Bioactive cellulose acetate nanofiber loaded with annatto support skeletal muscle cell attachment and proliferation.

Electrospinning emerged as a promising technique to produce scaffolds for cultivated meat in function of its simplicity, versatility, cost-effectiveness, and scalability. Cellulose acetate (CA) is a biocompatible and low-cost material that support cell adhesion and proliferation. Here we investigated CA nanofibers, associated or not with a bioactive annatto extract (CA@A), a food-dye, as potential scaffolds for cultivated meat and muscle tissue engineering. The obtained CA nanofibers were evaluated concerning its physicochemical, morphological, mechanical and biological traits. UV-vis spectroscopy and contact angle measurements confirmed the annatto extract incorporation into the CA nanofibers and the surface wettability of both scaffolds, respectively. SEM images revealed that the scaffolds are porous, containing fibers with no specific alignment. Compared with the pure CA nanofibers, CA@A nanofibers showed increased fiber diameter (420 ± 212 nm vs. 284 ± 130 nm). Mechanical properties revealed that the annatto extract induces a reduction of the stiffness of the scaffold. Molecular analyses revealed that while CA scaffold favored C2C12 myoblast differentiation, the annatto-loaded CA scaffold favored a proliferative state of these cells. These results suggest that the combination of cellulose acetate fibers loaded with annatto extract may be an interesting economical alternative for support long-term muscle cells culture with potential application as scaffold for cultivated meat and muscle tissue engineering.

Influence of ethylenediaminetetraacetic acid irrigation on the regenerative endodontic procedure in an immature rat molar model.

AIM: To analyse the influence of ethylenediaminetetraacetic acid (EDTA) on the repair process in immature rat molars after a regenerative endodontic procedure (REP). METHODOLOGY: The lower first molars of 12 4-week-old Wistar rats underwent pulpectomy in the mesial root and were divided into the following groups: sodium hypochlorite (NaOCl; n = 6) - the mesial canals were irrigated with 2.5% NaOCl for 5 min, and NaOCl-EDTA (n = 6) - the canals were irrigated with 2.5% NaOCl, followed by 17% EDTA for 5 min each. After evoking bleeding using a size 10 K-file, the cavities were sealed. Three molars on the untreated side were randomly used as control (control-15 d; n = 3), and three molars from the other three rats untreated were used as immediate control (n = 3). After 15 days (NaOCl, NaOCl-EDTA and control-15 d groups) or immediately (control-immediate), the animals were euthanized, and the teeth were subjected to histologic evaluation of tissue regeneration and presence of collagen fibres. Mann-Whitney U-test was used (p < .05). RESULTS: The experimental groups had newly formed cementum-like tissue and increased root length and thickness. Half of the specimens in NaOCl-EDTA group showed apical foramen closure, whilst the NaOCl group had partial apical closure. The experimental groups showed inflammatory infiltrate extending mainly to the medium third of the root canal. These parameters were similar between experimental groups (p > .05). Newly formed connective tissue in the pulp space was significantly higher in the NaOCl-EDTA group than in NaOCl group (p < .05). Regarding the collagen fibres, the NaOCl-EDTA group had more collagen fibres in the root tip, but there was no significant difference compared to NaOCl group, and both groups showed greater amount of immature fibres in this area; in the centre of the apical third of root canal, there was equivalence between mature and immature fibres from both groups (p > .05). CONCLUSIONS: Ethylenediaminetetraacetic acid irrigation improved newly formed intracanal connective tissue after REP in immature molars of rats; however, EDTA did not influence cementum-like tissue formation, apical closure, inflammatory infiltrate and maturation of collagen fibres.

Transcriptomic characterization of the molecular mechanisms induced by RGMa during skeletal muscle nuclei accretion and hypertrophy.

BACKGROUND: The repulsive guidance molecule a (RGMa) is a GPI-anchor axon guidance molecule first found to play important roles during neuronal development. RGMa expression patterns and signaling pathways via Neogenin and/or as BMP coreceptors indicated that this axon guidance molecule could also be working in other processes and diseases, including during myogenesis. Previous works from our research group have consistently shown that RGMa is expressed in skeletal muscle cells and that its overexpression induces both nuclei accretion and hypertrophy in muscle cell lineages. However, the cellular components and molecular mechanisms induced by RGMa during the differentiation of skeletal muscle cells are poorly understood. In this work, the global transcription expression profile of RGMa-treated C2C12 myoblasts during the differentiation stage, obtained by RNA-seq, were reported. RESULTS: RGMa treatment could modulate the expression pattern of 2,195 transcripts in C2C12 skeletal muscle, with 943 upregulated and 1,252 downregulated. Among them, RGMa interfered with the expression of several RNA types, including categories related to the regulation of RNA splicing and degradation. The data also suggested that nuclei accretion induced by RGMa could be due to their capacity to induce the expression of transcripts related to 'adherens junsctions' and 'extracellular-cell adhesion', while RGMa effects on muscle hypertrophy might be due to (i) the activation of the mTOR-Akt independent axis and (ii) the regulation of the expression of transcripts related to atrophy. Finally, RGMa induced the expression of transcripts that encode skeletal muscle structural proteins, especially from sarcolemma and also those associated with striated muscle cell differentiation. CONCLUSIONS: These results provide comprehensive knowledge of skeletal muscle transcript changes and pathways in response to RGMa.

