Anna Christina Medeiros Fossati

Biocompatibility of RealSeal, its primer and AH Plus implanted in subcutaneous connective tissue of rats

Objective This study tested rat connective tissue response to RealSeal, RealSeal primer or AH Plus after 7, 15, 30, 60 and 90 days of implantation. Material and methods Thirty Wistar rats had subcutaneous sockets created on their back and received four implants each of polyethylene tubes containing one of the materials tested according to the groups: AH (AH Plus Sealer); RS (RealSeal Sealer); RP (RealSeal Primer); CG (control group – empty tube). After histological processing, sections were analyzed to identify the presence of neutrophils, lymphocytes and plasma cells, eosinophils, macrophages and giant cells, as well as fibrous capsule and abscesses, by an examiner using light microscope. Kruskal- Wallis and multiple-comparisons test were used for statistical analysis. Significance level was set at 5%. Results Lymphoplasmacytic infiltrate scores significantly higher than those of the control group were observed at 14 and 60 days in AH group, and at 90 days in RS group (p<0.05). There were no differences in terms of presence of macrophages, giant cells, eosinophils, neutrophils or fibrosis. AH Plus group scored higher for abscesses at 7 days than after any other period (p=0.031). RP group scored higher for lymphoplasmacytic infiltrate at 14 days than at 90 days (p=0.04). Conclusion The main contribution of this study was to demonstrate that issues involved with tissue tolerance of a Resilon-containing sealer, RealSeal Sealer, cannot be attributed to its primer content.

Nanofiber Scaffolds Support Bone Regeneration Associated with Pulp Stem Cells

Currently, there are a number of alternatives for bone grafting, though when used correctly they present physical, chemical or biological limitations, which justifies the pursuit for new alternatives for bone regeneration. This study gives a report on the potential for bone regeneration in the use of biodegradable nanofibers from poly (lactic-co-glycolic acid) (PLGA) in association with human mesenchymal stem cells from dental pulp of deciduous teeth (SCDT). Five samples of SCDT were seeded with scaffolds (test) or without scaffolds (control) for cell adhesion and viability assay. To evaluate the ability of the association in promoting bone formation, critical defects were made in the calvarium of rats (n=20), which were then divided into the following groups: I--sham group; II--implant of scaffolds; III--scaffolds/ SCDT; and IV--scaffolds/SCDT. They were kept for 13 days in osteogenic media. After 60 days, the histomorphometric analysis was performed. It was observed that the adherence and viability of SCDT in the control and test group were similar throughout the experiment (p>0.05). The association of scaffolds/SCDT maintained in osteogenic media, showed greater bone formation than the other groups (p<0.05). The study demonstrated that the association of SCDT seeded in biodegradable PLGA scaffolds has the ability to promote bone regeneration in rats, which is a promising alternative for application in regenerative medicine.

Distribution of small Rho GTPases in the developing rat submandibular gland.

During the rat submandibular gland (SMG) development, organogenesis and cytodifferentiation depend on the actin cytoskeleton, which is regulated by small Rho GTPases. These proteins link cell surface receptors to pathways that regulate cell motility, polarity, gene expression, vesicular trafficking, proliferation and apoptosis. The aim of this study was to evaluate, by immunohistochemistry, the distribution pattern of RhoA, RhoB, RhoC, Rac1 and Cdc42 during cytodifferentiation of the rat SMG and in male adults. All GTPases were found in epithelial and mesenchymal tissues throughout gland development. Rac1 appeared to be important for parenchyma expansion at the beginning of cytodifferentiation, while RhoC, Cdc42 and the inactive phosphorylated form of Rac1 seemed associated with lumen formation and cell polarization in terminal tubules. RhoA and RhoB labeling was evident throughout development. All GTPases were differentially expressed in the adult gland, suggesting that they play specific roles during differentiation and function of the rat SMG.

BH3-mimetic small molecule inhibits the growth and recurrence of adenoid cystic carcinoma

