Fat is an essential component of meat which contributes to its sensory characteristics. Therefore, producing cultivated fat is essential to replicate the texture, flavor, and juiciness of conventional meat. One of the challenges in obtaining cultivated fat is that once adipocytes reach differentiation in culture, they tend to float. In this study, we tested whether immortalized pre-adipocytes could be viable, grow, and differentiate when cultivated onto a fibrous scaffold produced by the electrospun of cellulose acetate. Our results demonstrated that the cells attach, proliferate, colonize, and differentiate into mature adipocytes in the three-dimensional fibrous structure during the culture period. Moreover, when layers of the scaffold containing differentiated cells were stacked, it acquired a characteristic similar to conventional animal fat. Therefore, this research suggests that fibrous scaffolds produced using cellulose acetate are a promising substrate for producing cultivated fat.

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.

Introduction: Mineralized bovine bone grafts have been widely used as substitutes for bone losses in dental clinics. Studies have shown that exposing the organic components of the bone matrix at surgical sites accelerates bone deposition. Objective: The present study evaluated the bone repair progress of an intraoral bone defect in rats after grafting with mineralized (MBB) and demineralized bovine bone (DBB). Materials and Methods: An intraoral bone defect was created after extraction of the right maxillary first molar, drilling the area of the alveoli of the four distal roots using a diamond tip. The defect was filled with the MBB or DBB graft. Grafting effects were evaluated after 1, 7, 14, 21, and 49 days, using radiographic and histological data. Results: After 14 days, all groups showed full mucosa epithelialization at the surgical site. Radiographic data showed an improvement in bone deposition in defects grafted with the organic matrix. This data was confirmed by histological analysis. A higher level of bone maturation of the neoformed trabeculae and a faster reabsorption rate were also observed to be a feature of the DBB graft. Conclusion: The present in vivo data revealed that the DBB graft may represent an alternative to mineralized biomaterials.

Synthetic polymers scaffolds often need to be coated with extracellular matrix (ECM) proteins to improve cell adhesion. For cultivated meat applications, coating should be avoided since it is necessary to eliminate expensive and animal-derived components. As cellulose acetate nanofibers is a low-cost cellulose-derived material, that induces cell adhesion and proliferation, we investigated its use associated with a bioactive annatto extract, a food-dye and potential meat preservative, as scaffolds for cultivated meat. Here, the bioactive electrospun nanofibers were evaluated through morphological, mechanical and biological characterizations. The results revealed that the scaffolds were porous with no specific alignment and average fiber diameter of 420±212 nm. Molecular analyzes revealed that in contrast to cellulose acetate scaffold, annatto-loaded cellulose acetate scaffold favor a proliferative state of C2C12 mouse skeletal myoblasts. SEM microscopy images suggests that the nanofiber substrates can sustain long-term culture of the cells, up to 28 days. 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 a scaffold for cultivated meat and muscle tissue engineering.

Our work presents a comparative study of morphological characteristics and the osteogenic potential of MC3T3-E1 cells on different modified surfaces of titanium: nanostructured TiO2 with 20 and 100 nm nanotube diameter, and sandblasting and acid etching, commercially known as SLA. Nanostructured TiO2 surface was prepared by anodizing of titanium plates, while SLA surface was provided by commercial supplier. Surfaces were characterized by SEM, EDS,AFM, and water contact angle measurements. In order to evaluate cell response, in vitro tests of MTT, alkaline phosphatase and staining with alizarin red were performed. From the results of in vitro tests, 100 nm nanotubular surface showed lower levels of cell mineralization, differentiation and adhesion. In general, 20 nm TiO2 nanotubular and SLA surfaces promoted similar response from osteoblasts. As a result, 20 nm nanotubular surface proved to be a possible alternative to SLA surface with potential for use in oral implantology market.

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.

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.

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.

Background: Collagen has been used as the material of choice for absorbable biomaterials. The cross-link of collagen membranes, used in Guided Tissue Regeneration (GTR), increases their bioabsorption time and structural stability, however, membranes without cross-linking are excellent hemostatic agents, due to its porosity and enable greater cell colonization. Aim/Hypothesis: In a healing process, collagen matrices have conductive properties for repair. The objective of this study was to evaluate the differences in the topography of different scaffolds commercially used in dental surgical practice. Materials and Methods: This study evaluated, using scanning electron microscopy (SEM), the surface topography of five collagen membranes, commercially available: Bio-Gide® (BG), Surgidry Filme® (SF), Surgidry Esponja® (SE), Lyostypt® (LY) and Jason® (JS). Results: The results showed considerable differences between the biomaterials, with and without cross-linking, varying in the design of its topography and pores according to the treatment of collagen. Conclusions and Clinical Implications: We conclude that, despite being directed to the use of standardized regenerative surgical techniques, the structure and topography of these biomaterials differ considerably. As perspectives, further studies should be conducted, involving cell culture on these collagenous scaffolds, to better understand the influence of these structural differences on cellular behaviour. Keywords: Electron Scanning Microscopy, Collagen, Biomaterial, Scaffold

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.

BackgroundThe 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.ResultsThe D. albiventris transcriptome was obtained from whole neonates harvested at birth (P0), at five days of age (P5) and at ten 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, 2,077 were found to be DE between P0 and P5, 13,780 between P0 and P10, and 1,453 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.ConclusionsThe 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.