Chitosan-based biomaterial and hyaluronic acid on the repair of intrabuccal bone defects in rats.

Aims: This experimental study aimed to evaluate the effects of a three-dimensional matrix of chitosan-gelatin (CG) associated with 1% hyaluronic acid (HA) on gingival healing and repairing of intrabuccal bone defects in rats. Materials and methods: Standardized bone defects were created in the region of the upper 1st molars of rats. Study groups were created according to bone defects (n=6/group) treatment: Control group (CO); blood clot; HA group; CG group, and HA+CG group. After 7 and 21 days, the animals were sacrificed for histological and histomorphometric analysis. Bone formation was quantified as the percentage of newly synthesized collagen, visualized by Gomori's trichromic. Clinical/macroscopic evaluation was based on predetermined scores of gingival healing. Results: Treatment with HA improved gingival healing at day 7, but no statistical differences were found among groups at day 21. The morphometric analysis demonstrated better results after the treatment of bone defects with both HA and CG on day 21. The three-dimensional structure of CG prevented the invasion of epithelial tissue into the defect, preserving its original volume. Conclusions: Isolated use of a chitosan-gelatin osteoconductive matrix promoted greater bone deposition and preserved the volume of the surgical site, irrespective of the presence of hyaluronic acid.

RGMa can induce skeletal muscle cell hyperplasia via association with neogenin signalling pathway.

Although originally discovered inducing important biological functions in the nervous system, repulsive guidance molecule a (RGMa) has now been identified as a player in many other processes and diseases, including in myogenesis. RGMa is known to be expressed in skeletal muscle cells, from somites to the adult. Functional in vitro studies have revealed that RGMa overexpression could promote skeletal muscle cell hypertrophy and hyperplasia, as higher efficiency in cell fusion was observed. Here, we extend the potential role of RGMa during C2C12 cell differentiation in vitro. Our results showed that RGMa administrated as a recombinant protein during late stages of C2C12 myogenic differentiation could induce myoblast cell fusion and the downregulation of different myogenic markers, while its administration at early stages induced the expression of myogenic markers with no detectable morphological effects. We also found that RGMa effects on skeletal muscle hyperplasia are performed via neogenin receptor, possibly as part of a complex with other proteins. Additionally, we observed that RGMa-neogenin is not playing a role as an inhibitor of the BMP signalling in skeletal muscle cells. This work contributes to placing RGMa as a component of the mechanisms that determine skeletal cell fusion via neogenin receptor.

In vitro analysis of the influence of mineralized and EDTA-demineralized allogenous bone on the viability and differentiation of osteoblasts and dental pulp stem cells.

Grafting based on both autogenous and allogenous human bone is widely used to replace areas of critical loss to induce bone regeneration. Allogenous bones have the advantage of unlimited availability from tissue banks. However, their integration into the remaining bone is limited because they lack osteoinduction and osteogenic properties. Here, we propose to induce the demineralization of the allografts to improve these properties by exposing the organic components. Allografts fragments were demineralized in 10% EDTA at pH 7.2 solution. The influence of the EDTA-DAB and MAB fragments was evaluated with respect to the adhesion, growth and differentiation of MC3'T3-E1 osteoblasts, primary osteoblasts and dental pulp stem cells (DPSC). Histomorphological analyses showed that EDTA-demineralized fragments (EDTA-DAB) maintained a bone architecture and porosity similar to those of the mineralized (MAB) samples. BMP4, osteopontin, and collagen III were also preserved. All the cell types adhered, grew and colonized both the MAB and EDTA-DAB biomaterials after 7, 14 and 21 days. However, the osteoblastic cell lines showed higher viability indexes when they were cultivated on the EDTA-DAB fragments, while the MAB fragments induced higher DPSC viability. The improved osteoinductive potential of the EDTA-DAB bone was confirmed by alkaline phosphatase activity and calcium deposition analyses. This work provides guidance for the choice of the most appropriate allograft to be used in tissue bioengineering and for the transport of specific cell lineages to the surgical site.

