Characterization of a Local Renin-Angiotensin System in Rat Gingival Tissue

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Histological evaluation of experimentally induced subluxation in rat molars and its implications on the management of orthodontic treatment

The purpose of this study was to evaluate the histological alterations occurred in the periradicular region of rat molars after intentional subluxation using an experimental method to induce dentoalveolar trauma. Eighteen adult male Wistar rats (Rattus norvegicus albinus) were selected for the study. The dentoalveolar trauma was experimentally induced by the application of an occlusogingival force on the occlusal surface of the maxillary right first molar using a tensiometer secured on a fully articulated support with adjustable steel shafts. The animals were assigned to six groups (n = 3), according to the intensity of the force applied to induce trauma: Group I (GI, control) - no force application; Groups II-VI (GII-GVI) - the animals were subjected to 600, 700, 800, 900 and 1000 cN force, respectively. After experimental induction of trauma, the animals were sacrificed by anesthetic overdose and the right maxillas were removed and processed for histological analysis under light microscopy. In the animals of GII, GIII and GIV, the histological alterations were similar to those described for GI. GVI (1000 cN) presented the most severe alterations, with the occurrence of buccal bone plate fracture, alveolar fracture and root fracture, which are not present in mild traumatic injuries like subluxation. The 900 cN force (GV) was capable to produce clinical and histological alterations in the gingival and periodontal tissues compatible with those observed in subluxation.

Influence of ovariectomy on healing of autogenous bone block grafts in the mandible: A histomorphometric study in an aged rat model

Purpose: The aim of this study was to evaluate quantitatively and qualitatively the influence of estrogen deficiency on autogenous bone block grafts in aged variectomized rats. Materials and Methods: Fifty 12-month-old female Wistar rats were used in the study. They were divided into 2 groups, an ovariectomized group and a sham-operated group. After 30 days the animals received autogenous block bone grafts on the angle of the mandible, harvested from the calvaria. The animals were euthanized at 7, 14, or 28 days postoperatively. Results: Histologic analysis showed that at 7 days postsurgery, the interface between graft and recipient site in the sham-operated group appeared filled by a granulation tissue with angiogenic activity, whereas the ovariectomized group still exhibited a blood clot and a granulation tissue in organization. on the 14th postoperative day, the interface in the shamoperated group was partially filled by newly formed bone establishing a union between the graft and the recipient site. The interface in the ovariectomized group was typically filled by granulation tissue with discrete osteogenic activity in most specimens. on the 28th postoperative day, the graft in the sham-operated group appeared histologically integrated to the mandible. However, the interface in the ovariectomized group appeared partially filled by newly formed bone, with areas of interposed connective tissue. The statistical analysis revealed that bone neoformation was significantly greater in the sham-operated group (57.41% at 14 days and 68.35 at 28 days) in comparison with the ovariectomized group (40.82% at 14 days and 53.09 at 28 days) at the 5% level. Conclusion: The estrogen depletion caused by the ovariectomy hindered the healing process of autogenous block bone grafts placed in the mandibles of aged rats.

The effect of BMP-2 on the osteoconductive properties of beta-tricalcium phosphate in rat calvaria defects

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of platelet-rich plasma on bone healing of autogenous bone grafts in critical-size defects

AimThis study histologically analysed the effect of autogenous platelet-rich plasma (PRP), prepared according to a new semiautomatic system, on healing of autogenous bone (AB) grafts placed in surgically created critical-size defects (CSD) in rabbit calvaria.Material and MethodsSixty rabbits were divided into three groups: C, AB and AB/PRP. A CSD was created in the calvarium of each animal. In Group C (control), the defect was filled by blood clot only. In Group AB (autogenous bone graft), the defect was filled with particulate autogenous bone. In Group AB/PRP (autogenous bone graft with platelet-rich plasma), it was filled with particulate autogenous bone combined with PRP. All groups were divided into subgroups (n=10) and euthanized at 4 or 12 weeks post-operatively. Histometric and histologic analyses were performed. Data were statistically analysed (anova, t-test, p < 0.05).ResultsGroup C presented significantly less bone formation compared with Group AB and AB/PRP in both periods of analysis (p < 0.001). At 4 weeks, Group AB/PRP showed a statistically greater amount of bone formation than Group AB (64.44 +/- 15.0% versus 46.88 +/- 14.15%; p=0.0181). At 12 weeks, no statistically significant differences were observed between Groups AB and AB/PRP (75.0 +/- 8.11% versus 77.90 +/- 8.13%; p > 0.05). It is notable that the amount of new bone formation in Group AB/PRP at 4 weeks was similar to that of Group AB at 12 weeks (p > 0.05).ConclusionWithin its limitation, the present study has indicated that (i) AB and AB/PRP significantly improved bone formation and (ii) a beneficial effect of PRP was limited to an initial healing period of 4 weeks.

