Bioactive glass containing 90% SiO2 in hard tissue engineering: An in vitro and in vivo characterization study.

Bioactive glass has been proved to have many applications in bioengineering due to its bone regenerative properties. In this work, an innovative, highly resorbable bioactive glass containing 90% SiO2 (BG90) to be used as a bone substitute was developed. The BG90 was synthetized by the sol-gel process with the dry step at room temperature. The biomaterial showed in vitro and in vivo bioactivities even with silica content up to 90%. Moreover, the BG90 presented high porosity and surface area due to its homogenously interconnected porous network. In vitro, it was observed to have high cell viability and marked osteoblastic differentiation of rat bone marrow-derived cells when in contact with BG90 ion extracts. The BG90 transplantation into rat tibia defects was analysed at 1, 2, 3, 4, 7, and 10 weeks post-operatively and compared with the defects of negative (no graft) and positive (autogenous bone graft) controls. After 4 weeks of grafting, the BG90 was totally resorbed and induced higher bone formation than did the positive control. Bone morphogenetic protein 2 (BMP-2) expression at the grafting site peaked at 1 week and decreased similarly after 7 weeks for all groups. Only the BG90 group was still exhibiting BMP-2 expression in the last experimental time. Our data demonstrated that the BG90 could be an attractive candidate to provide useful approaches in hard-tissue bioengineering.

Distinct healing pattern of maxillary sinus augmentation using the vitroceramic Biosilicate®: Study in rabbits.

OBJECTIVES: To follow healing process of augmented maxillary sinus in rabbits analyzing the histological pattern of bone tissue formation, along with the osteogenic activity and vascularization using a bioactive vitroceramic in comparison to deproteinized bovine bone associated or not with autogenous bone graft. DESIGN: Forty five male adult New Zealand rabbits, 5 months of age, mean weight of 4 Kg, underwent bilateral sinus augmentation surgeries to be divided in five groups: G - (Control) particulate autogenous bone graft (AG), BO - deproteinized bovine bone, BO+G - deproteinized bovine bone + AG, BSi -vitroceramic, and BSi + G - vitroceramic +AG. After 15, 45 and 90 days, all animals were euthanized for specimen's removal to be analyzed under light microscopy, histomorphometry, and immunohistochemistry for Runx2 and VEGF labeling. RESULTS: G, BO and BO+G groups healed uneventfully, allowing the formation of mature remodeling bone at day 90, regarding the association of AG with the biomaterial. On the other hand, BSi and BSi + G groups showed an important cellular reaction and granulation/fibrous tissue formation from the first to the last period of observation. Runx-2 and VEGF immunolabeling were coherent with this result. However, histomorphometry did not reveal significant differences considering new bone formation. CONCLUSIONS: Reconstructed maxillary sinuses using Biosilicate® permitted satisfactory new bone formation in comparison to the deproteinized bovine bone and AG. However, the presence of granulation/fibrous tissue and inflammatory cells associated to the degrading biomaterial indicate that further studies should be careful performed considering the immunological aspect of this new biomaterial.

Origin of axonal proteins: Is the axon-schwann cell unit a functional syncytium?

The structural homeostasis is challenging for neurons, whose axons extend up to meters in large animals, and the axoplasmic mass reaches over a thousand times that of the cell body. Thus, the protein demand may overcome the capacity of the cell body to supply the right protein species, to the right place, in the right time. In this context, a body of evidence indicates that glial cells support the axonal maintenance and regenerative responses by diverse mechanisms of intercellular communication. We showed recently that Schwann cells (SC) transfer ribosomes to axons and also enhance regeneration by means of extracellular vesicles known as exosomes that contain mRNAs, miRNAs and proteins. These findings strongly suggest that the nucleus of the SC supports the machinery for protein synthesis of the axon and participates in the specification of the phenotype of the underlying axon. That the genetic programs of many nuclei modulate the axoplasm on a local basis is akin to a syncytium but at variance with it, the nuclei belong to satellite cells. We propose that the SC-axon unit is a functional syncytium. This intercellular organization opens a novel understanding of the nervous system and a new avenue of research into its physiology and disorders © 2016 Wiley Periodicals, Inc.

Serum amyloid A in the placenta and its role in trophoblast invasion.