Collection and culture of human connective tissue cells from gingival explant technique for oral tissue bioengineering / Colección de células de tejido conectivo humano a partir de la técnica de explante gingival para bioingeniería de tejidos orales

SUMMARY: Cell culture is an important tool in medical, odontological and biological research laboratories, supporting cell therapies and tissue bioengineering strategies. Gingival fibroblasts present structural function, being able to modulate their metabolic capacity, which is reflected in the tissue morphology. The possibility of culturing fibroblasts in vitro, in monolayer or on three-dimensional scaffolds, for subsequent transplants in vivo opens important perspectives for the periodontal surgical clinic. The objective of the present article is to present a method of obtaining and cultivating viable human gingival fibroblasts for in vitro research. Explants derived from periodontal surgical discards were used, grown in 25 cm2 bottles to obtain a primary cell culture. After observing the proliferation and growth of the fibroblasts that interconnected and formed a monolayer network, involving the periphery of the explants, it was possible to remove the explants, to make the passage and the new subcultures were obtained in a ratio of 1:1. After 7 days, the amount of viable cells was analyzed in triplicate, using the Neubauer chamber technique, in cell culture bottles of 25 mm2 (T25) and 75 mm2 (T75). Fibroblasts were described and subclassified morphologically. The results showed a growth pattern in both bottles, but with a larger number in bottles of 75 cm2. Cells with fibroblastic morphology were subclassified into reticular and fusiform, being predominant those with fusiform morphology. In conclusion, culture of explant of human gingival connective tissue is a viable method for obtaining gingival connective tissue cells suitable for laboratory tests in cell culture, aiming at obtaining constructs for gingival tissue engineering.

RESUMEN: El cultivo celular es una herramienta importante en los laboratorios de investigación médica, odontológica y biológica, que apoyan las terapias celulares y las estrategias de bioingeniería de tejidos. Los fibroblastos gingivales presentan una función estructural, pudiendo modular su capacidad metabólica, que se refleja en la morfología tisular. La posibilidad de cultivar fibroblastos in vitro, en monocapa o en andamios tridimensionales, para trasplantes posteriores in vivo abre perspectivas importantes para la clínica de cirugía periodontal. El objetivo del presente artículo es presentar un método para obtener y cultivar fibroblastos gingivales humanos viables para investigación in vitro. Se utilizaron explantes derivados de los descartes quirúrgicos periodontales, crecidos en frascos de 25 cm2 para obtener un cultivo de células primarias. Después de observar la proliferación y el crecimiento de los fibroblastos que se interconectaron y formaron una red de monocapa, que involucraba la periferia de los explantes, fue posible eliminar los explantes, hacer el pasaje y los nuevos subcultivos se obtuvieron en una proporción de 1:1. Después de 7 días, la cantidad de células viables se analizó por triplicado, utilizando la técnica de cámara de Neubauer, en botellas de cultivo celular de 25 mm2 (T25) y 75 mm2 (T75). Los fibroblastos fueron descritos y sub-clasificados morfológicamente. Los resultados mostraron un patrón de crecimiento en ambas botellas, pero con un número mayor en botellas de 75 cm2. Las células con morfología fibroblástica se subclasificaron en reticulares y fusiformes, predominando aquellas con morfología fusiforme. En conclusión, el cultivo de explante de tejido conectivo gingival humano es un método viable para obtener células de tejido conectivo gingival adecuadas para pruebas de laboratorio en cultivos celulares, con el objetivo de obtener construcciones para la ingeniería del tejido gingival.

Didelphis albiventris: an overview of unprecedented transcriptome sequencing of the white-eared opossum.

BACKGROUND: The white-eared opossum (Didelphis albiventris) is widely distributed throughout Brazil and South America. It has been used as an animal model for studying different scientific questions ranging from the restoration of degraded green areas to medical aspects of Chagas disease, leishmaniasis and resistance against snake venom. As a marsupial, D. albiventris can also contribute to the understanding of the molecular mechanisms that govern the different stages of organogenesis. Opossum joeys are born after only 13 days, and the final stages of organogenesis occur when the neonates are inside the pouch, depending on lactation. As neither the genome of this opossum species nor its transcriptome has been completely sequenced, the use of D. albiventris as an animal model is limited. In this work, we sequenced the D. albiventris transcriptome by RNA-seq to obtain the first catalogue of differentially expressed (DE) genes and gene ontology (GO) annotations during the neonatal stages of marsupial development. RESULTS: The D. albiventris transcriptome was obtained from whole neonates harvested at birth (P0), at 5 days of age (P5) and at 10 days of age (P10). The de novo assembly of these transcripts generated 85,338 transcripts. Approximately 30% of these transcripts could be mapped against the amino acid sequences of M. domestica, the evolutionarily closest relative of D. albiventris to be sequenced thus far. Among the expressed transcripts, 2077 were found to be DE between P0 and P5, 13,780 between P0 and P10, and 1453 between P5 and P10. The enriched GO terms were mainly related to the immune system, blood tissue development and differentiation, vision, hearing, digestion, the CNS and limb development. CONCLUSIONS: The elucidation of opossum transcriptomes provides an out-group for better understanding the distinct characteristics associated with the evolution of mammalian species. This study provides the first transcriptome sequences and catalogue of genes for a marsupial species at different neonatal stages, allowing the study of the mechanisms involved in organogenesis.

