Tania Mary Cestari

Alloxan-induced diabetes triggers the development of periodontal disease in rats.

Background Periodontal disease in diabetic patients presents higher severity and prevalence; and increased severity of ligature-induced periodontal disease has been verified in diabetic rats. However, in absence of aggressive stimuli such as ligatures, the influence of diabetes on rat periodontal tissues is incompletely explored. The aim of this study was to evaluate the establishment and progression of periodontal diseases in rats only with diabetes induction. Methodology/Principal Findings Diabetes was induced in Wistar rats (n = 25) by intravenous administration of alloxan (42 mg/kg) and were analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. The hemimandibles were removed and submitted to radiographical and histopathological procedures. A significant reduction was observed in height of bone crest in diabetic animals at 3, 6, 9 and 12 months, which was associated with increased numbers of osteoclasts and inflammatory cells. The histopathological analyses of diabetic rats also showed a reduction in density of collagen fibers, fibroblasts and blood vessels. Severe caries were also detected in the diabetic group. Conclusions/Significance The results demonstrate that diabetes induction triggers, or even co-induces the onset of alterations which are typical of periodontal diseases even in the absence of aggressive factors such as ligatures. Therefore, diabetes induction renders a previously resistant host into a susceptible phenotype, and hence diabetes can be considered a very important risk factor to the development of periodontal disease.

Radiographic and histological study of perennial bone defect repair in rat calvaria after treatment with blocks of porous bovine organic graft material

Over the last few years, various bone graft materials of bovine origin to be used in oromaxillofacial surgeries have entered the market. In the present study, we determined the capacity of a block organic bone graft material (Gen-ox, Baumer SA, Brazil) prepared from bovine cancellous bone to promote the repair of critical size bone injuries in rat calvaria. A transosseous defect measuring approximately 8mm in diameter was performed with a surgical trephine in the parietal bone of 25 rats. In 15 animals, the defects were filled with a block of graft material measuring 8mm in diameter and soaked in the animals own blood, and in the other 10 animals the defects were only filled with blood clots. The calvariae of rats receiving the material were collected 1, 3 and 6 months after surgery, and those of animals receiving the blood clots were collected immediately and 6 months after surgery. During surgery, the graft material was found to be of easy handling and to adapt perfectly to the receptor bed after soaking in blood. The results showed that, in most animals treated, the material was slowly resorbed and served as a space filling and maintenance material, favoring angiogenesis, cell migration and adhesion, and bone neoformation from the borders of the lesion. However, a foreign body-type granulomatous reaction, with the presence of numerous giant cells preventing local bone neoformation, was observed in two animals of the 1-month subgroup and in one animal of the 3-month subgroup. These cases were interpreted as resulting from the absence of demineralization and the lack of removal of potential antigen factors during production of the biomaterial. We conclude that, with improvement in the quality control of the material production, block organic bone matrix will become a good alternative for bone defect repair in the oromaxillofacial region due to its high osteoconductive capacity.

Proteomic Analysis of Liver in Rats Chronically Exposed to Fluoride

Fluoride (F) is a potent anti-cariogenic element, but when ingestion is excessive, systemic toxicity may be observed. This can occur as acute or chronic responses, depending on both the amount of F and the time of exposure. The present study identified the profile of protein expression possibly associated with F-induced chronic hepatotoxicity. Weanling male Wistar rats (three-weeks old) were divided into three groups and treated with drinking water containing 0, 5 or 50 mg/L F for 60 days (n=6/group). At this time point, serum and livers were collected for F analysis, which was done using the ion-sensitive electrode, after hexamethyldisiloxane-facilitated diffusion. Livers were also submitted to histological and proteomic analyses (2D-PAGE followed by LC-MS/MS). Western blotting was done for confirmation of the proteomic data A dose-response was observed in serum F levels. In the livers, F levels were significantly increased in the 50 mg/L F group compared to groups treated with 0 and 5 mg/L F. Liver morphometric analysis did not reveal alterations in the cellular structures and lipid droplets were present in all groups. Proteomic quantitative intensity analysis detected 33, 44, and 29 spots differentially expressed in the comparisons between control vs. 5 mg/L F, control vs. 50 mg/L F, and 5 mg/L vs. 50 mg/L F, respectively. From these, 92 proteins were successfully identified. In addition, 18, 1, and 5 protein spots were shown to be exclusive in control, 5, and 50 mg/L F, respectively. Most of proteins were related to metabolic process and pronounced alterations were seen for the high-F level group. In F-treated rats, changes in the apolipoprotein E (ApoE) and GRP-78 expression may account for the F-induced toxicity in the liver. This can contribute to understanding the molecular mechanisms underlying hepatoxicity induced by F, by indicating key-proteins that should be better addressed in future studies.

