Comparison between fractal analysis and radiopacity evaluation as a tool for studying repair of an osseous defect in an animal model using biomaterials.

OBJECTIVES: To evaluate bone repair of an osseous defect in a rat animal model through fractal analysis and radiopacity analysis in radiographic images. METHODS: 120 rats were subjected to extraction of their first molar and divided into four groups (n = 6/group) according to the material used for bone grafting: mineralized bovine bone, demineralized bovine bone (DBB), blood clot (BC - control) or Bio-Oss® (BO). The animals were sacrificed after 1, 7, 14, 21 and 49 days and subjected to radiographic evaluation. For fractal analysis (FA), a square regionof interest of 30 × 30 pixels was used, and radiopacity was measured as the mean gray scale (MGS) value for three points of 5 × 5 pixels in the apical, medial and coronal regions of the defect. Histomorphometric evaluation was realized as the gold standard for bone neo-formation and maturation of the new osseous matrix. RESULTS: Histomorphometric evaluation suggested that DBB showed faster mineralized deposition and resulted in more mature bone at the final time point of evaluation. Mineralized bovine bone and Bio-Oss presented similar results. The mineralized groups did not show significant differences in bone maturation. The radiopacity analysis revealed a significant difference (p < 0.05) between the DBB and blood clot groups at the final time point. FA did not show any significant differences at the final time point. CONCLUSIONS: Mean gray scale seemed to be more effective for the quantification of bone repair than FA in the demineralized group in this animal model. Results for the mineralized groups did not reveal a significant difference, leading to the conclusion that both methods are effective.

Enhancement of Bone Healing by Local Administration of Carbon Nanotubes Functionalized with Sodium Hyaluronate in Rat Tibiae.

It has been reported that carbon nanotubes (CNTs) serve as nucleation sites for the deposition of bone matrix and cell proliferation. Here, we evaluated the effects of multi-walled CNTs (MWCNTs) on bone repair of rat tibiae. Furthermore, because sodium hyaluronate (HY) accelerates bone restoration, we associated CNTs with HY (HY-MWCNTs) in an attempt to boost bone repair. The bone defect was created by a 1.6-mm-diameter drill. After 7 and 14 days, tibiae were processed for histological and morphometric analyses. Immunohistochemistry was used to evaluate the expression of vascular endothelial growth factor (VEGF) in bone defects. Expression of osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and collagen I (Col I) was assessed by real-time PCR. Histomorphometric analysis showed a similar increase in the percentage of bone trabeculae in tibia bone defects treated with HY and HY-MWCNTs, and both groups presented more organized and thicker bone trabeculae than nontreated defects. Tibiae treated with MWCNTs or HY- MWCNTs showed a higher expression of VEGF. Treatment with MWCNTs or HY-MWCNTs increased the expression of molecules involved in the bone repair process, such as OCN and BMP-2. Also, HY- and MWCNT-treated tibiae had an increased expression of Col I. Thus, it is tempting to conclude that CNTs associated or not with other materials such as HY emerged as a promising biomaterial for bone tissue engineering.

Rapid maxillary expansion: Do banded teeth develop more external root resorption than non-banded anchorage teeth?

OBJECTIVE: To compare external root resorption (ERR) when bands and wires are used as orthodontic anchorage during rapid maxillary expansion (RME). MATERIALS AND METHODS: Histologic analysis was performed on 108 sites from 18 maxillary first premolars and on 36 sites from six mandibular first premolars in nine subjects (mean age = 15.2 ± 1.4 years) 3 months after RME. Maxillary teeth were pooled into two groups (n = 54 each) according to the type of orthodontic anchorage (band group [BG] vs wire group [WG]). Anchorage type was randomly chosen in a split-mouth design. Mandibular first premolars, which were not subjected to orthodontic forces, were used as the control group (CG). RESULTS: All premolars in the BG and WG showed ERR at the level of the cementum and dentin. Repair with cementum cells was observed in all resorption areas, but complete repair was rarely found. No statistically significant difference was found between the BG and WG with regard to the ERR. No association was found between the root height position (middle or cervical third) and the incidence of ERR. Buccal root surfaces showed a higher amount of ERR compared with the palatal and interproximal surfaces. ERR was not found in any teeth in the CG. CONCLUSION: All maxillary first premolars subjected to RME showed ERR and partial cementum repair. Banded teeth did not develop more ERR than nonbanded anchorage teeth.

