2 days; at 7, 15 and 30 days, no inflammation was noted similar to the controls (P > 0.05). Osteocalcin was absent in the Ble at 0 h, moderate at 2 days and increased thereafter, differing from the controls at all two periods (P < 0.05). Osteopontin was higher principally at 7 and 15 days in Ble groups, but differing with control groups from 2 days after bleaching (P < 0.05). The Ble group had more ROS-positive cells in the pulp at 7 and 15 days (P < 0.05). Tertiary dentine was observed at 7 days, increasing thereafter (P < 0.05). CONCLUSIONS: Post-bleaching pulp repair was associated with increased osteocalcin over time. Osteopontin also participated in this process, and anti-ROS was involved in cellular defence against H2 O2 .

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.

Concentration-dependent effect of bleaching agents on the immunolabelling of interleukin-6, interleukin-17 and CD5-positive cells in the dental pulp.

AIM: To evaluate lymphocyte-like cell activation (CD5-positive cells) and the expression of interleukin (IL)-6 and IL-17 in the pulp after tooth bleaching with two concentrations of hydrogen peroxide (H2 O2 ). METHODOLOGY: The right and left maxillary molars from 40 rats were treated randomly with bleaching gel with 20% H2 O2 (BLUE group, 1 application of 50 min), 35% H2 O2 (MAXX group, three applications of 15 min), or placebo gel (control). After 2 and 30 days, the rats were killed (n = 10), and the jaws were processed for histological and immunohistochemistry analysis of the pulp tissue. The scores of inflammation and immunolabelling (IL-6/IL-17) were submitted to Mann-Whitney and Kruskal-Wallis followed Dunn tests, respectively; anova tests were used for comparisons of number of CD5-positive cells and pulp chamber area values (P < 0.05). RESULTS: At 2 days, 60% of specimens of the BLUE group were associated with moderate inflammation in pulp horns, and in the MAXX group with necrosis (P < 0.05). At 30 days, the pulp was organized, and tertiary dentine was formed. The MAXX group had superior immunolabelling of IL-17 at 2 days differing significantly from other groups (P < 0.05). At 2 days, 90% of the specimens of the BLUE group had moderate immunolabelling of IL-6, and 50% of the MAXX group had severe immunolabelling, both significantly different from the control (P < 0.05). There was no significant difference between the groups at 30 days (P > 0.05). CD5-positive cells were present at 2 and 30 days, particularly in the bleached groups (P < 0.05), without significant difference between time periods (P > 0.05). CONCLUSIONS: IL-6 and IL-17 participated in inflammation in the pulp tissue of rats after tooth bleaching, particularly at 2 days. The immunolabelling was greater with increasing H2 O2 concentration. This process was accompanied by the prolonged activation of CD5-positive cells.

The effect of dental bleaching on pulpal tissue response in a diabetic animal model: a study of immunoregulatory cytokines.

AIM: To evaluate the influence of tooth bleaching on immunoregulatory cytokines production (IL-6, Tumour necrosis factor (TNF)-α and IL-17) in the pulp tissue of normoglycaemic and diabetic rats. METHODOLOGY: Twenty-eight rats were divided into normoglycaemic and diabetic rats (n = 14). Diabetes mellitus (DM) was induced with a single dose of alloxan diluted in citrate buffer via intramuscular injection. After DM confirmation, all rats were sedated and tooth bleaching was performed using 35% hydrogen peroxide on the right maxillary molars for 30 min. Left molars were used as controls. Bleaching resulted in four hemimaxillae groups: normoglycaemic (N), N-bleached (NBle), diabetic (D) and D-bleached (DBle). After 2 and 30 days, rats were euthanized and hemimaxillae processed for analysis by haematoxylin and eosin and immunohistochemistry. Results within and between animals were submitted to Wilcoxon signed-rank and Mann-Whitney tests (P < 0.05). RESULTS: At 2 days, the NBle group had mild, and the DBle had severe inflammatory infiltration in the pulpal tissue (P < 0.05). TNF-α and IL-6 cytokines were associated with increased immunolabelling in the bleached groups compared to nonbleached (P < 0.05). However, IL-17 had increased immunolabelling in the NBle compared to the N and DBle group (P < 0.05). At 30 days, reactionary dentine was observed in the coronal pulp of all bleached teeth and no inflammation was present (P > 0.05). TNF-α cytokines had increased immunolabelling in the DBle group compared to the D group (P < 0.05). However, for IL-6 and IL-17, no difference was observed in this period (P > 0.05). CONCLUSIONS: Tooth bleaching increased IL-6 and TNF-α in the pulp tissue regardless of diabetes mellitus; however, diabetic rats had higher TNF-α levels for longer periods. Tooth bleaching influenced the increase in IL-17 in the early periods in normoglycaemic rats.