Effects of strontium ranelate treatment on osteoblasts cultivated onto scaffolds of trabeculae bovine bone.

Blocks of Bovine bone have shown promising results as implantable scaffolds to promote bone regeneration. Strontium ranelate (SrR) is both an antiresorptive and an anabolic drug that has been indicated for oral administration to treat osteoporosis. Few studies, however, have investigated the local effects of SrR and its use in association with biomaterials thus far. In this work, we investigated SrR effects in cultures of primary osteoblasts (PO, from Wistar rats calvaria) and immortalized osteoblasts (IO, from MC3T3-E1 cell line) cultivated as a monolayer or in association with scaffolds of bovine bone in mineralized (MBB) and demineralized (DBB) forms. The optimum dose to induce SrR effects on cell viability was established as 0.1 mM. Our results suggested that the local administration of SrR is biocompatible and non-cytotoxic. In addition, SrR appeared to accelerate primary osteoblast cell differentiation by enhancing alkaline phosphatase activity, the expression of osteogenic differentiation markers, the synthesis of the organic matrix, and a decrease of Ca2+ ions in mineralized nodules. DBB was found to be a better scaffold material to promote PO and IO cell proliferation. Exposing the proteins of the demineralized bone matrix might improve scaffold osteoconductive properties. Our results indicated the importance of further investigation of the administration of SrR at sites of bone repair. The association of SrR and bone grafts suggests the possibility of using SrR as a co-adjuvant for bone tissue bioengineering and in bone regeneration therapies.

Perfil de prescrição de antimicrobianos nas unidades básicas de saúde conveniadas com a universidade de Itaúna/MG / Antimicrobial prescription profile in the basic health units agreed with the university of Itaúna/MG

INTRODUÇÃO: O uso abusivo de antibacterianos está intimamente relacionado ao desenvolvimento de resistência bacteriana, considerada, atualmente, um problema de saúde pública mundial. Segundo a Organização Mundial de Saúde, em 2001, mais da metade das prescrições de antimicrobianos foram inapropriadas e dois terços de sua utilização foram feitas sem prescrição médica. Assim, o uso racional desse medicamento requer uma seleção criteriosa e bom senso clínico do prescritor. OBJETIVOS: Analisar o perfil de prescrição de antimicrobianos nas Unidades Básicas de Saúde da Família (UBSF) do município de Itaúna-MG, conveniados com a Universidade de Itaúna (UIT) e contribuir para que futuras intervenções possam ser conduzidas promovendo do uso racional dos antimicrobianos na atenção primária. MÉTODOS: Estudo transversal de prontuários médicos de pacientes atendidos nas UBSF de Itaúna/MG conveniadas com a UIT, realizado entre março de 2013 e março de 2014, para os quais foram prescritos antibióticos. RESULTADOS: A classe de antimicrobianos mais prescrita foi a das penicilinas seguido pelas quinolonas e macrolídeos. Quanto à duração do tratamento, o período de cinco a dez dias foi observado na maioria das prescrições. As principais indicações clínicas foram infecção das vias aéreas superiores não especificadas, amigdalite, otite, sinusite, infecção do trato urinário entre outros. A solicitação de culturas foi realizada em apenas 5,5% dos casos. CONCLUSÃO: A análise do perfil das prescrições revelou a necessidade de reciclagem da equipe e adoção de protocolos clínicos. Tais medidas permitirão a uniformização das condutas, otimizando as prescrições e reduzindo o risco do uso inapropriado de antimicrobianos. (AU)