The novel Mas agonist, CGEN-856S, attenuates isoproterenol-induced cardiac remodeling and myocardial infarction injury in rats.

CGEN-856S is a novel Mas agonist. Herein, we examined the effects of this peptide on isoproterenol (ISO)-induced cardiac remodeling and myocardial infarction (MI) injury. We also sought to determine whether CGEN-856S activates the underlying mechanisms related to Mas receptor activation. Heart hypertrophy and fibrosis were induced by ISO (2 mg·kg(-1)·day(-1)) in Wistar rats. After a 7-day treatment period with CGEN-856S (90 µg·kg(-1)·day(-1)) or vehicle, the cardiomyocyte diameter was evaluated in left ventricular sections stained with hematoxylin and eosin, and immunofluorescence labeling and quantitative confocal microscopy were used to quantify the deposition of type I and III collagen and fibronectin in the left ventricles. MI was induced by coronary artery ligation, and CGEN-856S (90 µg·kg(-1)·day(-1)) or saline was administered for 14 days. The Langendorff technique was used to evaluate cardiac function, and left ventricular sections were stained with Masson's trichrome dye to quantify the infarct area. Using Chinese hamster ovary cells stably transfected with Mas cDNA, we evaluated whether CGEN-856S alters AKT and endothelial nitric oxide synthase (eNOS) phosphorylation. CGEN-856S reduced the degree of ISO-induced hypertrophy (13.91±0.17 µm vs. 12.41±0.16 µm in the ISO+CGEN-856S group). In addition, the Mas agonist attenuated the ISO-induced increase in collagen I, collagen III, and fibronectin deposition. CGEN-856S markedly attenuated the MI-induced decrease in systolic tension, as well as in +dT/dt and -dT/dt. Furthermore, CGEN-856S administration significantly decreased the infarct area (23.68±2.78% vs. 13.95±4.37% in the MI+CGEN-856S group). These effects likely involved the participation of AKT and NO, as CGEN-856S administration increased the levels of p-AKT and p-eNOS. Thus, our results indicate that CGEN-856S exerts cardioprotective effects on ISO-induced cardiac remodeling and MI-mediated heart failure in rats through a mechanism likely involving the eNOS/AKT pathway.

Mesenchymal stem cells associated with porous chitosan-gelatin scaffold: a potential strategy for alveolar bone regeneration.

Tissue engineering has emerged as a novel treatment for replacement of lost bone tissue. This study evaluated the effects of a chitosan-gelatin scaffold seeded with bone marrow mesenchymal stem cells (BMMSCs) in the healing process of tooth sockets in rats. BMMSCs isolated from transgenic rats expressing enhanced green fluorescent protein (eGFP) were expanded and seeded on a chitosan-gelatin scaffold. These constructs were cultured for three days and characterized by scanning electronic microscopy (SEM) and energy dispersion spectroscopy (EDS). Receptor rats received the implant in the left sockets, after upper first-molar extraction. Right alveoli served as control. Animals were sacrificed at days 5, 21, and 35 post-graft for examination. Morphometry demonstrated increased bone mineralization after 21 and 35 days in transplanted sockets. Migration, differentiation, and fate of eGFP-labeled BMMSCs were monitored by immunohistochemistry. Tartrate-resistant acid phosphatase staining (TRAP) was carried out at 21 days, to identify the involvement of osteoclastic cells in the scaffold resorption. The biomaterial was resorbed by TRAP-negative giant cells in a typical foreign body reaction. Immunohistochemical findings showed that BMMSCs contributed to bone, epithelial, and vascular repair. Together, results indicate that BMMSCs loaded in the chitosan-gelatin scaffold is a strategy for tissue development in bone engineering.

Gestational and postnatal protein deficiency affects postnatal development and histomorphometry of liver, kidneys, and ovaries of female rats' offspring.