Background Absorbable collagen biomaterials have biocompatibility and ability to promote wound repair, in addition to their natural biological potential, which enables a favorable environment for regeneration, and therefore, their use in the technique is highly preferred among clinical professionals. However, the degree of rapid reabsorption of collagen, as well as the associated specific inflammatory process, is a constant concern for clinicians and researchers. Aim/Hypothesis Histological evaluation of the behavior of two absorbable biomaterials composed of type I collagen, with different cross-links grafted to the dorsal muscular tissue of rats. Material and Methods The sample consisted of 13 animals, Holtzman rats (male 250 g), divided into 5 experimental groups, with sacrifice periods of 2 h (n = 1), 1, 5, 10 and 15 days (n = 3). 5 mm2 of the biomaterial, cross-link (CL) and no cross-link (NCL) collagen membrane, was inserted in the dorsal muscular tissue of the animals, being used the Masson's trichrome dye for the histological analysis. Results NCL showed integrity at 5 days and complete degradation at 10 days, when the remission of the inflammatory process detected during its degradation was observed, without presenting foreign body reactions. The CL membrane presented an inflammatory process with intermittent peaks of low and high intensity, and also with no foreign body reaction, but with minimal degradation, maintaining its semi-intact structure isolated by fibrous connective tissue at 15 days. Conclusion and Clinical Implications In the evaluated period, it was concluded that the NCL degradation was better biocompatible, with the CL being more stable. The stability over a longer period is desirable in the GTR technique. Further studies for CL over a longer period of time are needed to evaluate its complete degradation and temporal biocompatibility.

Background Cell culture is an important tool in medical 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 human fibroblasts in vitro, in monolayer or on three-dimensional scaffolds, for subsequent transplants in vivo opens important perspectives for the periodontal regeneration. Aim/Hypothesis The objective is present a method to obtaining and culture of the viable human gingival fibroblasts for in vitro research Material and Methods 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 sub classified morphologically. Results The results showed a growth pattern in both bottles, but with a larger number in bottles of 75 cm2. Cells with fibroblastic morphology were sub classified into reticular and fusiform, being predominant those with fusiform morphology. Conclusion and Clinical Implications Culture of the 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.

Background Fresh and frozen allogeneic bone grafts, from Bone Bank Allograft, are configured as alternative resources to biomaterials and autogenous grafts. Aim/Hypothesis The present study evaluated histologically five samples of fresh, treated and frozen human bone tissue obtained from the UNIOSS® bone bank (Brazil, Campinas-SP). Material and Methods The cortico-medullary samples, from different donors, were legally obtained by an accredited professional and qualified in Implantology. The fragments were removed prior to the surgical procedure of reconstructive bone grafting. The samples were fixed in 10% formalin, demineralized in 10% EDTA, paraffin embedded and stained in HE, Masson and Gomori trichrome. Results Microscopy revealed a characteristic trabecular aspect. Cell remains as well as intercellular matrix residues were observed in the medullary spaces. In some samples cartilaginous tissue and chondrocyte remains were observed. Conclusion and Clinical Implications We concluded that the morphology of the collagen matrix of the bones from bone bank allograft may contribute to cell adhesion for bone repair. However, the eventual presence of hyaline cartilage and cell resources, in these bone fragments, may influence the repair process. These findings justify, at least partially, the controversy regarding the clinical results and the lack of consensus of the professionals regarding this type of therapy for bone reconstruction.

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 cm(2) 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 mm(2) (T25) and 75 mm(2) (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 cm(2). 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.

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.

The treatment of intrabuccal bone defects and the search for new agents to optimize regeneration procedures are extremely valuable. The present experimental study aimed to evaluate the effects of orally administered Strontium Ranelate (SrR), in the repair of intrabuccal bone defects in rats. Twenty Lewis rats, divided in 4 groups (2 control and 2 test groups) were used and evaluated at 14 and 42 days. Standardized bone defects in the distal alveolar region of the first superior molar were created in all animals. Test groups received a daily dose of SrR (625 mg/kg), and control groups received a placebo. Bone neoformation within the defects were evaluated through histological and morphometric analysis. At 14 days, histological analysis revealed similar healing patterns between groups. However, at 42 days, the test group presented healing patterns with better tissue organization, compatible with slightly advanced bone maturation. At 14 days, morphometric analysis revealed a higher rate of bone deposition in the test group when compared to the control group (P<0.05). At 42 days, no significant differences between groups were observed in relation to morphometric parameters. SrR seemed to accelerate the process of bone neoformation.

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. (C) 2016 Elsevier GmbH. All rights reserved.

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.

During pregnancy the viviparous vertebrates develop a complex system of nutritional membranes surrounding the fetus. In place of the union or apposition of the fetal membranes with the uterine lining is formed the placenta. The placental types may be categorized into several complementary levels that reflect placental characteristics, being the swine placenta classified as chorioallantoic, diffuse, pleated, epitheliochorial and cross to counter-current. Structures, as the yolk sac, have function even before the appearance of the chorioallantoic placenta. The areola is also an accessory structure of the placenta, which may be found in ungulates. The extraembryonic membranes are linked intimately in the placentation, and these are important in swine early pregnancy, since the definitive placenta starts developing by the 18th day of gestation. Modifications in the swine placenta, like the presence of areolas, might have arisen as domestic species adaptions in order to supply nourishing needs during the development of concept.