Introduction: The overuse of antibacterials is closely related to the development of bacterial resistance, currently considered a problem of public health worldwide. According to the World Health Organization, in 2001 over half of the antimicrobial prescriptions were inappropriate and two thirds of its use were made without prescription. Thus, the rational use of this drug requires careful selection and clinical judgment of the prescriber. Objectives: To analyze the antimicrobial prescription profile in the Basic Health Units of family of Itaúna-MG, that have agreements with the University of Itaúna (UIT) and contribute to future interventions that can be conducted to promote the rational use of antimicrobials in primary care. Methods: Cross-sectional study of medical records of patients treated in Basic Health Units of family Itaúna / MG with agreement with the UIT, held between March 2013 and March 2014, for which antibiotics were prescribed. Results: The most prescribed class of antimicrobials was the penicillins followed by quinolones and macrolides. About the duration of treatment, five to ten days was observed in the majority of prescriptions. The main clinical indications were infection of the upper airways unspecified, tonsillitis, otitis, sinusitis, urinary tract infection, among others. The request of cultures was performed in only 5.5% of cases. Conclusion: The analysis of the profile of prescriptions revealed the need for retraining of staff and adoption of clinical protocols. These measures will enable to uniform the procedures, optimizing the regulations and reducing the risk of inappropriate use of antimicrobials. (AU)

FGF2, FGF3 and FGF4 expression pattern during molars odontogenesis in Didelphis albiventris.

Odontogenesis is guided by a complex signaling cascade in which several molecules, including FGF2-4, ensure all dental groups development and specificity. Most of the data on odontogenesis derives from rodents, which does not have all dental groups. Didelphis albiventris is an opossum with the closest dentition to humans, and the main odontogenesis stages occur when the newborns are in the pouch. In this study, D. albiventris postnatals were used to characterize the main stages of their molars development; and also to establish FGF2, FGF3 and FGF4 expression pattern. D. albiventris postnatals were processed for histological and indirect immunoperoxidase analysis of the tooth germs. Our results revealed similar dental structures between D. albiventris and mice. However, FGF2, FGF3 and FGF4 expression patterns were observed in a larger number of dental structures, suggesting broader functions for these molecules in this opossum species. The knowledge of the signaling that determinates odontogenesis in an animal model with complete dentition may contribute to the development of therapies for the replacement of lost teeth in humans. This study may also contribute to the implementation of D. albiventris as model for Developmental Biology studies.

Rat dental pulp stem cells: isolation and phenotypic characterization method aiming bone tissue bioengineering

ABSTRACT: Dental pulp stem cells (DPSC) have been showing a considerable potential for regenerative medicine. Pulps were collected from lower incisors (n=2) through direct access of the tooth pulp chamber. The isolated cells were cultured in alfa-MEM 10% FBS, in standard culture conditions. At the third passage, DPSC were characterized by flow cytometry (MHCI, CD54, CD73, CD90, CD45, CD11 and CD34); RT-PCR for Nanog gene; and their differentiation capacity in osteogenic, adipogenic and chondrogenic cell lines. Isolated cells exhibited adhesion capacity to plastic; fusiform morphology, and 80% confluence reached in approximately 3 days. These cells have also revealed positive expression for CD54, CD73 and CD90 markers; and negative expression for CD11, CD34 and CD45. Nanog expression was detected by RT-PCR, expected for a mesenchymal stem cell profile. DPSC chondrogenic differentiation was confirmed by positive staining in Alcian Blue; lipidic droplets stained with oil red confirmed their capacity to differentiate in adipogenic fate; while mineralized beads, stained with alizarin red, confirmed their differentiation in osteogenic phenotype. These results indicate the viability of the isolation and expansion of rat DPSC following this method, and osteogenic differentiation potential opens new perspectives for in vivo studies and the use of these cells in cellular therapies and tissue bioengineering, aiming bone repair.

Repulsive Guidance Molecules a, b and c Are Skeletal Muscle Proteins, and Repulsive Guidance Molecule a Promotes Cellular Hypertrophy and Is Necessary for Myotube Fusion.

Repulsive guidance molecules (RGMs) compose a family of glycosylphosphatidylinositol (GPI)-anchored axon guidance molecules and perform several functions during neural development. New evidence has suggested possible new roles for these axon guidance molecules during skeletal muscle development, which has not been investigated thus far. In the present study, we show that RGMa, RGMb and RGMc are all induced during skeletal muscle differentiation in vitro. Immunolocalization performed on adult skeletal muscle cells revealed that RGMa, RGMb and RGMc are sarcolemmal proteins. Additionally, RGMa was found to be a sarcoplasmic protein with a surprisingly striated pattern. RGMa colocalization with known sarcoplasmic proteins suggested that this axon guidance molecule is a skeletal muscle sarcoplasmic protein. Western blot analysis revealed two RGMa fragments of 60 and 33 kDa, respectively, in adult skeletal muscle samples. RGMa phenotypes in skeletal muscle cells (C2C12 and primary myoblasts) were also investigated. RGMa overexpression produced hypertrophic cells, whereas RGMa knockdown resulted in the opposite phenotype. RGMa knockdown also blocked myotube formation in both skeletal muscle cell types. Our results are the first to show an axon guidance molecule as a skeletal muscle sarcoplasmic protein and to include RGMa in a system that regulates skeletal muscle cell size and differentiation.