Pre- and postnatal protein deficiency may lead to decreased foetal intra-uterine development and postnatal growth, which is common in developing countries. The present study aimed to investigate the consequences of a low-protein intake during gestation and postnatally on adult female rats' offspring. Female rats were given either a control or a protein-deficient diet throughout the gestation and lactation periods. A subset of females was killed at day 20 of pregnancy for foetal and placental measurements. Another subset of females farrowed and the number, length, and weight of the offspring were measured. After weaning, the offspring received the same diet as their dams until 70 days of age. They were sacrificed, and some organs were weighed and collected for histomorphometrical analyses. Placental weight and size and foetal weight were lower in protein-deficient dams. The weight and length of pups at birth were also lower in the deficient group. The organs to body weight ratio were higher in the deficient animals at 70 days of age. The protein-deficient female offspring had a smaller ovarian area, greater numbers of primordial follicles and developing follicles per square millimetres of ovarian cortex, and no corpora lutea. The liver showed smaller nuclear diameter of the hepatocytes and height of the hepatocytes cords. The kidneys showed smaller cortical area with reduced glomerular number and diameter. These results provide the first evidence of the histomorphological changes of the association between gestational and postnatal protein deficiency in female rats' offspring.

Immunization with rP22 induces protective immunity against Schistosoma mansoni: effects on granuloma down-modulation and cytokine production.

Schistosomiasis remains a significant public health problem in tropical countries and it is recognized as the most important human helminth infection in terms of morbidity and mortality. Although the existing antischistosomal drugs are highly effective, they do not prevent against reinfection or granuloma formation. Therefore, vaccine strategies are essential for the control of schistosomiasis. Our group recently identified the recombinant (r) P22 protein, a component of the adult worm protein fraction PIII that has been shown to engender protective and immunomodulatory effects on murine schistosomiasis. A cDNA clone encoding rP22 was isolated from a Schistosoma mansoni adult worm cDNA library using anti-PIII rabbit serum; it exhibited complete identity with S. mansoni Sm21.7 EF-hand antigen. Confocal microscopy revealed that rP22 is a tegument protein localized on the surface of S. mansoni miracidia and adult worms. Mice immunized with rP22 exhibited a 51% and 22.5% decrease in adult worm burden and in hepatic eggs, respectively. Additionally, rP22 vaccine produced a reduction in 60% of liver granuloma size and 71% of fibrosis in mice, suggesting that rP22 might contribute to down-modulate granulomatous hypersensitivity to S. mansoni eggs. Protective immunity in mice was associated with high titers of rP22-specific IgG antibodies; elevated production of IFN-γ, TNF-α and IL-10; and a reduced level of IL-4. In conclusion, these findings indicate that rP22-based vaccines could be useful to elicit protection and reduce pathology associated to schistosomiasis.

An oral formulation of angiotensin-(1-7) produces cardioprotective effects in infarcted and isoproterenol-treated rats.

In this study we evaluated the cardiac effects of a pharmaceutical formulation developed by including angiotensin (Ang)-(1-7) in hydroxypropyl ß-cyclodextrin (HPßCD), in normal, infarcted, and isoproterenol-treated rats. Myocardial infarction was produced by left coronary artery occlusion. Isoproterenol (2 mg/kg, IP) was administered daily for 7 days. Oral administration of HPßCD/Ang-(1-7) started immediately before infarction or associated with the first dose of isoproterenol. After 7 days of treatment, the rats were euthanized, and the Langendorff technique was used to analyze cardiac function. In addition, heart function was chronically (15, 30, 50 days) analyzed by echocardiography. Cardiac sections were stained with hematoxylin/eosin and Masson trichrome to evaluate cardiac hypertrophy and damage, respectively. Pharmacokinetic studies showed that oral HPßCD/Ang-(1-7) administration significantly increased Ang-(1-7) on plasma whereas with the free peptide it was without effect. Oral administration of HPßCD/Ang-(1-7) (30 µg/kg) significantly reduced the deleterious effects induced by myocardial infarction on systolic and diastolic tension, ±dT/dt, perfusion pressure, and heart rate. Strikingly, a 50% reduction of the infarcted area was observed in HPßCD/Ang-(1-7)-treated rats. Furthermore, HPßCD/Ang-(1-7) attenuated the heart function impairment and cardiac remodeling induced by isoproterenol. In infarcted rats chronically treated with HPßCD/Ang-(1-7), the reduction of ejection fraction and fractional shorting and the increase in systolic and diastolic left ventricular volumes observed in infarcted rats were attenuated. Altogether, these findings further confirm the cardioprotective effects of Ang-(1-7). More importantly, our data indicate that the HPßCD/Ang-(1-7) is a feasible formulation for oral administration of Ang-(1-7), which can be used as a cardioprotective drug.