RUNX-2, OPN and OCN expression induced by grey and white mineral trioxide aggregate in normal and hypertensive rats.

AIM: To investigate whether hypertension affects mineralization associated with white and grey mineral trioxide aggregate (MTA Angelus® ) implanted subcutaneously into rats by assaying osteoblastic biomarkers. METHODOLOGY: Polyethylene tubes containing grey MTA Angelus® , white MTA Angelus® , intermediate restorative material (IRM; positive control) or an empty tube (negative control) were implanted into the dorsal connective tissue of spontaneous hypertensive (n = 12) and Wistar (normotensive; n = 10) rats. Half of the rats in each group were killed after 7 days, and the remaining after 30 days. Tubes with surrounding tissue were removed, and immunostaining was performed to detect RUNX-2, OPN and OCN proteins. The normality of data was analysed using the Shapiro-Wilk test. Comparison of two independent groups was performed using the Mann-Whitney U-test, to detect a significant difference. A post hoc test accounting for multiple comparisons was performed following Tukey's test (P < 0.05). RESULTS: Under hypertensive conditions after 30 days, both MTA materials were associated with immunolabelling for RUNX-2 from low to moderate, which was less than that observed at normal blood pressure and the 7-day groups (P < 0.05). The expression of OPN and OCN proteins under both MTA conditions was considered low after both 7 and 30 days for the hypertensive condition, and was less than that in animals with normal blood pressure after 30 days (P < 0.05). No immunostaining for any biomarkers in the control and IRM groups was observed (P < 0.05). CONCLUSION: Hypertension decreased the immunostaining of RUNX-2, OPN and OCN biomarkers in response to MTA. Thus, hypertension can jeopardize the mineralization ability of MTA and may have a negative impact on endodontic treatment outcomes.

Teneurin-2 presence in rat and human odontoblasts.

Teneurins are transmembrane proteins consisting of four paralogues (Ten-1-4), notably expressed in the central nervous system during development. All teneurins contain a bioactive peptide in their carboxyl terminal named teneurin C-terminal associated peptide (TCAP). The present study analyzed the detailed distribution of teneurin-2-like immunoreactive (Ten-2-LI) cells in developing and mature rat molar teeth, as well as in mature human dental pulps. Ten-2 and TCAP-2 genic expressions were also evaluated in rat and human dental pulps. Finally, Ten-2-LI cells were analyzed during the repair process after dentin-pulp complex injury in rat lower molar teeth. For this, histological sections of rat molar teeth and human dental pulps were submitted to immunohistochemical techniques, while total RNA from developing rat teeth and mature human dental pulps were submitted to conventional RT-PCR. Ten-2-LI cells were evident in the initial bell stage of rat molar teeth development, especially in ectomesenchymal cells of the dental papilla. Ten-2-LI odontoblasts showed strong immunoreactivity in rat and human mature teeth. Ten-2 and TCAP-2 genic expressions were confirmed in rat and human dental pulps. Dentin-pulp complex injury resulted in a decrease of Ten-2-LI odontoblasts after traumatic injury. Interestingly, Ten-2-LI cells were also evident in the pulp cell-rich zone in all postoperative days. In conclusion, Ten-2-LI presence in rat and human odontoblasts was demonstrated for the first time and Ten-2/TCAP-2 genic expressions were confirmed in rat and human dental pulps. Furthermore, it was revealed that Ten-2-LI rat odontoblasts can be modulated during the regenerative process.

The effect of dental bleaching on pulpal tissue response in a diabetic animal model.