Effect of low-level laser therapy on the healing process after tooth replantation: a histomorphometrical and immunohistochemical analysis

Success of tooth replantation is limited because part of the replanted tooth is lost because of progressive root resorption. This study used histomorphometry and immunohistochemistry to evaluate the effect of low-level laser therapy (LLLT) on the healing process of rat teeth replanted after different extra-oral periods, simulating immediate and delayed replantation. Sixty Wistar rats (Rattus norvegicus albinus) had their maxillary right incisors extracted and randomly assigned to six groups (n = 10): C4, C30 and C45, in which the teeth were replanted 4 min (immediate), 30 min (delayed) and 45 min (delayed) after extraction, respectively, and L4, L30 and L45, in which the teeth were replanted after the same extra-alveolar times, but the root surfaces and the alveolar wounds were irradiated with a gallium-aluminum-arsenate (GaAlAs) diode laser before replantation. The animals were sacrificed after 60 days. The anatomic pieces containing the replanted teeth were obtained and processed for either histomorphometrical analysis under optical microscopy or immunohistochemical expression of receptor activator of nuclear factor Kappa-B (RANK), and its ligand (RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phosphatase (TRAP) proteins. Areas of external replacement and inflammatory root resorption were observed in all groups, without statistically significant differences (P > 0.05). Ankylosis was more frequent in L30 than in C30 (P < 0.05). RANKL immunostaining predominated over RANK and OPG immunostaining in both groups with immediate tooth replantation (P < 0.05). For the 45-min extra-alveolar time, however, there was greater evidence of RANK immunostaining compared to RANKL for both control and laser-treated groups (P < 0.05). Positive TRAP immunostaining predominated in L4 and L30 (P < 0.05). In conclusion, under the tested conditions, the treatment of the root surface and the alveolar wound with LLLT did not improve the healing process after immediate and delayed tooth replantation in rats.

Bone regeneration in surgically created defects filled with autogenous bone: an epifluorescence microscopy analysis in rats

Although the search for the ideal bone substitute has been the focus of a large number of studies, autogenous bone is still the gold standard for the filling of defects caused by pathologies and traumas, and mainly, for alveolar ridge reconstruction, allowing the titanium implants installation. Objectives: The aim of this study was to evaluate the dynamics of autogenous bone graft incorporation process to surgically created defects in rat calvaria, using epifluorescence microscopy. Material and methods: Five adult male rats weighing 200-300 g were used. The animals received two 5-mm-diameter bone defects bilaterally in each parietal bone with a trephine bur under general anesthesia. Two groups of defects were formed: a control group (n=5), in which the defects were filled with blood clot, and a graft group (n=5), in which the defects were filled with autogenous bone block, removed from the contralateral defect. The fluorochromes calcein and alizarin were applied at the 7th and 30th postoperative days, respectively. The animals were killed at 35 days. Results: The mineralization process was more intense in the graft group (32.09%) and occurred mainly between 7 and 30 days, the period labeled by calcein (24.66%). Conclusions: The fluorochromes showed to be appropriate to label mineralization areas. The interfacial areas between fluorochrome labels are important sources of information about the bone regeneration dynamics.