Effects of passive smoke inhalation on the vocal cords of rats

UNLABELLED Few studies have demonstrated the pathologic reactions yielded by smoke inhalation on the airway in rats. AIM The aim of this study was to analyze the possible histopathological effects produced by chronic cigarette smoke inhalation on the vocal folds of rats. STUDY DESIGN Experimental. MATERIAL AND METHOD 36 male rats (Rattus norvergicus Wistar strain), aged 60 days, were kept in cages and exposed to inhalation of the smoke produced by 10 cigarettes lit 3 times a day, 7 days a week, for periods of 25, 50 and 75 days, and their respective controls. Thereafter the animals were killed and their larynxes were dissected and submitted to histological processing for achievement of histological sections, which were stained with Hematoxylin and Eosin and analyzed by light microscopy. RESULTS The rats exposed to smoke displayed smaller (p< 0,05) body mass than the control group. There was hyperplasia and squamous metaplasia in the free edge of the vocal fold and squamous hyperplasia on the middle portion of the vocal fold in all 3 study periods. Moreover, the 50-day group revealed keratinizing metaplasia in this area. Morphological alterations in other areas of the larynx and inflammatory reaction of the lamina propria were also not observed. CONCLUSION It was concluded that the passive inhalation of cigarette smoke yields important morphological changes in the vocal fold epithelium, which may progress to neoplasia.

Efeitos da inalação passiva da fumaça de cigarro sobre as pregas vocais de ratos

Few studies have demonstrated the pathologic reactions yielded by smoke inhalation on the airway in rats. AIM: The aim of this study was to analyze the possible histopathological effects produced by chronic cigarette smoke inhalation on the vocal folds of rats. STUDY DESIGN: Experimental. MATERIAL AND METHOD: 36 male rats (Rattus norvergicus Wistar strain), aged 60 days, were kept in cages and exposed to inhalation of the smoke produced by 10 cigarettes lit 3 times a day, 7 days a week, for periods of 25, 50 and 75 days, and their respective controls. Thereafter the animals were killed and their larynxes were dissected and submitted to histological processing for achievement of histological sections, which were stained with Hematoxylin and Eosin and analyzed by light microscopy. RESULTS: The rats exposed to smoke displayed smaller (p< 0,05) body mass than the control group. There was hyperplasia and squamous metaplasia in the free edge of the vocal fold and squamous hyperplasia on the middle portion of the vocal fold in all 3 study periods. Moreover, the 50-day group revealed keratinizing metaplasia in this area. Morphological alterations in other areas of the larynx and inflammatory reaction of the lamina propria were also not observed. CONCLUSION: It was concluded that the passive inhalation of cigarette smoke yields important morphological changes in the vocal fold epithelium, which may progress to neoplasia.

Bone repair and augmentation using block of sintered bovine‐derived anorganic bone graft in cranial bone defect model

OBJECTIVE To histomorphometrically investigate the repair of critical size defects (CSDs) and bone augmentation in cranial walls using block of sintered bovine-derived anorganic bone (sBDAB) graft. MATERIAL AND METHODS Forty guinea-pigs were divided into test (n=20) and CSD control (n=20) groups. In each animal, a full-thickness bone defect with 9.5 mm diameter was made in the frontal bone. The defects were filled with an sBDAB block soaked in blood in the test group and with blood clot in the CSD control group. The skulls were collected at 0 h (n=2) and 30, 90 and 180 days (n=6/group and period) postoperatively. The volume density and total volume of newly formed bone, sBDAB, blood vessels and connective tissue, vertical thickness of removed bone plug, sBDAB block and graft area were evaluated. RESULTS The vertical thickness of the adapted sBDAB block was 3.8 times higher than that of the removed bone plug and did not show significant difference between periods, filling in average 29.8% of the total graft region. The sBDAB block exhibited complete osseointegration with the borders of the defect at 90 days. At 90 and 180 days, the vertical thickness of the graft was 279% in the average, and the total volume of bone augmentation was, respectively, 78.8% and 148.5% higher compared with the removed bone plug. The defects of the CDS control group showed limited osteogenesis and filling by connective tissue plus tegument. CONCLUSION The sBDAB block can be used to promote repair of CSDs and bone augmentation in the craniomaxillofacial region, due to its good osteoconductive and slow resorptive properties.

Lack of repair of rat skull critical size defect treated with bovine morphometric protein bound to microgranular bioabsorbable hydroxyapatite

The ability of a pool of bovine bone morphogenetic proteins bound to synthetic microgranular hydroxyapatite (BMPb-HA) to stimulate bone repair was determined in rat critical size defects. An 8-mm diameter defect was created in the calvaria of 25 rats. In 15 rats, the defects were filled with BMPb-HA homogenized with blood (experimental group), and in 10 rats the defects were filled only with blood clots (control). The calvariae of experimental rats were collected 1, 3 and 6 months after surgery and of the control rats at the end of surgery and 6 months thereafter. The morphometric results obtained in the radiographs showed an absence of new bone formation at 1 and 3 months post-surgery and, histologically, the defects were filled with fibrous connective tissue and numerous foci of a foreign body-type granulomatous reaction around hydroxyapatite agglomerates. At the end of 6 months, the number and size of the granulomatous foci decreased and the area of the defects was reduced by 22% compared to the 0-hour control due to the formation of new bone at their borders, although the mean area was similar to the 6-month control. We conclude that the use of BMPb-HA in the treatment of critical size bone defects of the rat skull leads to the formation of a foreign body-type granulomatous reaction that markedly inhibits new bone formation, suggesting that synthetic microgranular hydroxyapatite does not represent a good carrier for BMP-induced bone formation.