AIM: To evaluate pulpal tissue response after dental bleaching in normal and alloxan-induced diabetic rats. METHODOLOGY: Twenty-eight rats were divided into two groups of normoglycaemic and diabetic rats (n = 14). Diabetes mellitus (DM) was induced with alloxan. After DM confirmation, all rats were anaesthetized and dental bleaching was performed with 35% hydrogen peroxide (H2 O2 ) on the right maxillary molars for 30 min. Left molars were used as controls. Bleaching resulted in four hemimaxillae groups: normoglycaemic (N), N-bleached (NBle), diabetic (D) and D-bleached (DBle). After 2 or 30 days, the animals were euthanized and the hemimaxillae were removed, processed for histopathological analysis and stained with haematoxylin-eosin (HE), Masson's trichrome (MT) and picrosirius red (PSR). Results obtained within animals (normoglycaemic or diabetic rats) were submitted to Wilcoxon or paired t-tests, and between animal (normoglycaemic and diabetic rats), to Mann-Whitney test or t-tests. RESULTS: At 2 days, the NBle group had a mild inflammatory infiltration in the pulpal tissue, whilst the DBle had severe inflammation or necrosis (P < 0.05). At 30 days, no inflammation was present. However, a significant difference in pulp chamber area reduction by reactionary dentine deposition was found between the NBle and DBle groups (P < 0.05). At 2 days, fewer immature collagen fibres and more mature collagen fibres were noted in the NBle, D and DBle groups; this was significantly different when compared to the N group (P < 0.05). At 30 days, significantly fewer immature collagen fibres and more mature collagen fibres were noted in NBle compared with DBle group (P < 0.05). CONCLUSIONS: The inflammatory tissue response in rats' teeth after dental bleaching was greater in diabetic rats. Additionally, the increase in reactionary dentine deposition and mature collagen fibres observed in diabetic rats needs further evaluation to confirm the present results.

Effect of low-level laser therapy as an adjuvant in the treatment of periodontitis induced in rats subjected to 5-fluorouracil chemotherapy.

BACKGROUND AND OBJECTIVE: This study aimed to assess the effect of multiple sessions of a low-level laser therapy (LLLT) adjuvant to scaling and root planing (SRP) on the treatment of experimental periodontitis (EP) in rats treated with 5-fluorouracil (5-FU). MATERIAL AND METHODS: A total of 120 rats were divided into five groups: no treatment (NT); treatment with 5-FU (60 and 40 mg/kg) and no local periodontal treatment (5FU); treatment with 5-FU and SRP (5FU-SRP); treatment with 5-FU, SRP and one LLLT session (660 nm; 0.035 W; 4.2 J; 120 s) (5FU-SRP-1LLLT); and treatment with 5-FU, SRP and four LLLT sessions (0, 24, 48 and 72 h) (5FU-SRP-4LLLT). EP was induced in the mandibular molars through ligature placement. The alveolar bone loss (ABL) area in the furcation region was analysed histometrically. TRAP, proliferating cell nuclear antigen, RANKL, osteoprotegerin and activated caspase-3 patterns were analysed by immunolabeling. Prostaglandin E2 was quantified using an ELISA, and tumour necrosis factor α and interleukin-6 were assessed using the multiplex method. The prevalence rates of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia and Fusobacterium nucleatum were assessed using the PCR method. The data were subjected to statistical analysis (α = 5%). RESULTS: 5FU, 5FU-SRP and 5FU-SRP-1LLLT treatment groups showed higher ABL compared with the NT group (p < 0.05), whereas the 5FU-SRP-4LLLT group showed lower ABL compared with the 5FU group on day 7 and decreased RANKL immunolabeling (p < 0.05). CONCLUSION: Treatment with 5-FU worsened EP, and multiple LLLT sessions adjuvant to SRP seemed to improve periodontitis in rats subjected to 5-FU chemotherapy.

Effect of the probiotic Saccharomyces cerevisiae on ligature-induced periodontitis in rats.