Three-dimensional culture of rat BMMSCs in a porous chitosan-gelatin scaffold: A promising association for bone tissue engineering in oral reconstruction.

OBJECTIVE: this study investigated the in vitro effects of a chitosan-gelatin scaffold on growth and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMMSCs) in three-dimensional (3D) cultures and evaluated the biomaterial biocompatibility and degradability after its grafting into tooth sockets of rats. DESIGN: a porous chitosan-gelatin scaffold cross-linked by glutaraldehyde was synthesised and characterised by light (LM), scanning electronic microscopy (SEM), energy dispersion spectroscopy (EDS) and X-ray diffraction (XRD). Rat BMMSCs were isolated, expanded and seeded onto scaffold using Dulbecco's Modified Eagle's Medium (DMEM) with or without an osteogenic supplement. Cell viability by MTT assay, alkaline phosphatase (ALP) activity and morphological LM and SEM analysis were performed after 1, 3, 8 and 14 days in culture. Free-cell scaffolds were implanted into tooth sockets of Lewis rats after upper first molars extraction. Fifteen male recipient rats were sacrificed after 5, 21 and 35 days for histological analysis. RESULTS: scaffold characterisation revealed the porous structure, organic and amorphous content. This biomaterial promoted the adhesion, spreading and in vitro viability of the BMMSCs. Osteogenic-supplemented media did not improve the cellular response compared to DMEM. The biomaterial presented high biocompatibility and slow biodegradation in vivo. Remains of biomaterial were still observed at 21 and 35 days after implantation. However, on the 21st day, alveolar bone and epithelial healing were completely established. CONCLUSIONS: these results indicate that chitosan-gelatin support the adhesion and osteogenic differentiation of rat BMMSCs and offer adequate physico-chemical and biological properties for use as scaffolds in bone tissue engineering-related strategies.

Effects of single wall carbon nanotubes and its functionalization with sodium hyaluronate on bone repair.

AIMS: Sodium hyaluronate (HY) accelerates the repair of bone defects. However, the weak stability of HY formulations in aqueous environments has hindered its wide utilization. The functionalization of carbon nanotubes (SWCNT) with HY (HY-SWCNT) results in a reinforced hydrogel with an increased stability. Nevertheless, the biological effects of HY-SWCNT have not been explored. Thus, our objective was to evaluate whether this biomaterial preserves the bioactivity of the HY. MAIN METHODS: Wistar rats were subjected to molar extraction and the sockets were treated with SWCNT (50-400 microg/mL), 1% HY, HY-SWCNT (50-400 microg/mL) or carbopol (vehicle). After seven days of surgery, histological and morphometric analyses were performed to evaluate the trabecular bone formation and the number of cell nuclei in the sockets. Expression of collagen types I and III was determined by immunohistochemistry. KEY FINDINGS: Treatment with SWCNT did not alter the bone deposition, as well as the cell nuclei counting. Additionally, no significant evidence of toxicity was observed in SWCNT-treated sockets. Contrastingly, both HY and HY-SWCNT induced a marked increase in the bone formation (HY: 10.10+/-1.99%; HY-SWCNT 100 microg/mL: 10.90+/-1.13%; control: 3.69+/-1.17%) and decreased the cell nuclei amount in the sockets. Moreover, collagen type I expression was more pronounced in HY- and HY-SWCNT-treated sockets. No significant differences were viewed in the expression of collagen type III. SIGNIFICANCE: Our results indicate that SWCNT is a feasible material to deliver HY to bone defects. Importantly, the functionalization of SWCNT with HY preserved the beneficial biological properties of HY in the healing process, thereby suggesting that HY-SWCNT scaffolds are potentially useful biomaterials for the restoration of bone defects.

Epidermal growth factor receptors, testosterone levels and parotid gland changes in rats infected with Trypanosoma cruzi.