Effect of PDGF-BB, IGF-I growth factors and their combination carried by liposomes in tooth socket healing.

This work evaluated the bone-forming potential of the platelet-derived growth factor isoform BB (PDGF-BB), insulin-like growth factor I (IGF-I), and mixed PDGF-BB/IGF-I delivered in liposomes compared with phosphate buffered saline (PBS), in the healing process of rat tooth sockets. One hundred and twelve Wistar rats were randomized into 7 groups of 16 animals each and were evaluated at 3, 7, 14 and 21 days after extraction of the maxillary second molars. The left sockets were treated with PBS (P), empty liposome (L), IGF-I in PBS (IP), IGF-I in liposome (IL), PDGF-BB in PBS (PDP), PDGF-BB in liposome (PDL) and both growth factors (GFs) together within liposomes (PDIL). The right sockets were filled with blood clot (BC). Histological and histomorphometric analyses were used to evaluate the formation of new bone and blood vessels. Immunohistochemistry was performed to evaluate the expression of osteocalcin and vascular endothelial growth factor (VEGF) during bone repair. Data were tested statistically using a Tukey's test according to a Dunn's analysis and Mann-Whitney U test followed by Kruskal-Wallis one-way analysis. Results were considered significant when p<0.05. A significantly higher percentage of bone trabeculae and a higher number of blood vessels were observed in the IL, PDL and PDIL groups (p<0.05). However, these GF-liposome groups had statistically similar results. Immunohistochemical assays first detected osteocalcin and VEGF expression at 3 days followed by a peak at 7 days. Lower immunoreactivity levels were observed in the BC, L, P, IP and PDP groups compared with the IL, PDL and PDIL groups (p<0.05). The results suggest that GFs carried by liposomes, either in isolated or mixed forms, enhanced the healing process in rat tooth sockets. The differential expression of the osteogenic markers VEGF and osteocalcin in the early phases of bone healing support these findings.

Effect of PDGF-BB, IGF-I Growth Factors and their Combination Carried by Liposomes in Tooth Socket Healing

This work evaluated the bone-forming potential of the platelet-derived growth factor isoform BB (PDGF-BB), insulin-like growth factor I (IGF-I), and mixed PDGF-BB/IGF-I delivered in liposomes compared with phosphate buffered saline (PBS), in the healing process of rat tooth sockets. One hundred and twelve Wistar rats were randomized into 7 groups of 16 animals each and were evaluated at 3, 7, 14 and 21 days after extraction of the maxillary second molars. The left sockets were treated with PBS (P), empty liposome (L), IGF-I in PBS (IP), IGF-I in liposome (IL), PDGF-BB in PBS (PDP), PDGF-BB in liposome (PDL) and both growth factors (GFs) together within liposomes (PDIL). The right sockets were filled with blood clot (BC). Histological and histomorphometric analyses were used to evaluate the formation of new bone and blood vessels. Immunohistochemistry was performed to evaluate the expression of osteocalcin and vascular endothelial growth factor (VEGF) during bone repair. Data were tested statistically using a Tukey's test according to a Dunn's analysis and Mann-Whitney U test followed by Kruskal-Wallis one-way analysis. Results were considered significant when p<0.05. A significantly higher percentage of bone trabeculae and a higher number of blood vessels were observed in the IL, PDL and PDIL groups (p<0.05). However, these GF-liposome groups had statistically similar results. Immunohistochemical assays first detected osteocalcin and VEGF expression at 3 days followed by a peak at 7 days. Lower immunoreactivity levels were observed in the BC, L, P, IP and PDP groups compared with the IL, PDL and PDIL groups (p<0.05). The results suggest that GFs carried by liposomes, either in isolated or mixed forms, enhanced the healing process in rat tooth sockets. The differential expression of the osteogenic markers VEGF and osteocalcin in the early phases of bone healing support these findings.