BACKGROUND AND OBJECTIVE: This study assessed the effects of the local use of Saccharomyces cerevisiae as monotherapy and as an adjuvant to the mechanical treatment of ligature-induced periodontitis in rats. MATERIAL AND METHODS: Periodontitis was induced in 72 rats via the installation of a ligature around the mandibular first molar. After 7 d, the ligature was removed and the rats were placed in one of the following groups: no treatment (C; n = 18); scaling and root planing (SRP; n = 18); local irrigation with probiotics (PRO; n = 18); and SRP followed by local irrigation with probiotics (SRP/PRO; n = 18). Six rats from each group were killed at 7, 15 and 30 d. The histological characteristics, alveolar bone loss (ABL) and immunolabeling of tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1ß), interleukin-10 (IL-10) and TRAP on the furcation area of the first molar were assessed. RESULTS: The PRO group showed features of acceleration of the tissue-repair process during the entire experiment. On day 15, there was less ABL in the SRP/PRO group compared with the C group. There were fewer TRAP-positive cells in the SRP and SRP/PRO groups at 30 d. There was less immunostaining for TNF-α in the PRO and SRP/PRO groups and less immunostaining for IL-1ß in the PRO group. However, there was more immunostaining for IL-10 in the PRO group on day 15. CONCLUSION: Local use of the probiotic did not result in any adverse effects on periodontal tissues. When used as monotherapy or as an adjuvant, the probiotic was effective at controlling periodontitis in rats.

Raloxifene modulates regulators of osteoclastogenesis and angiogenesis in an oestrogen deficiency periapical lesion model.

AIM: To analyse the local regulatory mechanisms of osteoclastogenesis and angiogenesis during the progression of periapical lesions in female rats with oestrogen deficiency and treatment with raloxifene (RLX). METHODOLOGY: Female Wistar rats were distributed into groups: SHAM-veh, subjected to sham surgery and treated with a vehicle; OVX-veh, subjected to ovary removal and treated with a vehicle; and OVX-RLX, subjected to ovary removal and treated with RLX. Vehicle or RLX was administered orally for 90 days. During treatment, the dental pulp of mandibular first molars was exposed to the oral environment for induction of periapical lesions, which were analysed after 7 and 30 days. After the experimental periods, blood samples were collected for measurement of oestradiol, calcium, phosphorus and alkaline phosphatase. The rats were euthanized and the mandibles removed and processed for immunohistochemical detection of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), hypoxia-inducible factor-1 alpha (HIF-1α) and bone-specific alkaline phosphatase (BALP). Data were compared using Kruskal-Wallis followed by Dunn test (nonparametric values) and anova followed by the Tukey's test (parametric values). RESULTS: The plasma concentration of oestradiol showed hypo-oestrogenism in the rats subjected to ovary removal. On day 7, alkaline phosphatase activity, calcium and phosphorus were higher in the OVX-RLX group than in the OVX-veh group (P < 0.001), but immunolabelling for RANKL and HIF-1α was lower in OVX-RLX group (P < 0.001). On day 30, the OVX-veh group had higher immunolabelling for RANKL than the OVX-RLX group (P < 0.05). There were no significant differences in the immunoreactivity of OPG and BALP between any groups at either time-point (P > 0.05). CONCLUSION: RLX therapy reversed the increased levels of the local regulators of both osteoclastogenesis and angiogenesis induced by oestrogen deficiency.

Effect of the concentration of phenothiazine photosensitizers in antimicrobial photodynamic therapy on bone loss and the immune inflammatory response of induced periodontitis in rats.

BACKGROUND AND OBJECTIVE: Antimicrobial therapy can suppress periodontal pathogens and increase the effectiveness of conventional mechanical treatment. The aim of this study was to assess bone loss and the immune inflammatory response of rats under the influence of two photosensitizing agents (MB and TBO) at two different concentrations in antimicrobial photodynamic therapy (aPDT), used as an adjuvant therapy in the treatment of periodontitis. MATERIAL AND METHODS: Periodontitis was induced in the mandibular first molars of 162 rats. The animals were divided into nine groups: G1 - scaling and root planing (SRP); G2 - SRP plus 100 µg/mL of methylene blue (MB); G3 - SRP plus 10 mg/mL of MB; G4 - SRP plus 100 µg/mL of toluidine blue (TBO); G5 - SRP plus 10 mg/mL of TBO; G6 - SRP plus 100 µg/mL of MB and laser; G7 - SRP plus 10 mg/mL of MB and laser; G8 - SRP plus 100 µg/mL of TBO and laser; and G9 - SRP plus 10 mg/mL of TBO and laser. Six animals from each group were euthanized 7, 15, or 30 d after treatment. Bone loss (BL) in the furcation region was evaluated using histomorphometric and immunohistochemical analyses to detect the receptor activator of nuclear factor-Κappa B ligand (RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phosphatase (TRAP). RESULTS: There was significantly less BL in animals treated with aPDT using low concentrations of MB and TBO at 7, 15 and 30 d. Immunohistochemical analysis revealed decreased RANKL and increased OPG in the aPDT groups and decreased TRAP-positive cells in G6 and G8. CONCLUSIONS: aPDT, using low concentrations of MB and TBO, was the most effective adjuvant therapy to SRP, acting indirectly as a downregulator of the molecular mechanisms that control bone resorption in periodontitis.

