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.

Demineralised human dentine matrix stimulates the expression of VEGF and accelerates the bone repair in tooth sockets of rats.

OBJECTIVE: In this study we investigated the possible use of human demineralised dentine matrix (DHDM), obtained from the extracted teeth, as bone graft material and evaluated the expression of vascular endothelial growth factor (VEGF) induced by this material in the healing process of tooth sockets of rats. DESIGN: To evaluate bone regeneration and expression of VEGF induced by DHDM, thirty-two male Wistar rats weighing approximately 200 g were used. After maxillary second molar extraction, the left sockets were filled with DHDM and the right sockets were naturally filled by blood clot (control). The animals were sacrificed at 3, 7, 14 and 21 days after surgery and upper maxillaries were processed for histological, morphometric and immunohistochemical analyses. DHDM was used to evaluate the mechanical effect of bone graft material into sockets. Expression of VEGF was determined by immunohistochemistry in all groups. RESULTS: Our results demonstrated a significant increase in the newly formed bone tissue in sockets of 7, 14 and 21 days and a significant increase in VEGF expression at days 7 and 14 on treated sockets. CONCLUSIONS: Our results showed that DHDM increases the expression of VEGF and accelerates the healing process in rats tooth sockets, by stimulating bone deposition and also vessels formation. These results suggest that DHDM has osteoinductive/osteoconductive potential and may represent an efficient grafting material on guided bone regeneration.

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.

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.

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.

Polymorphism in the Msx1 gene associated with hypodontia in a Brazilian family.

Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions. With the large number of genes involved in the odontogenesis process, the opportunity for mutations to disrupt this process is high. Mutational analysis has revealed genes that are major causes of non-syndromic hypodontia. The most common permanent missing teeth are the third molars, second premolars, and maxillary lateral incisors. Although hypodontia does not represent a serious public health problem, it may cause masticatory and speech dysfunctions and esthetic problems. Msx1 (Muscle Segment Box) is believed to play an important role in tooth development. To further investigate the role of the gene in human hypodontia, we analyzed genotypes in a family with hypodontia using the SSCP assay. Examinations of all affected and unaffected members of the family studied indicated that 5 of the 10 family members had hypodontia, and it was possible to observe polymorphisms/mutation by SSCP as bands with an anomalous migration pattern in individuals with hypodontia. Our data suggest that Msx1 gene polymorphism is associated with hypodontia.

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.

PPAR-gamma agonist rosiglitazone prevents inflammatory periodontal bone loss by inhibiting osteoclastogenesis.

Rosiglitazone (RGZ), an oral anti-hyperglycemic agent used for non-insulin-dependent diabetes mellitus, is a high-affinity synthetic agonist for peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Both in vitro and in vivo experiments have also revealed that RGZ possesses anti-inflammatory properties. Therefore, in the present study, we investigated the anti-inflammatory effects of RGZ in a rat model of periodontal disease induced by ligature placed around the mandible first molars of each animal. Male Wister rats were divided into four groups: 1) animals without ligature placement receiving administration of empty vehicle (control); 2) animals with ligature receiving administration of empty vehicle; 3) animals with ligature receiving administration with oral RGZ (10 mg/kg/day); and 4) animals with ligature receiving administration of subcutaneous RGZ (10 mg/kg/day). Thirty days after induction of periodontal disease, the animals were sacrificed, and mandibles and gingival tissues were removed for further analysis. An in vitro assay was also employed to test the inhibitory effects of RGZ on osteoclastogenesis. Histomorphological and immunohistochemical analyses of periodontal tissue demonstrated that RGZ-treated animals presented decreased bone resorption, along with reduced RANKL expression, compared to those animals with ligature, but treated with empty vehicle. Corresponding to such results obtained from in vivo experiments, RGZ also suppressed in vitro osteoclast differentiation in the presence of RANKL in MOCP-5 osteoclast precursor cells, along with the down-regulation of the expression of RANKL-induced TRAP mRNA. These data indicated that RGZ may suppress the bone resorption by inhibiting RANKL-mediated osteoclastogenesis elicited during the course of experimental periodontitis in rats.

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.

Protective effect and granuloma down-modulation promoted by RP44 antigen a fructose 1,6 bisphosphate aldolase of Schistosoma mansoni.

A Schistosoma mansoni adult worm cDNA expression library was screened using rabbit IgG against PIII, an adult worm protein fraction, already known to possess protective and immunomodulating effects to a challenge infection in mice. A positive cDNA clone was selected and characterized. The cDNA screened encodes a protein (P44) with an ORF of 1089 bp and an amino acid sequence of 363 residues with a predictable molecular weight of 44 kDa. The P44 amino acid sequence exhibits 100% identity to the fructose 1,6 bisphosphate aldolase of S. mansoni, 66% to Homo sapiens and 66% to Mus musculus. The cDNA was cloned into a pGEX-4T-3 vector and expressed in Escherichia coli as a fusion protein (GST/P44). Mice vaccinated with recombinant P44 were able to develop high levels of IgG or IgG1 and displayed low levels of IgG2a isotype. Moreover, immunization of mice with this antigen induced a significant protection of 57% against a challenge infection and significant decrease in hepatic granuloma formation. Our results demonstrate that granuloma modulation can be targeted for pathology elimination through vaccination. This represents an advance in schistosome vaccinology and allows for the development of a therapeutic as well as a prophylactic vaccine.

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.

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.

IL-12 enhances protective immunity in mice engendered by immunization with recombinant 14 kDa Schistosoma mansoni fatty acid-binding protein through an IFN-gamma and TNF-alpha dependent pathway.

Herein, we tested the ability of IL-12 to enhance protection induced by recombinant Sm14 (rSm14). Mice immunization with three doses of 25 microg of rSm14 was able to induce 25% of protection in mice against challenge. However, co-administration of exogenous IL-12 enhanced protective immunity engendered by rSm14 from 25 to 42.2%. Higher levels of IgG2a and TNF-alpha were observed in mice immunized with rSm14 plus IL-12 compared to animals vaccinated with rSm14 alone. Regarding other cytokines, significant amounts of IFN-gamma were measured in splenocyte culture supernatants of rSm14/IL-12 or rSm14 vaccinated mice and no IL-4 was detected. In an attempt to determine the role of IFN-gamma and TNF-alpha in IL-12 induced immunity, IFN-gamma and TNFR-p55 knockout mice were immunized with rSm14/IL-12 and no protection was achieved. Therefore, protection induced by rSm14/IL-12 was shown to be dependent on endogenous IFN-gamma and TNF-alpha. Although, rSm14 immunization induced partial protection, reduction of hepatic granuloma area was only observed when IL-12 was co-administered.

Route of jejunal mucosa absorption of trypsin demonstrated by immunofluorescence.

Previous studies have demonstrated the absorption of porcine trypsin in isolated jejunal loops from male Wistar rats by open-loop perfusion. The possible routes of absorption were examined in the study reported here. Trypsin (0.5 mg/ml) was dissolved in tyrode solution and perfused at a rate of 0.5 ml/min, at 37 degrees C, for 40 min. Using immunoperoxidase and immunofluorescence techniques, strong reactivity towards anti-TLCK-trypsin antibody was demonstrated through out the enterocyte cytosol. The present data indicate that trypsin was absorbed by enterocytes, probably through a transcellular route.