It has been demonstrated that parotid glands of rats infected with Trypanosoma cruzi present severe histological alterations; changes include reduction in density and volume of the acini and duct systems and an increase in connective tissue. We evaluated the association between morphological changes in parotid glands, circulating testosterone levels and epidermal growth factor receptor (EGF-R) expression in experimental Chagas disease in rats. Animals at 18 days of infection (acute phase) showed a significant decrease in body weight, serum testosterone levels and EGF-R expression in the parotid gland compared with a control group. Since decreases in body weight could lead to a reduction in circulating testosterone concentration, we believe that the reduction in EGF-R expression in parotid glands of infected rats is due to alterations in testosterone levels and atrophy of parotid glands is caused by changes in EGF-R expression. Additionally, at 50 days (chronic phase) of infection parotid glands showed a normal histological aspect likely due to the normalization of the body weight. These findings suggest that the testosterone-EGF-R axis is involved in the histological changes.

Local delivery of EGF-liposome mediated bone modeling in orthodontic tooth movement by increasing RANKL expression.

AIMS: It has long been demonstrated that epidermal growth factor (EGF) has catabolic effects on bone. Thus, we examined the role of EGF in regulating mechanically induced bone modeling in a rat model of orthodontic tooth movement. MAIN METHODS: The maxillary first molars of rats were moved mesially using an orthodontic appliance attached to the maxillary incisor teeth. Rats were randomly divided into 4 groups: (G1) administration of PBS (phosphate buffer saline) solution (n=24); (G2) administration of empty liposomes (n=24); (G3) administration 20ng of EGF solution (n=24); and (G4) 20ng of EGF-liposomes solution (n=24). Each solution was injected in the mucosa of the left first molar adjacent to the appliance. At days 5, 10, 14 and 21 after drug administration, 6 animals of each group were sacrificed. Histomorphometric analysis was used to quantify osteoclasts (Tartrate-resistant acid phosphatase (TRAP)+cells) and tooth movement. Using immunohistochemistry assay we evaluated the RANKL (receptor activator of nuclear factor kappaB ligand) and epidermal growth factor receptor (EGFR) expression. KEY FINDINGS: The EGF-liposome administration showed an increased tooth movement and osteoclast numbers compared to controls (p<0.05). This was correlated with intense RANKL expression. Both osteoblasts and osteoclasts expressed EGFR. SIGNIFICANCE: Local delivery of EGF-liposome stimulates osteoclastogenesis and tooth movement.

Delay in maturation of the submandibular gland in Chagas disease correlates with lower DNA synthesis.

It has been demonstrated that the acute phase of Trypanosoma cruzi infection promotes several changes in the oral glands. The present study examined whether T. cruzi modulates the expression of host cell apoptotic or mitotic pathway genes. Rats were infected with T. cruzi then sacrificed after 18, 32, 64 or 97 days, after which the submandibular glands were analyzed by immunohistochemistry. Immunohistochemical analyses using an anti-bromodeoxyuridine antibody showed that, during acute T. cruzi infection, DNA synthesizing cells in rat submandibular glands were lower than in non-infected animals (p < 0.05). However, after 64 days of infection (chronic phase), the number of immunolabeled cells are similar in both groups. However, immunohistochemical analysis of Fas and Bcl-2 expression did not find any difference between infected and non-infected animals in both the acute and chronic stages. These findings suggest that the delay in ductal maturation observed at the acute phase of Chagas disease is correlated with lower expression of DNA synthesis genes, but not apoptotic genes.

Delay in maturation of the submandibular gland in Chagas disease correlates with lower DNA synthesis

It has been demonstrated that the acute phase of Trypanosoma cruzi infection promotes several changes in the oral glands. The present study examined whether T. cruzi modulates the expression of host cell apoptotic or mitotic pathway genes. Rats were infected with T. cruzi then sacrificed after 18, 32, 64 or 97 days, after which the submandibular glands were analyzed by immunohistochemistry. Immunohistochemical analyses using an anti-bromodeoxyuridine antibody showed that, during acute T. cruzi infection, DNA synthesizing cells in rat submandibular glands were lower than in non-infected animals (p < 0.05). However, after 64 days of infection (chronic phase), the number of immunolabeled cells are similar in both groups. However, immunohistochemical analysis of Fas and Bcl-2 expression did not find any difference between infected and non-infected animals in both the acute and chronic stages. These findings suggest that the delay in ductal maturation observed at the acute phase of Chagas disease is correlated with lower expression of DNA synthesis genes, but not apoptotic genes.

Sodium hyaluronate accelerates the healing process in tooth sockets of rats.