Este trabalho avaliou o potencial de formação óssea do fator de crescimento derivado de plaquetas na isoforma BB (PDGF-BB), fator de crescimento semelhante à insulina I (IGF-I), e a mistura PDGF-BB/IGF-I administrada em lipossomas comparando com tampão fosfato salino (PBS), no processo de cicatrização de alvéolos dentários de ratos. Cento e doze ratos Wistar foram distribuídos aleatoriamente em 7 grupos de 16 animais cada e foram avaliados aos 3, 7, 14 e 21 dias após a extração dos segundos molares maxilares. Os alvéolos esquerdos foram tratados com PBS (P), lipossomas vazios (L), IGF-I em PBS (IP), IGF-I em lipossomas (IL), PDGF-BB em PBS (PDP), PDGF-BB em lipossomas (PDL) e ambos os fatores de crescimento (GFs) em associação dentro de lipossomas (PDIL). Os alvéolos direitos foram preenchidos com coágulo sanguíneo (BC). As análises histomorfométrica e histológica foram utilizadas para avaliar a formação de novo osso e vasos sanguíneos. Imunohistoquímica foi realizada para avaliar a expressão de osteocalcina e o fator de crescimento endotelial vascular (VEGF) durante o reparo ósseo. Os dados foram testados estatisticamente utilizando o teste de Tukey em acordo com análise de Dunn e o teste Mann-Whitney U seguido pela análise de um passo de Kruskal-Wallis. Os resultados foram considerados significantes quando p<0,05. Uma percentagem altamente significativa de osso trabecular e alto número de vasos sanguíneos foram observados nos grupos IL, PDL e PDIL (p<0,05). Todavia, esses grupos lipossoma-GF tiveram resultados similares estatisticamente. Ensaios de imunohistoquímica inicialmente detectaram a expressão de osteocalcina e VEGF aos 3 dias, seguida por um pico aos 7 dias. Niveis mais baixos de imunorreatividade foram observados em BC, L, P, PI e PDP quando comparados com os grupos IL, PDL e PDIL (p<0,05). Os resultados sugerem que GFs carreados por lipossomas, na forma isolada ou em combinação, aceleram o processo de cicatrização em alvéolos dentários de rato. A expressão diferencial dos marcadores osteogênicos VEGF e osteocalcina, nas fases iniciais de cicatrização óssea, confirma esses achados.

TGF-ß1 and BMP-4 carried by liposomes enhance the healing process in alveolar bone.

OBJECTIVE: In this work we evaluated the bone-forming potential of BMP4, TGFß1 and BMP4/TGFß1 mixed by performing histological and morphometric analysis. We also evaluated the immunolabelling of fibronectin (FN) and collagen type III (Col III), two determinant proteins for the early phase of bone repair. DESIGN: Histological, histomorphometric and immunohistochemistry analysis were used to evaluate new bone and blood vessels formation as well as fibronectin and collagen type III expression. 112 male Wistar rats weighing 250-300g had their maxillary second molar extracted. Sockets filled with blood clot (BC) or treated with L (empty liposome), P (PBS), BP (BMP-4 in PBS) and TP (TGF-ß1 in PBS), as well as with BL (BMP-4 in liposome) and TL (TGF-ß1 in liposome) administered isolated or in association (BTL) were obtained. The animals were sacrificed at 3, 7, 14 and 21 days after surgery. RESULTS: An increased percentage of bone trabeculae, and a higher number of blood vessels were observed in groups BL or TL administered isolated or in association when compared to groups BC, L, P, BP and TP. Fibronectin and collagen type III analysis revealed enhanced expression firstly detected at 3 days followed by a peak at 7 days. Lower levels of immunoreactivity were observed in the sockets filled with blood clot, and treated with L, P, BP and TP when compared with sockets from groups BL, TL and BTL. CONCLUSION: The present study indicates growth factors carried by liposomes, either in isolated or associated forms, as successful enhancers of the healing process in rat tooth sockets. We also conclude that the expression of fibronectin and collagen type III increases during the early phases of bone repair.

Subclinical failures of direct pulp capping of human teeth by using a dentin bonding system.

INTRODUCTION: The goal of this study was to examine the adhesive interface of pulp tissue to investigate subclinical failures after direct pulp capping (DPC) of human teeth by using a dentin adhesive system. METHODS: The pulps of 12 caries-free first premolars scheduled for extraction for orthodontic reasons were exposed and capped with the Single Bond adhesive system. The adhesive technique was carefully performed to ensure complete coverage of the exposed area and a satisfactory clinical aspect. After 1 (n = 6) and 30 days (n = 6), the teeth were extracted for evaluation of the adhesive interface under light microscopy and scanning electron microscopy. Brown-Brenn staining was used to detect bacteria. RESULTS: The clinical aspect of direct pulp capping during the operation was satisfactory, and all patients were asymptomatic in the postoperative phase. Brown-Brenn staining revealed no bacterial microleakage at both time points. A hybrid layer was seen on all walls but decreased gradually toward the area of pulp exposure. In contrast to clinical data, light microscopy and scanning electron microscopy revealed important subclinical bond failures near the area of exposed pulp. Some frequent findings were gaps between the restoration and the dentin substrate; unpolymerized monomers under the adhesive layer; interface breaks with blood extravasation between the layers of the adhesive system; rupture of the odontoblast layer; and multinucleated giant cells close to the bonding agent. CONCLUSIONS: The Single Bond adhesive system should not be used for direct pulp capping of human teeth because subclinical adhesive failures invariably occur at its interface with the pulp tissue.