The serum amyloid A (SAA) protein is known to function in the acute phase response and immunoregulation. Recently, SAA has been shown to be involved in cell proliferation, differentiation and migratory behavior in different cell types. Here, we evaluated whether exogenous SAA could influence trophoblast invasion and differentiation using both the trophoblast-like BeWo cell line and fully differentiated human extravillous trophoblast cells (EVT) isolated from term placentae. SAA stimulated BeWo cell invasion, as measured in Matrigel invasion assays, and induced metalloprotease mRNA expression and activity. Given that BeWo cells express Toll-like receptor 4 (TLR4), a known receptor for SAA, we examined the role of TLR4 in SAA-induced invasion using a TLR4 neutralizing antibody. We also tested whether SAA could affect markers of trophoblast syncytialization in BeWo cells. We observed that SAA decreased ßhCG secretion and did not influence trophoblast syncytialization. Using EVT cells isolated from human term basal plates, we confirmed that SAA at 1 and 10 µg/mL doubled EVT invasion in a TLR4-dependent manner, but at 20 µg/mL inhibited EVT cells invasiveness. In addition, we observed that SAA was expressed in both BeWo cells and human term placentae, specifically in the syncytiotrophoblast, decidual cells and EVT. In conclusion, SAA was identified as a molecule that functions in the placental microenvironment to regulate metalloprotease activity and trophoblast invasion, which are key processes in placentation and placental homeostasis.

Reação do tecido subcutâneo de rato à implantação do MTA branco e Ultrapex com e sem própolis / Reaction of connective tissues rats to implantation of white MTA and Ultrapex with or without propolis

O objetivo deste estudo foi comparar a reação do tecido subcutâneo de rato, frente a implantação de MTA branco-Angelus® e Ultrapex® com e sem própolis. Tubos de polietileno, preenchidos com MTA branco, MTA branco+própolis, Ultrap ex® e Ultrapex®+própolis foram implantados em tecido subcutâneo de 21 ratos Wistar (Rattus norvegicus). Os animais foram mortos aos 30, 45 e 60 dias. O tecido contendo o implante foi removido e processado histologicamente.Cortes seriados de 5μm foram corados pela hematoxilina e eosina em que se avaliou a intensidade do infiltrado inflamatório, característica da cápsula fibrosa e dispersão do material. A esses dados foi aplicado o teste do Qui-quadrado o qual se observou diferença estatisticamente significante entre os quatro materiais experimentais. O MTA branco+própolis foi o que apresentou uma menor reação tecidual, seguido do MTA branco, Ultrapex®+própolis e Ultrapex®. A adição da própolis melhorou o comportamento biológico dos materiais em teste

The aims of this study was to compare the reaction of white MTA-Angelus® and Ultrapex® with and without propolis. Polyethylene tubes filled with white MTA, white MTA+propolis, Ultrapex, Ultrapex®+propolis were implanted in subcutaneous tissue of 21 Wistar rats (Rattus norvegicus). The animals were killed at 30, 45 and 60 days. The tissue containing the implant was removed and processed histologically. Serial sections of 5μm were stained with hematoxylin and eosin. They were evaluated the inflammatory infiltrate, and characteristic of fibrous capsule anddispersal of the material. The data was applied to the Chi-square where there was a statistically significant difference among the four experimental materials. White MTA + propolis showed a more favorable tissue reaction, followed by white MTA, Ultrapex® + propolis and Ultrapex®. The propolis has improved the biological behavior of materials under test

Biocompatibility evaluation of 3 facial silicone elastomers.

The failure of facial prostheses is caused by limitations in the properties of existing materials, especially the biocompatibility. This study aimed to evaluate the biocompatibility of maxillofacial silicones in subcutaneous tissue of rats. Thirty Wistar rats received subcutaneous implants of 3 maxillofacial silicone elastomers (LIM 6050, MDX 4-4210, and industrial Silastic 732 RTV). A histomorphometric evaluation was conducted to analyze the biocompatibility of the implants. Eight areas of 60.11 mm(2) from the surgical pieces were analyzed. Mesenchymal cells, eosinophils, and foreign-body giant cells were counted. Data were submitted to analysis of variance and Tukey test. Initially, all implanted materials exhibited an acceptable tissue inflammatory response, with tissue reactions varying from light to moderate. Afterward, a fibrous capsule around the silicone was observed. The silicones used in the current study presented biocompatibility and can be used for implantation in both medical and dental areas. Their prosthetic indication is conditioned to their physical properties. Solid silicone is easier to adapt and does not suffer apparent modifications inside the tissues.

Decreased CD4 and wide-ranging expression of other immune receptors after HIV-envelope-mediated formation of syncytia in vitro.