OBJECTIVES To evaluate the anti-tumor effect of BM-1197, a new potent and highly specific small molecule inhibitor of Bcl-2/Bcl-xL, in preclinical models of human adenoid cystic carcinoma (ACC). METHODS Low passage primary human adenoid cystic carcinoma cells (UM-HACC-2A,-2B,-5,-6) and patient-derived xenograft (PDX) models (UM-PDX-HACC) were developed from surgical specimens obtained from 4 patients. The effect of BM-1197 on cell viability and cell cycle were evaluated in vitro using this panel of low passage ACC cells. The effect of BM-1197 on tumor growth, recurrence and tumor cell apoptosis in vivo was evaluated with the PDX model of ACC (UM-PDX-HACC-5). RESULTS Exposure of low passage primary human ACC cells to BM-1197 mediated an IC50 of 0.92-2.82 μM. This correlated with an increase in the fraction of apoptotic cells (p<0.0001) and an increase in caspase-3 activity (p<0.0001), but no noticeable differences in cell cycle (p>0.05). In vivo, BM-1197 inhibited tumor growth (p=0.0256) and induced tumor cell apoptosis (p=0.0165) without causing significant systemic toxicities, as determined by mouse weight over time. Surprisingly, weekly BM-1197 decreased the incidence of tumor recurrence (p=0.0297), as determined by Kaplan-Meier analysis. CONCLUSION These data demonstrated that single agent BM-1197 induces apoptosis and inhibits tumor growth in preclinical models of adenoid cystic carcinoma. Notably, single agent BM-1197 inhibited tumor recurrence, which is considered a major clinical challenge in the clinical management of adenoid cystic carcinoma. Collectively, these results suggest that patients with adenoid cystic carcinoma might benefit from therapy with a BH3-mimetic small molecule.

Estudo da morfo e citodiferenciação da glândula submandibular remanescente de ratos após excisão parcial de um de seus lobos

Maintaining good health depends on, amongst other factors, of an adequate salivary flow1,2,3. Damage caused by obstruction, trauma or surgical removal of the salivary glands may lead to alterations in the saliva production. Any reduction in the salivary flow may result in harmful consequences to the living organism. This project was developed in order to enlighten the knowledge about the mechanisms that involve the regeneration of the rat submandibular gland (SMG) submitted to a partial lobe excision. STUDY DESIGN: Experimental. MATERIAL AND METHOD: Archived sections of rat submandibular glands of 15, 16, 17, 18 and 19 days of fetal life, and twenty male rats aging 30 or 60 days were utilized. Following anesthesia, the inferior third of the left lobe of the SMG of each animal was removed. Each animal was then left for recovery during 2, 3, 7 or 15 days, when were euthanazied and the glands removed, fixed in Methacarn solution, parafinated, and with sections of 5 ¼m under microtomy. Staining was either done by hematoxilin/eosin or by Periodic Schiff Acid (PAS). RESULTS: It was observed that the regenerative process occurred early and in all the specimens studied. It was similar to the aspect verified in the normal glandular development, and more pronounced at the 30-day rat. The cytodifferentiation represented by the neutral mucin labeling by the PAS, initially discrete, later on reaching its highest peak, and finally reducing its expression, having its place of initial establishment being changed. CONCLUSION: Based on the results, it was possible to conclude that the regenerative process of the rat excised SMG is stable, permanent and gradual, following determined stages.

Atorvastatin inhibits osteoclastogenesis and arrests tooth movement

Introduction In addition to their cholesterol‐lowering effects, the statin class of drugs appears to enhance osteogenesis and suppress bone resorption, which could be a clinical concern during orthodontic treatment. In this animal study, we aimed to determine whether atorvastatin (ATV) affects orthodontic tooth movement (OTM) through osteoclast inhibition. Furthermore, we analyzed the potential adverse effects of ATV on long‐bone turnover and endochondral ossification. Methods Rats were administered ATV (15 mg/kg) or saline solution via gavage (n = 12 animals/group), starting 2 weeks before initial OTM. Tooth displacement was measured after 7, 14, and 21 days. Histologic sections of the maxilla and femur were obtained after 14 and 21 days of OTM and stained (hematoxylin and eosin; TRAP assay) for histomorphometric analysis. Results ATV was associated with significant (P <0.05) reductions in OTM and osteoclast counts. Independently of drug administration, OTM increased the number of osteoclasts and reduced the bone‐volume ratio compared with the control maxillae without OTM. Long‐term statin administration did not appear to affect femoral endochondral ossification. Conclusions This experimental study showed that the long‐term use of ATV can significantly promote osteoclast inhibition and slow the OTM in the first week in rats. Under physiologic conditions, the drug did not affect bone turnover and endochondral ossification. HighlightsAtorvastatin transiently affects orthodontic tooth movement.During orthodontic movement, atorvastatin inhibits osteoclastogenesis transiently.Pharmacologic bone modulation could be a strategy to regulate tooth movement.Long‐term atorvastatin administration did not affect endochondral ossification.