Phenotypic alterations of neuropeptide Y and calcitonin gene-related peptide-containing neurons innervating the rat temporomandibular joint during carrageenan-induced arthritis.

The aim of this study was to identify immunoreactive neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) neurons in the autonomic and sensory ganglia, specifically neurons that innervate the rat temporomandibular joint (TMJ). A possible variation between the percentages of these neurons in acute and chronic phases of carrageenan-induced arthritis was examined. Retrograde neuronal tracing was combined with indirect immunofluorescence to identify NPY-immunoreactive (NPY-IR) and CGRP-immunoreactive (CGRP-IR) neurons that send nerve fibers to the normal and arthritic temporomandibular joint. In normal joints, NPY-IR neurons constitute 78±3%, 77±6% and 10±4% of double-labeled nucleated neuronal profile originated from the superior cervical, stellate and otic ganglia, respectively. These percentages in the sympathetic ganglia were significantly decreased in acute (58±2% for superior cervical ganglion and 58±8% for stellate ganglion) and chronic (60±2% for superior cervical ganglion and 59±15% for stellate ganglion) phases of arthritis, while in the otic ganglion these percentages were significantly increased to 19±5% and 13±3%, respectively. In the trigeminal ganglion, CGRP-IR neurons innervating the joint significantly increased from 31±3% in normal animals to 54±2% and 49±3% in the acute and chronic phases of arthritis, respectively. It can be concluded that NPY neurons that send nerve fibers to the rat temporomandibular joint are located mainly in the superior cervical, stellate and otic ganglia. Acute and chronic phases of carrageenan-induced arthritis lead to an increase in the percentage of NPY-IR parasympathetic and CGRP-IR sensory neurons and to a decrease in the percentage of NPY-IR sympathetic neurons related to TMJ innervation.

Celecoxib treatment does not alter recruitment and activation of osteoclasts in the initial phase of experimental tooth movement.

In a previous study, we reported that the short-term treatment with celecoxib, a non-steroidal anti-inflammatory drug (NSAID) attenuates the activation of brain structures related to nociception and does not interfere with orthodontic incisor separation in rats. The conclusion was that celecoxib could possibly be prescribed for pain in orthodontic patients. However, we did not analyze the effects of this drug in periodontium. The aim of this follow-up study was to analyze effects of celecoxib treatment on recruitment and activation of osteoclasts and alveolar bone resorption after inserting an activated orthodontic appliance between the incisors in our rat model. Twenty rats (400-420 g) were pretreated through oral gavage with celecoxib (50 mg/kg) or vehicle (carboxymethylcellulose 0.4%). After 30 min, they received an activated (30 g) orthodontic appliance, set not to cause any palate disjunction. In sham animals, the appliance was immediately removed after introduction. All animals received ground food and, every 12 h, celecoxib or vehicle. After 48 h, they were anesthetized and transcardiacally perfused through the aorta with 4% formaldehyde. Subsequently, maxillae were removed, post-fixed and processed for histomorphometry or immunohistochemical analyses. As expected, incisor distalization induced an inflammatory response with certain histological changes, including an increase in the number of active osteoclasts at the compression side in group treated with vehicle (appliance: 32.2 ± 2.49 vs sham: 4.8 ± 1.79, P<0.05) and celecoxib (appliance: 31.0 ± 1.45 vs sham: 4.6 ± 1.82, P<0.05). The treatment with celecoxib did not modify substantially the histological alterations and the number of active osteoclasts after activation of orthodontic appliance. Moreover, we did not see any difference between the groups with respect to percentage of bone resorption area. Taken together with our previous results we conclude that short-term treatment with celecoxib can indeed be a therapeutic alternative for pain relieve during orthodontic procedures.