Spontaneous Periodontitis Development in Diabetic Rats Involves an Unrestricted Expression of Inflammatory Cytokines and Tissue Destructive Factors in the Absence of Major Changes in Commensal Oral Microbiota

Diabetes mellitus is a heterogeneous group of disorders, in which hyperglycemia is a main feature. The objective was to evaluate the involvement of RAGE, inflammatory cytokines, and metalloproteinases in spontaneous periodontitis triggered by diabetes induction. Immunohistochemical procedures for MMP-2, MMP-9, TNF-α, IL-1β, IL-6, RANKL, and RAGE were performed in rats after 1, 3, 6, 9, and 12 months of diabetes induction. Total DNA was extracted from paraffin-embedded tissues and evaluated by Real-TimePCR for 16S total bacterial load and specific periodontopathogens. Our data did not demonstrate differences in microbiological patterns between groups. In diabetic groups, an increase in RAGE-positive cells was detected at 6, 9, and 12 months, while TNF-alpha-stained cells were more prevalent at 6 and 12 months. In experimental groups, IL-β-positive cells were increased after 12 months, IL-6 stained cells were increased at 9 and 12 months, and RANKL-positive cells at 9 months. Diabetes resulted in widespread expression of RAGE, followed by expression of proinflammatory mediators, without major alterations in oral microbial profile. The pervasive expression of cytokines suggests that spontaneous periodontitis development may be independent of microbial stimulation and may be triggered by diabetes-driven imbalance of homeostasis.

Microscopic and radiographic analysis of the effect of particle size of demineralized bovine cancellous bone matrix on the repair of bone defects in femurs of rabbits

The bone tissue has a great regenerative potential, with ability to completely restore its structure and original functions. In some situations, though, bone defects cannot be self-repaired, thus requiring the use of grafts for a correct treatment and good prognosis. This work aimed at microscopically analyzing the effect of the particle size of demineralized bovine cancellous bone matrix in micro and macrogranular forms on the repair of bone defects in femurs of rabbits, with blood clot used as control. At 1, 3 and 6 months after implantation of the materials, the animals were killed and the anatomic specimens were removed. A foreign body-type granulomatous reaction containing macrophages and multinucleated giant cells in contact with the implanted particles was observed. These results suggest a failure in demineralization and/or interruption of the antigenic potential during production of the biomaterial. It is concluded that the size of the particles did not influence the evolution of the repair process of bone defects, acting only as bone-filler substances, and that the material implanted should be improved by quality control during production, since it may represent a good alternative for bone graft.

Effects of low-level laser therapy on autogenous bone graft stabilized with a new heterologous fibrin sealant.

Autogenous bone grafts are used to repair bone defects, and the stabilization is needed for bone regeneration. Laser photobiomodulation is a modality of treatment in clinical practice for tissue regeneration, and it has therapeutic effects as an anti-inflammatory, analgesic and modulating cellular activity. The aim of the present study was to evaluate the effects of low-level laser therapy (LLLT) on an autogenous bone graft integration process stabilized with a new heterologous fibrin sealant. Forty rats were divided into two groups: Autogenous Fibrin Graft (AFG, n=20), in which a 5mm dome osteotomy was conducted in the right parietal bone and the graft was adhered to the left side using fibrin sealant; and Autogenous Fibrin Graft Laser (AFGL, n=20), which was subjected to the same procedures as AFG with the addition of LLLT. The treatment was performed immediately following surgery and then three times a week until euthanasia, using an 830nm laser (30mW, 6J/cm(2), 0.116cm(2), 258.6mW/cm(2), 2.9J). Five animals from each group were euthanized at 10, 20, 30 and 40days postoperative, and the samples were submitted to histomorphological and histomorphometric analysis. Partial bone regeneration occurred, with new bone tissue integrating the graft to the recipient bed and small areas of connective tissue. Comparative analysis of the groups at the same intervals revealed minor interfaces in group AFGL, with statistically significant differences (p<0.05) at all of the analyzed intervals (10days p=0.0087, 20days p=0.0012, 30days p<0.0001, 40days p=0.0142). In conclusion, low-level laser therapy stimulated bone regeneration and accelerated the process of integration of autogenous bone grafts.