Atorvastatin-induced osteoclast inhibition reduces orthodontic relapse

Introduction: The statin class of drugs enhances osteogenesis and suppresses bone resorption, which could be a plausible biologic mechanism for mitigation of orthodontic relapse. We aimed to determine whether atorvastatin (ATV) might affect orthodontic relapse and osteoclastogenesis by modulating expression of RANKL and osteoprotegerin (OPG), crucial molecules involved in bone turnover. Furthermore, we analyzed the adverse effects of ATV on femur turnover and endochondral ossification. Methods: Wistar rats were subjected to orthodontic tooth movement for 21 days, followed by removal of the appliance and ATV or saline solution administration. Up to 7, 14, and 21 days of ATV administration, tooth relapse was measured, and maxilla and femur sections were obtained and prepared for hematoxylin and eosin, TRAP, and immunohistochemical (RANKL and OPG) staining. Results: ATV decreased tooth relapse (P = 0.03) and osteoclast counts (P = 0.04), which were positively correlated (P = 0.006). Statin administration increased periodontal expression of OPG (P = 0.008), but not of RANKL protein. ATV administration also enhanced growth plate cartilage thickness. Conclusions: Statin‐induced OPG overexpression reduces relapse after orthodontic tooth movement, in a phenomenon correlated with decreased osteoclast counts. This phenomenon sheds light on OPG as a molecular target that modulates maxillary bone metabolism and orthodontic relapse. HighlightsPharmacologic bone modulation is a feasible strategy to reduce orthodontic relapse.Atorvastatin stimulates periodontal OPG expression and inhibits osteoclastogenesis.There is a positive correlation between osteoclast numbers and orthodontic relapse.Atorvastatin affects endochondral ossification of the long bones.

Long- and short-term diabetes mellitus type 1 modify young and elder rat salivary glands morphology

OBJECTIVE In this study we performed a temporal analysis of the effects of Diabetes Mellitus on morphology and laminin deposition in salivary glands of young (2 months-old) and aging (12 months-old) male Wistar rats, using immunohistochemistry. MATERIALS AND METHODS The animals were divided in control and diabetic (Streptozotocin induced) groups and euthanized after short and long-term diabetes induction. RESULTS Short-term induction led to vacuolization of parotid acinar cells and increased laminin deposition in both animal ages. In young rats, no difference was observed between short or long-term diabetes regarding laminin deposition, but parotid acinar cells vacuolization was more discrete after long-term diabetes. A slight decrease of submandibular gland convoluted granular ducts was observed in young and elder diabetic animal ages. In diabetic aging rats was observed an increase of laminin content only in the parotid gland. CONCLUSIONS These results suggest that some Diabetes Mellitus effects on salivary glands are not progressive over time, possibly due to the existence of adaptive mechanisms in response to chronic hyperglycemia. They also show that the duration of the disease was more relevant to the morphological effects than the age, although it is known that aging per se affects salivary gland morphology and function.

X-ray irradiation alters the actin cytoskeleton in murine lacrimal glands

Abstract Objective. The aim of this study was to evaluate the effects of X radiation on the distribution of filamentous actin (F-actin) in the mouse exorbital lacrimal gland. Materials and methods. Mice were divided into groups that received no radiation (n = 6) or one single exposure of 36 mGy of X radiation (n = 12). The animals were sacrificed after 4, 8 or 24 h. The lacrimal glands were stained with Hematoxylin/Eosin or Rhodamine-phalloidin and the filamentous actin arrangement was analyzed by confocal microscopy. Results. After 4 h of X-ray exposure there was an apparent increase in acini area and a decrease in the cortical F-actin content in secretory cells. This effect decreased gradually over time, returning to values close to the control after 24 h. Conclusion. This study shows that a 36mGy diagnostic X-ray dose affected reversibly the mouse exorbital lacrimal gland, suggesting that radiation used in diagnosis may induce changes on cell morphology due to actin remodeling.

Chronic alcohol consumption promotes alterations on salivary gland regeneration process

The aim of this study is to investigate the histological effect of alcohol ingestion on the regeneration of the submandibular gland (SMG) in rats. Twelve 60‐day‐old male Wistar rats were randomized into two experimental groups. Test group (TG) animals ingested 40° GL of alcohol for 45 days before surgery, being its concentration gradually increased 10° GL/week for 4 weeks to achieve the final concentration of 40° GL. The control group (CG) received water during the whole experimental period. One‐third of the left SMG lobe was removed. Three and seven days after, the whole gland was excised and analyzed. In the TG, the inflammatory process was pronounced when comparing the CG on day 3. The inverse aspect was observed on day 7, associated with an advanced parenchyma development. Changes in laminin expression and glycoproteins production were observed in the TG, causing advanced morphogenesis and delay in cytodifferentiation during the salivary gland regeneration, probably due to alcohol effects. Animals who received ethanol showed alterations in the pattern of glandular regeneration. Microsc. Res. Tech. 76:1125–1130, 2013.