OBJECTIVE: In this study we evaluated the effects of sodium hyaluronate (HY) in the healing process of tooth sockets of rats. DESIGN: Immediately after the extraction of the upper first molars of male Holtzman rats, right sockets were treated with 1% HY gel (approximately 0.1 ml), while left sockets were used as control (blood clot). The animals were sacrificed at 2, 7, and 21 days after tooth extraction and upper maxillaries processed for histological and morphometric analysis of the apical and medium thirds of the sockets. Carbopol, an inert gel, was used to evaluate the mechanical effect of gel injection into sockets. Expression of bone morphogenetic protein-2 (BMP-2) and osteopontin (OPN) was determined by immunohistochemistry at 1, 2, 3, 4, 5, and 7 days after tooth extraction. RESULTS: Histological analysis showed that HY treatment induced earlier trabecular bone deposition resulting in a bone matrix more organized at 7 and 21 days after tooth extraction. Also, HY elicited significant increase in the amount of bone trabeculaes at 7 and 21 days after tooth extraction (percentage of trabecular bone area at 7 days: 13.21+/-4.66% vs. 2.58+/-1.36% in the apical third of control sockets) and in the vessels counting at 7 days. Conversely, the number of cell nuclei was decreased in HY-treated sockets. Additionally, expression of BMP-2 and OPN was enhanced in HY-treated sockets compared with control sockets. CONCLUSIONS: These findings suggest that HY accelerates the healing process in tooth sockets of rats stimulating the expression of osteogenic proteins.

Direct capping of human pulps with a dentin bonding system and calcium hydroxide: an immunohistochemical analysis.

Pulp capping is a treatment where a protective agent is applied to an exposed pulp to allow the maintenance of its vitality and function. The present study analyzed the immunohistochemical expression of fibronectin and type III collagen in human dental pulps submitted to direct pulp capping with calcium hydroxide [Ca(OH)2] or the Single Bond adhesive system (SBAS). The results demonstrated that both proteins were not expressed in the SBAS group, although in the group capped with Ca(OH)2 a diffuse labeling in the extracellular matrix was initially observed, followed by a late expression in the odontoblast-like layer and beneath the dentin bridge. It seems that application of adhesive systems in direct contact with healthy pulps will not lead to expression of proteins that are believed to be essential for pulpal repair. Moreover, Ca(OH)2 showed good biocompatibility properties with the dental pulp tissue, inducing the expression of reparative molecules, and therefore remains the material of choice for the treatment of accidental pulp exposures.

The nonpeptide angiotensin-(1-7) receptor Mas agonist AVE-0991 attenuates heart failure induced by myocardial infarction.

The nonpeptide AVE-0991, which has been reported as a selective ligand for the angiotensin-(1-7) [ANG-(1-7)] receptor Mas, has actions similar to those attributed to the cardioprotective product of the renin-angiotensin system, ANG-(1-7). In this study, we evaluated the cardiac effects of AVE-0991 in normal and infarcted male Wistar rats. Myocardial infarction was induced by left coronary artery ligation. At the end of the treatment, the Langendorff technique was used to analyze cardiac function. Left ventricle serial sections were dyed with Gomori trichrome stain to quantify the infarcted area. In normal hearts, AVE-0991 produced a significant decrease in perfusion pressure and an increase in systolic tension, rate of tension rise and fall (+/-dT/dt), and heart rate. These effects were completely blocked by the perfusion of the hearts with a solution containing the selective ANG-(1-7) antagonist A-779. N(G)-nitro-l-arginine methyl ester treatment abolished the AVE-0991-induced vasodilation in isolated hearts. AVE-0991 significantly attenuated the decrease in systolic tension (sham operated, 13.00 +/- 1.02 g; infarction, 7.18 +/- 0.66 g; AVE treated, 9.23 +/- 1.05 g, n = 5), +dT/dt, -dT/dt, and heart rate induced by myocardial infarction. Infarction-induced vasoconstriction was completely prevented by AVE-0991 treatment. Furthermore, AVE-0991 significantly decreased the infarcted area (6.98 +/- 1.01 vs. 3.94 +/- 1.04 mm(2) in AVE-treated rats). These data indicate that the compound AVE-0991 produces beneficial effects in isolated perfused rat hearts involving the ANG-(1-7) receptor Mas and the release of nitric oxide. In addition, our results indicate that AVE-0991 attenuates postischemic heart failure.

Renal function in transgenic rats expressing an angiotensin-(1-7)-producing fusion protein.