Analysis of the healing process in delayed tooth replantation after root canal filling with calcium hydroxide, Sealapex and Endofill: a microscopic study in rats

The major concern in the therapeutics of tooth replantation refers to the occurrence of root resorption and different approaches have been proposed to prevent or treat these complications. The purpose of this study was to evaluate tissue response to delayed replantation of anterior rat teeth treated endodontically using calcium hydroxide, Sealapex, and Endofill without the placement of gutta-percha cones. Thirty rats had their right upper incisor extracted and maintained in dry storage for 60 min. After removal of the dental papilla, enamel organ, pulp tissue, and periodontal ligament remnants, the teeth were immersed in 2% sodium fluoride phosphate acidulated, pH 5.5, for 10 min. The root canals were dried with absorbent paper points and the teeth were assigned to three groups (n = 10) according to the filling material. Group I - calcium hydroxide and propyleneglycol paste, Group II - Sealapex, and Group III - Endofill. The sockets were irrigated with saline and the teeth were replanted. Replacement resorption, inflammatory resorption and ankylosis were observed in all groups. Although the occurrence of inflammatory resorption was less frequent in Group I, there were no statistically significant differences among the groups. It may be concluded that compared to the paste, filling the root canals with Sealapex and Endofill sealers without the placement of gutta-percha cones did not provide better results.

Delayed replantation of rat teeth after use of reconstituted powdered milk as a storage medium

Minimal extraoral dry storage period and moist storage for the avulsed tooth are identified as key steps for the treatment protocol of tooth replantation. Among the possible moist storage media, bovine milk has stood out because of its capacity of preserving the integrity of the periodontal ligament (PDL) fibers. This condition has attracted the attention to investigate the use of powdered milk, which is one of the presentation forms of bovine milk, as a feasible storage medium in cases of delayed tooth replantation. The aim of this study was to evaluate the healing process after delayed replantation of rat teeth stored in reconstituted powdered milk and long shelf-life (ultra high temperature) whole milk. Forty maxillary right rat incisors were assigned to four groups (n = 10): group I - the teeth were extracted and immediately replanted into theirs sockets; group II - the teeth were stored for 60 min in 200 ml of freshly reconstituted powdered milk; group III - the teeth were stored for 60 min in 200 ml of long shelf-life whole milk; group IV - the teeth were kept dry for the same time. All procedures were performed at room temperature. Next, the root canals of teeth in groups II, III, and IV were instrumented, filled with a calcium hydroxide-based paste, and replanted into their sockets. All animals received systemic antibiotic therapy and were killed by anesthetic overdose 60 days after replantation. The pieces containing the replanted teeth were removed, fixed, decalcified, and paraffin-embedded. Semi-serial 6-mu m-thick sections were obtained and stained with hematoxylin and eosin for histomorphological analysis. There was statistically significant difference (P < 0.05) between groups I and IV regarding the presence of replacement resorption and PDL remnants on root surface. The powdered milk and long shelf-life whole milk presented similar results to each other and may be indicated as storage media for avulsed teeth.

Evaluation of Rat Alveolar Bone Response to Angelus MTA or Experimental Light-cured Mineral Trioxide Aggregate Using Fluorochromes

Introduction: The aim of this study was to evaluate the rat alveolar bone response after the implantation of experimental light-cured mineral trioxide aggregate (MTA) or Angelus MTA (Angelus, Londrina, Parana, Brazil) by histological and fluorescence analysis. Methods: Thirty Wistar Albino rats were divided into three groups. In the control group, empty polyethylene tubes were inserted into the rat alveolar sockets immediately after extraction. In the other groups, the tubes were filled with light-cured MTA or Angelus MTA. Five animals from each group were injected with calcein on day 7, alizarin on day 14, and oxytetracycline on day 21. on day 30, these animals were killed, and the right hemimaxillas were removed and histologically processed. Half of the maxillas were processed and stained with hematoxylin and eosin. The remaining maxillas were processed for fluorescence analysis and stained with Stevenel blue and alizarin red. New bone was histomorphometrically evaluated using a Merz grid. Results: The light-cured MTA presented a similar response when compared with Angelus MTA; it was characterized by a mild inflammatory response and complete bone healing. In the light-cured MTA group, the fluorescence areas were more evident at 21 days, showing an increase in bone formation. However, dystrophic mineralization was observed only with Angelus MTA. Conclusions: It was concluded that both materials present a similar inflammatory response and bone healing, but dystrophic mineralization was observed only with Angelus MTA. (J Endod 2011;37:250-254)