Influence of hyperbaric oxygen therapy on peri-implant bone healing in rats with alloxan-induced diabetes.

AIM: The influence of hyperbaric oxygen therapy (HBOT) on peri-implant bone healing in rats with alloxan-induced type-1 diabetes was studied. MATERIALS AND METHODS: Forty-eight male rats were randomly divided into six groups: (1) healthy rats (HR) that received no HBOT; (2) HR that underwent 10 sessions of HBOT before implant installation; (3) HR that underwent 10 sessions of HBOT after implant installation; (4) rats with induced diabetes (DR) without HBOT; (5) DR that underwent 10 sessions of HBOT before implant installation; (6) DR that underwent 10 sessions of HBOT after implant installation. A screw-shaped titanium implant was inserted into the femur. The animals were killed 28 days after implantation. The percentage of bone-to-implant contact (BIC) within the implant threads was evaluated. RESULTS: Lower BIC was observed in DR (35.35 ± 18.04) compared with the HR (69.07 ± 09.01) (p = 0.001). However, with HBOT, either before or after implantation, BIC was increased in DR. HBOT before implantation was p = 0.03; HBOT after implantation was p = 0.08. This increase reversed the negative effect of diabetes; therefore, the differences between DR and HR were not significant with HBOT (p ≥ 0.21). CONCLUSION: HBOT, either before or after implantation, increased the BIC in DR to the level of HR.

Ultrastructural evaluation of the hybrid layer after cementation of fiber posts using adhesive systems with different curing modes.

This in vitro study evaluated the adhesive interface of intraradicular fiber glass posts and root dentin using scanning electron microscopy (SEM). Forty-eight single-rooted premolars were randomly divided into 6 groups consisting of chemical, dual, or light cured adhesive systems combined with either chemical or dual cure resin cements. Scanning electron microscopic analysis showed the best results for continuity, density and morphology of the hybrid layer and resin tags for the combination of a self-cure adhesive with self-cure cement resin, followed by a dual-cure adhesive with self-cure cement resin, and finally a light-cure adhesive with self-cure cement. For the dual-cure resin cement, the same relation may be observed. The apical third was the most critical region for evaluated the criteria for all combinations of materials (Kruskal-Wallis and Friedman tests; p<0.001). Generally, the simplification of steps in the adhesive system and the polymerization reaction of resin adhesives and cements produced a direct effect on the quality of the adhesive post/dentin substrate interface.

Ultrastructural evaluation of the hybrid layer after cementation of fiber posts using adhesive systems with different curing modes

This in vitro study evaluated the adhesive interface of intraradicular fiber glass posts and root dentin using scanning electron microscopy (SEM). Forty-eight single-rooted premolars were randomly divided into 6 groups consisting of chemical, dual, or light cured adhesive systems combined with either chemical or dual cure resin cements. Scanning electron microscopic analysis showed the best results for continuity, density and morphology of the hybrid layer and resin tags for the combination of a self-cure adhesive with self-cure cement resin, followed by a dual-cure adhesive with self-cure cement resin, and finally a light-cure adhesive with self-cure cement. For the dual-cure resin cement, the same relation may be observed. The apical third was the most critical region for evaluated the criteria for all combinations of materials (Kruskal-Wallis and Friedman tests; p<0.001). Generally, the simplification of steps in the adhesive system and the polymerization reaction of resin adhesives and cements produced a direct effect on the quality of the adhesive post/dentin substrate interface.

Este estudo in vitro avaliou as interfaces adesivas de pinos intra-radiculares de fibra de vidro e a dentina radicular utilizando microscópio eletrônico de varredura (MEV). Quarenta e oito pré-molares unirradiculares foram divididos ao acaso em seis grupos, compostos por sistemas adesivos de cura química, dual ou fotopolimerizável, associado com cimentos resinosos de polimerização química ou dual. As análises microscópicas mostraram a maior continuidade, densidade e morfologia da camada híbrida e prolongamentos resinosos para a associação entre adesivos e cimentos auto-polimerizáveis seguido pelo grupo do adesivo de dupla polimerização e cimento de resina auto-polimerizável, e finalmente pelo adesivo fotopolimerizável e cimento de resina auto-polimerizável . Para os cimentos resinosos de dupla polimerização a mesma relação pode ser observada. O terço apical mostrou ser o substrato mais crítico em relação aos critérios avaliados para todas as associações entre os materiais usados(testes de Kruskal-Wallis e Friedman p<0,001). De maneira geral, a simplificação dos passos do sistema adesivo e a reação de polimerização dos adesivos e cimentos resinosos produzem efeitos diretos na qualidade da interface adesivo pino/dentina.