Transgenic rats [TGR(A1-7)3292] present a chronic 2.5-fold increase in plasma Angiotensin-(1-7) [Ang-(1-7)] concentration. In the present study, we investigated the effects of this chronic elevation on renal function, vasopressin levels, kidney morphology, expression of Ang-(1-7) and vasopressin receptors in TGR(A1-7)3292. Urine volume and water intake were measured for 24 h. At the end of this period, plasma and urine samples were collected to evaluate renal function parameters and circulating vasopressin levels. Expression of renal V2 receptors and Mas was assessed by ribonuclease protection assay. Renal slices were processed for histological analysis. The urine flow of TGR(A1-7)3292 was significantly lower in comparison with Sprague-Dawley rats. The reduced urine volume of TGR(A1-7)3292 was accompanied by a significant increase in urinary osmolality and decrease free water clearance. Glomerular filtration rate, urinary sodium and potassium excretion were similar in both strains. No significant changes were observed in vasopressin levels as well as in V2 receptor and Mas mRNA expression in renal tissue. No changes in kidney structure of TGR(A1-7)3292 were detected. These data suggest that changes in circulating renin-angiotensin system produced by chronic increase of Ang-(1-7) levels can lead to adjustments in the water balance that are independent of vasopressin release and V2 receptor expression.

Direct pulp capping with a dentin bonding system in human teeth: a clinical and histological evaluation.

This study evaluated the pulpal response in human dental pulp to direct pulp capping with the Single Bond Adhesive System (SBAS) after 10% or 37% phosphoric acid etching and after capping with Calcium Hydroxide (CH). The degree of bleeding and hemostasis conditions was considered during the adhesive technique. The pulps of 78 sound premolars were capped with SBAS after 37% phosphoric acid etching (Group I) or 10% phosphoric acid etching (Group II) and CH (Group III-control). The cavities were restored with a resin composite (Charisma). After 1, 3, 7 and 30 days, the teeth were extracted and processed for light microscopical examination (H/E, AgNOR silver stain and Brown-Brenn). The patients were followed for postoperative symptomatology evaluation. Clinical results showed the possibility of hemostasis with saline solution only. There was no statistical difference between bleeding generated by 10% and 37% acid solutions. In some cases, contact of the pulp tissue with SASB started the bleeding process, thus damaging the adhesive technique. The histological response was similar in Groups I and II, without signs of cellular differentiation and dentin neoformation up to 30 days. Bacteria were not observed in any specimens. In the control group (CH) at day 7, the pulps exhibited cells with high synthetic activity (Ag-NOR-positive) underneath the area of coagulation necrosis. Dentin bridging was observed at the thirtieth day. The postoperative period was asymptomatic for all groups. In conclusion, SBAS should be avoided for vital pulp therapy, while CH remains the capping agent of choice for mechanically exposed human dental pulp.

Fibroblast growth factor 9: cloning and immunolocalisation during tooth development in Didelphis albiventris.

There are no reports in literature about functional roles of fibroblast growth factor 9 (FGF-9) in tooth development in animals with complete tooth pattern. The classical model for studying tooth development is the mouse, which has small number of teeth and distinctive incisor and molar patterns. The opossum Didelphis albiventris with five upper and four lower incisors, one canine, three premolars, and four molars, on each side of the jaw, seems to be a convenient model to test results obtained in the mouse. Molecular expression studies indicate that FGF-9 participates in murine tooth initiation and regulation of morphogenesis. Searching for similarities and differences in FGF-9 expression between the opossum and the mouse, amino acid sequence and expression pattern of FGF-9 in the developing first molars of D. albiventris were characterised. FGF-9 cDNA sequence was obtained using RT-PCR and expressed in bacterial system for recombinant protein production and analysis of immunoreactivity. FGF-9 expression during tooth development was investigated by immunoperoxidase method. FGF-9 protein consists in a 209-residue polypeptide with a predicted molecular mass of 23.5 kDa. FGF-9 amino acid sequence has 98% of sequence identity to human and 97% to rodents. During tooth development, epithelial FGF-9 expression was seen at the dental lamina stage. Mesenchymal expression was seen at the bud stage and at the cap stage. No significant expression was found in the enamel knot. While in rodents FGF-9 is involved in initiation and regulation of tooth shape, it is suggested that it is only involved in tooth initiation in D. albiventris.