Tânia Mary Cestari

The Role of Toll-Like Receptor 2 in the Recognition of Aggregatibacter actinomycetemcomitans

BACKGROUND Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) is a Gram-negative bacterium present in the oral cavity and is usually associated with localized aggressive periodontitis. Isolated antigens from A. actinomycetemcomitans can activate innate immune cells through Toll-like receptors (TLRs), which are molecules that recognize structural components conserved among microorganisms. In this study, we evaluate the role of TLR2 in the recognition of A. actinomycetemcomitans. METHODS Macrophages and neutrophils from knockout mice with targeted disruption of TLR2 (TLR2(-/-) mice) and wild-type mice were collected and used for the subsequent assays. The production of cytokines and chemokines was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of apoptotic cells was determined by flow cytometry. In addition, the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR2(-/-) mice were examined. RESULTS The results show that TLR2-deficient mice developed more severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly higher bone loss and inflammatory cell migration to periodontal tissues. The inflammatory cell influx into the peritoneal cavities of TLR2(-/-) mice was three-fold lower than that observed for the littermate controls. A significantly diminished production of the cytokines tumor necrosis factor-alpha and interleukin-1beta as well as the chemokine CC-ligand-5 in the peritoneal cavities of TLR2(-/-) mice was observed. In addition, a high frequency of apoptotic cells in the inflammatory exudates from TLR2(-/-) mice was observed. Phagocytosis and nitric oxide production was diminished in cells from TLR2(-/-) mice, facilitating the dissemination of the pathogen to the spleen. CONCLUSION The results of this study highlight the involvement of TLR2 in recognizing A. actinomycetemcomitans and its essential role in controlling A. actinomycetemcomitans infection.

Rat subcutaneous tissue response to macrogranular porous anorganic bovine bone graft

The ideal bone graft must present biocompatibility, osteoconductive and osteoinductive properties, resistance and plasticity. Xenogenic grafts of bovine cancellous bone origin are particularly interesting due to their biologically designed porous structure that enhance both cellular and vascular invasion. The purpose of this study was to evaluate the tissue response induced by bovine macrogranular porous anorganic bone implanted in rat subcutaneous tissue. Forty rats were assigned to 2 groups, as follows: the control group received empty collagen capsules and the test group received subcutaneous implants of the test material. Samples were collected after 10, 20, 30 and 60 days and processed histologically. Histological analysis showed at 10 days a granulomatous inflammatory infiltrate, rich in multinucleated giant cells and free of lymphocytes or plasma cells, similarly to mineralized allograft implanted in rat subcutaneous. In later periods, there was a significant decrease in the inflammatory infiltrate and an increase in fibrosis around graft particles. In conclusion, the test material induced a foreign body-type granuloma with subsequent fibrosis around the graft particles implanted in rat subcutaneous and did not elicit any immune response, thus being considered biocompatible.

Tissue response to a membrane of demineralized bovine cortical bone implanted in the subcutaneous tissue of rats

The treatment of persistent bone defects has encouraged the search for proper techniques or bone substitutes. In Dentistry, a common problem in the treatment of periodontal bone defects is the growth of tissues within the lesion, such as the junctional epithelium, which impair regeneration of these tissues. Guided tissue regeneration (GTR), based on the separation of the tissues by means of membranes or barriers, was developed in an attempt to improve periodontal regeneration. The aim of this study was to histologically evaluate the tissue response to a membrane of demineralized bovine cortical bone implanted in the subcutaneous tissue of rats. The study periods were 1, 3, 7, 15, 30 and 60 days after implantation. Analysis of the histological sections demonstrated a moderate to intense inflammatory response at 1 and 3 days, moderate at 7 and 15 days, and almost absent at 30 and 60 days. Resorption of the membrane began 15 days after implantation, and at 60 days only remnants could be detected in some animals. We concluded that the demineralized bovine cortical bone membrane was well tolerated by the tissues and is completely resorbed after 30-60 days by mononuclear cells and multinucleated giant cells, which disappear upon completion of the process.