Reaction of rat connective tissue to a new calcium hydroxide-based sealer

The aim of this study was to histopathologically examine the reaction of the connective tissue of rats to 2 calcium hydroxide-based sealers, Acroseal and Sealapex. Dentin tubes containing the materials and empty control tubes were implanted into the dorsal connective tissue of 36 Wistar albino rats. The animals were killed after 7 or 30 days, and the specimens were prepared for histologic analysis with hematoxylin and eosin, Von Kossa technique, and polarized light. Results were statistically analyzed using Kruskal-Wallis test. Both materials caused mild or moderate inflammatory reactions on the 7th day, but these reactions decreased by the 30th day with no significant difference at any time (P > .05). Mineralization of the subcutaneous tissue of the rats was observed only with Sealapex.

Bone healing in critical-size defects treated with either bone graft, membrane, or a combination of both materials: a histological and histometric study in rat tibiae

Objectives: The aim of the present investigation was to histologically analyze the effect of using lyophilized bovine bone (GenOx (R) organic matrix) with (or without) guided tissue regeneration (using a decalcified cortical osseous membrane [GenDerm (R)]) on bone healing in surgically created critical-size defects created in rat tibia.Material and methods: Surgical critical-size bone defects were created in 64 animals that were randomly divided into four groups: group I (control); group II (defect filled with GenOx (R)); group III (defect covered by GenDerm (R)); group IV (defect filled with GenOx (R) and covered by GenDerm (R)). Animals were killed at 30 or 90 days post-surgery. The specimens were embedded in paraffin, serially cut, and stained with hematoxylin and eosin for analysis under light microscopy. The formation of new bone in the cortical area of the defect was histomorphometrically evaluated.Results: All experimental groups demonstrated superior bone healing compared with the control group. However, group IV samples showed evidence of more advanced healing at both 30 and 90 days post-surgery as compared with the other experimental groups.Conclusions: The bovine organic bone graft GenOx (R) associated with GenDerm (R) this produced the best treatment results in the case of critical-size defects in rat tibia.

Expression of odontogenic ameloblast-associated protein, amelotin, ameloblastin, and amelogenin in odontogenic tumors: immunohistochemical analysis and pathogenetic considerations

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effect of cyclosporin A on alveolar bone homeostasis in a rat periodontitis model

Objectives: the administration of cyclosporin A has been associated with significant bone loss and increased bone remodeling. The present investigation was designed to evaluate the effects of cyclosporin A on alveolar bone of rats subjected to experimental periodontitis, using serum, stereometric and radiographic analysis.Methods: Twenty-four rats were divided into one of the following groups with six animals each: group I, control rats; group II, in which the animals received a cotton ligature around the lower first molars; group III, in which the rats received a cotton ligature around the lower first molars and were treated with 10 mg/(kg body weight day) of cyclosporin A; group IV, in which the rats were treated with 10 mg/(kg body weight day) of cyclosporin A. At the end of experimental period, at 30 days, animals were killed and the serum calcium and alkaline phosphatase levels were measured in all groups. The distance from the alveolar bone crest to the cemento-enamel junction was measured radiographically for each mesial surface of the lower first molars of each rat. After histological processing, the stereological parameters: volume densities of multinucleated osteoclasts (V-o), alveolar bone (V-b), marrow (V-m), and relation of eroded surface/bone surface (Es/Bs) were assessed at the mesial region of the alveolar bone.Results: Significant decreases in serum calcium were observed in those groups that received cyclosporin A therapy. No significant changes in serum alkaline phosphatase were observed. The therapy with cyclosporin A combined with the ligature placement decreased the V-b and increased the V-o, V-m and Es/Bs at the mesial surface of lower first molars. on the other hand, the radiographic data showed that cyclosporin A therapy diminished the alveolar bone loss at the mesial surface of the lower first molars.Conclusions: Therefore, within the limits of this study, we suggest that cyclosporin A at immunosuppressive levels can bring about an imbalance in the alveolar bone homeostasis in rats. However, in the presence of inflammatory stimulation, the inhibition of the immune system by cyclosporin A may decrease the initial periodontal breakdown.

Morphological characterization of rat incisor fluorotic lesions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)