Gerluza A.B. Silva

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 &bgr;-cyclodextrin (HP&bgr;CD), 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 HP&bgr;CD/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 HP&bgr;CD/Ang-(1-7) administration significantly increased Ang-(1-7) on plasma whereas with the free peptide it was without effect. Oral administration of HP&bgr;CD/Ang-(1-7) (30 &mgr;g/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 HP&bgr;CD/Ang-(1-7)–treated rats. Furthermore, HP&bgr;CD/Ang-(1-7) attenuated the heart function impairment and cardiac remodeling induced by isoproterenol. In infarcted rats chronically treated with HP&bgr;CD/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 HP&bgr;CD/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 Dulbeccos Modified Eagles 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.

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.

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.

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.

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.

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.

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.

Carbon nanotubes functionalized with sodium hyaluronate restore bone repair in diabetic rat sockets.

OBJECTIVE We evaluated the effects of sodium hyaluronate (HY) and carbon nanotubes functionalized with HY (HY-CNT) on bone repair in the tooth sockets of diabetic rats. MATERIALS AND METHODS Diabetes was induced by streptozotocin (50 mg kg(-1) i.v.), and the sockets were divided into normal control, diabetic control, diabetic treated with HY (1%), and diabetic treated with HY-CNT (100 μg ml(-1)) groups. The sockets were analyzed according to the percentage of bone formation and the number of cell nuclei. RESULTS The percentage of bone trabeculae was lower in diabetic control animals (11.16 ± 5.10% vs 41.92 ± 6.34% in normal animals) after 14 days. Treating diabetic animals with HY or HY-CNT significantly increased the percentage of neoformed trabeculae (HY: 29.43 ± 3.29%; HY-CNT: 36.90 ± 3.07%). Moreover, the sockets of diabetic animals had an increased number of cell nuclei and HY or HY-CNT reduced this parameter. CONCLUSION Our results indicate that HY and HY-CNT restore bone repair in the tooth sockets of diabetic rats, suggesting that these biomaterials are potential adjuvant therapies for the management of diabetes.