Tibial segmental bone defect treated with bone plate and cage filled with either xenogeneic composite or autologous cortical bone graft - An experimental study in sheep

Tibia segmental defect healing in sheep were clinically, radiographically and histologically evaluated. Twelve young sheep aged four to five months were divided into two groups, G1 and G2. A 3.5 cm long segmental defect was created in the right tibial diaphysis with maintenance of the periosteum. The bone defects in both groups were stabilized with a bone plate combined with a titanium cage. In G1 the cage was filled with pieces of autologous cortical bone graft. In G2 it was filled with a composite biomaterial which consisted of inorganic bovine bone, demineralized bovine bone, a pool of bovine bone morphogenetic proteins bound to absorbable ultra-thin powdered hydroxyapatiteand bone-derived denaturized collagen. Except for one G1 animal, all of them showed normal limb function 60 days after surgery. Radiographic examination showed initial formation of periosteal callus in both groups at osteo-tomy sites, over the plate or cage 15 days postoperatively. At 60 and 90 days callus remodeling occurred. Histological and morphometric analysis at 90 days after surgery showed that the quantity of implanted materials in G1 and G2 were similar, and the quantity of new bone formation was less (p = 0.0048) and more immature in G1 than G2, occupying 51 +/- 3.46% and 62 +/- 6.26% of the cage space, respectively. These results suggest that the composite biomaterial tested was a good alternative to autologous cortical bone graft in this experimental ovine tibial defect. However, additional evaluation is warranted prior to its clinical usage.

Expression of metalloproteinase 2 in the cell response to porous demineralized bovine bone matrix.

SummaryThe purpose of the study was to analyze the involvement of metalloproteinase 2 (MMP-2) and macrophages in the tissue and cell response to the organic graft material produced from bovine cancellous bone. Thirty adult male white Wistar rats (Rattus norvegicus) received implants of blocks of demineralized bovine bone matrix between the fasciae of the quadriceps muscle. The specimens collected at 3, 7, 14, 21 and 28 days after implantation (n = 6/period). Sections of 6 μm thick were stained with hematoxylin and eosin and immunolabeled with anti-MMP-2 and anti-CD68 using standard avidin–biotin–peroxidase method. The tissue response to the material was initially mediated by polymorphonuclear neutrophils, evolving to a mononuclear inflammatory infiltrate with macrophages and few lymphocytes and plasma cells and presence of inflammatory multinucleated giant cells (GC) in contact with the material that exhibited signs of resorption. The number of cells immunolabeled to MMP-2 was highest at day 7 (103.2 ± 39.1), but significantly decreased (F = 3.67; p = 0.044) until day 28 (45.9 ± 13.1). CD68 immunostaining also significantly decreased (F = 6.75; p = 0.007) from day 7 (49.5 ± 10.4) to day 28 (19.5 ± 8.9). A positive and statistically significant correlation was observed between the evolutions of these two variables. The material had been almost completely resorbed at day 28. Among cells present at the granuloma, anti-MMP-2 immunostaining was predominant and more intense in macrophages, yet lightly immunolabeled multinucleated giant cells were found in close contact with the material. Thus, considering the experimental limitations of this study, we concluded that MMP-2 produced by macrophages participates in the resorption of demineralized bovine bone.

Fluoride effects on ectopic bone formation in young and old rats.

This study evaluated the effect of fluoride on bone fluoride levels and on ectopic bone formation in young and old rats. Eighty male Wistar rats were assigned to four groups (n = 20/g), which differed according to the fluoride concentration in their drinking water (0, 5, 15 and 50 mg/l). When half of the rats were 90 days old, demineralized bone matrix (DBM) was implanted. The other rats received DBM implants when they were 365 days old. The animals were killed 28 days after. Fluoride in the femur surface, whole femur and plasma was analyzed with an electrode. The implants were analyzed histomorphometrically. Data were tested for statistically significant differences by ANOVA, Tukeys test, t-test and linear regression (p < 0.05). Increases in plasma, femur surface and whole femur fluoride concentrations were observed as water fluoride levels increased. There was also a trend for increase in plasma and femur fluoride concentrations as age increased. Significant positive correlations were found between plasma and femur surface, plasma and femur and femur surface and femur fluoride concentrations. The morphometric analyses indicated an increase in bone formation for younger rats that received 5 mg/l of fluoride in the drinking water. However, this was not statistically significant. The younger rats that received 50 mg/l of fluoride showed impairment in bone formation. Bone formation was not significantly affected among the older rats. The results suggest that lower doses of fluoride in the drinking water, which slightly increase plasma fluoride levels, may have an anabolic effect on bone formation in younger rats.

Microscopic analisys of porous microgranular bovine anorganic bone implanted in rat subcutaneous tissue

The tissue response to porous bovine anorganic bone implanted in rat connective tissue was evaluated by subjective light microscopy analysis. Forty rats were divided into two groups: control (empty collagen capsules) and test (collagen capsule filled with 0.1g biomaterial) and killed 10, 20, 30 and 60 days after implantation. At 10 days, intense chronic inflammatory infiltrate consisting mainly of macrophages and inflammatory multinucleated giant cells (IMGC) was observed. Neutrophils, plasma cells and lymphocytes were present in discrete amounts and slowly disappeared along the repair process. Porosity of the material was filled by reaction connective tissue exhibiting IMGC. The fibrosis was more intense after 60 days and clearly higher than the control group. Thus, the material did not cause any severe adverse reactions and did not stimulate the immune system. Based on the results it could be concluded that deproteinized bovine cancelous bone was well tolerated by rat connective tissue.

Experimental alveolitis in rats: microbiological, acute phase response and histometric characterization of delayed alveolar healing

The pathogenesis of alveolitis is not well known and therefore experimental situations that mimic some features of this disease should be developed. Objective In this study, the evolution of the experimentally induced infection in rat sockets is characterized, which leads to clinical signs of suppurative alveolitis with remarkable wound healing disturbs. Material and methods Non-infected (Group I) and experimentally infected sockets in Rattus novergicus (Group II) were histometrically evaluated regarding the kinetics of alveolar healing. In addition, the characterization of the present bacteria in inoculation material and the serum levels of C-reactive protein (CRP) were performed. The detected species were Capnocytophaga ochracea, Fusobacterium nucleatum ss nucleatum, Prevotella melaninogenica, Streptococcus anginosus, Treponema socranskii and Streptococcus sanguis. Results All experimentally infected rats developed suppurative alveolitis, showing higher levels of CRP in comparison to those non-infected ones. Furthermore, infected rats presented a significant delayed wound healing as measured by the histometric analysis (higher persistent polymorphonuclear infiltrate and lower density of newly formed bone). Conclusion These findings indicate that rat sockets with experimentally induced infection produced higher levels of serum CRP, showing the potential of disseminated infection and a disturb in the alveolar repair process in an interesting experimental model for alveolitis studies.

Dynamics of Subcutaneous Tissue Response to the Implantation of Tetracycline-Treated or Untreated Membrane of Demineralized Bovine Cortical Bone in Rats

This study aims to conduct a histological evaluation of tissue response to a membrane obtained from demineralized bovine cortical bone, associated or not, to tetracycline (TTC). TTC treated and untreated bovine membranes were implanted in the subcutaneous tissue of rats (n = 120). The animals were killed 1, 3, 7, 15, 30, and 60 days after surgery. The tissue around the material was fixed in 10% buffered formalin for 24 h. Sections of 6 mm were stained with hematoxylin and eosin. In general, moderate to intense inflammatory response was observed in the initial periods (1 and 3 days), moderate response in the 7- and 15-day periods, and that was remarkably reduced at 30 and 60 days. Resorptions of the membranes by mononuclear cells (fibroblasts and macrophages) and multinucleated giant cells were observed 15 days after implantation. Only the remnants of the material could be detected in some animals in 60 days. Both membranes were tolerated by the tissue and were completely resorbed after 30-60 days. While the association of TTC apparently accelerated the biodegradability of the membrane substrate, no significant differences were found in the tissue response behavior between the two groups tested.

Experimental dry socket: microscopic and molecular evaluation of two treatment modalities

PURPOSE To evaluate two treatment modalities of dry socket in rats and to discuss the first findings of the molecular analysis in this experimental model. METHODS 84 rats underwent a tooth extraction were divided in 4 groups: I-uninfected socket (control), II-infected socket without any treatment, III-infected socket treated with irrigation of 2% sodium iodide and 3% hydrogen peroxide solution, IV-infected socket submitted to curettage, irrigation with physiological saline solution and fulfilled with metronidazole paste as base. The groups were subdivided in postoperative sacrifice periods: 6/15/28 days. A quantitative and a qualitative microscopic analysis was performed. Also, a quantitative analysis was performed using a RealTimePCR to evaluate the genes expression in the wound healing: Collagen Type I/COL-I, vascular endothelial growth factor/VEGF, osteocalcin/OCN, alkaline phosphatase/ALP, runt-related transcription factor 2/RUNX2 and tumor necrosis factor alpha/TNF-α. RESULTS The group I showed higher bone formation, followed by groups IV, III, II respectively. The group II presented higher inflammatory infiltrate and the wound healing was delayed compared with other groups. It was obtained a significant positive correlation between bone neoformation and the expression of OCN and RUNX2, inflammatory infiltrate with TNF-α and a negative correlation between bone neoformation and TNF-α. CONCLUSION No significant difference was found between the treatments.