In human HIV infection, multinucleated cells (syncytia) are formed by fusion of HIV-infected cells with CD4+ cells. In order to examine possible functional implications of syncytia formation for the immune response, the expression of important surface molecules by T-cell syncytia and surrounding cells that remain unfused (bystander cells) was analyzed in cocultures of HIV-Env- and CD4-expressing E6 Jurkat T cells. Fusion partners were differentially labeled with lipophilic probes, and syncytia and bystander cells were identified by flow cytometry. The cellular phenotype and response to activation stimulus after fusion were analyzed with antibodies coupled to third-party fluorochromes. Cocultured unfused E6 cells showed a marked decrease in CD4 expression, suggesting the selective recruitment of cells strongly expressing CD4 into syncytia. However, the incorporated CD4 was not detected in the syncytia, whereas the range of expression of CD28, ICAM-1, CXCR4 and CD3 was wider than that of unfused cells. Limited expression of CD4 in the bystander unfused population, as well as in the newly formed syncytia, would result in limitation of further viral entry and a failure to identify these cells, and it could partially contribute to functional impairment and a decrease in the number of CD4+ T cells in AIDS. Most of the syncytia were viable and expressed CD25 and IL-2 in response to activation by phorbol myristate acetate (PMA) and ionomicyn. Thus, syncytia populations harboring widely heterogeneous levels of receptors would constitute a potential source of anomalous immune function.

Fungicidal activity of human monocyte-derived multinucleated giant cells induced in vitro by Paracoccidioides brasiliensis antigen.

Multinucleated giant cells (MGC) are characteristic cells in granulomatous disorders such as paracoccidioidomycosis (PCM) and also are formed in vitro from peripheral blood mononuclear cells by several stimuli. In this study, the authors investigated in vitro formation of MGC derived from monocytes of healthy individuals, stimulated with Paracoccidioides brasiliensis antigen (PbAg), compared with other stimuli such as IFN-gamma and supernatant of Con-A-stimulated peripheral blood mononuclear cells (CM-ConA). Besides, the fungicidal activity of monocytes and monocyte-derived MGC challenged with P. brasiliensis were compared, at a ratio of one fungus per 50 monocytes. Results demonstrated that PbAg, IFN-gamma, and CM-ConA stimuli were able to induce MGC generation, with fusion indices significantly higher than control cultures. Striking results were observed when MGC induced by PbAg and IFN-gamma presented higher fungicidal activity than monocytes, submitted to the same stimuli, showing a better capacity of these cells to kill P. brasiliensis. In summary, the results suggest that PbAg is able to induce MGC generation, and these cells presented higher fungicidal activity against P. brasiliensis than monocytes.

Invasive cells in the placentome of Andean populations of Mabuya: an endotheliochorial contribution to the placenta?

New world lizards of the genus Mabuya have the most specialized level of placentotrophy among reptiles known to date, and related to that, they have the most complex allantoplacenta characterized by a series of morphological specializations that converge with those known for eutherian mammals. One of these specializations is the placentome that is found in the embryonic pole of the incubation chamber. In the mature allantoplacenta, this structure is morphologically the most complex, which could support an important amount of nutrient exchange between mother and fetus. According to the relationship between the chorioallantois and the syncytial uterine epithelia, the placenta of Mabuya populations shows some interesting similarities to the synepitheliochorial type. Recently, cells of chorionic origin have been found invading the syncytial uterine epithelium, and in very close proximity with uterine blood vessels. In this study, we describe the relationship between these invasive chorionic cells, the uterine syncytium, and the subjacent blood vessels of several populations of this genus, by means of high resolution optical microscopy and transmission electron microscopy. Cell groups originating from the chorion, of variable size and shape, penetrate the uterine syncytial epithelium extending complex cytoplasmic projections that come in contact with uterine capillaries and form an extensive and complex double-membrane system that surrounds the capillary. The close relationship between the chorion and the maternal circulation suggests that the Mabuya placentome shows some characteristics of an endotheliochorial placenta. This finding constitutes so far the only documented example of an endotheliochorial placentation in Reptilia.

In vitro cell fusion between CD4(+) and HIV-1 Env(+) T cells generates a diversity of syncytia varying in total number, size and cellular content.

Syncytia formation in HIV infections is driven by the virus fusion-active molecules (Env) interacting with membrane components of hosts cells. HIV-syncytia are usually interpreted as pathogenic entities and although they may potentially vary in size, numbers and types of constituent cells, little is known about the extent and significance of their diversity. Here, we describe numerically the cell population dynamics and the diversity of syncytia produced in the in vitro cell-fusion between two Jurkat T cell lines, one CD4(+) and the other Env(+). Cell-fusion partners were differentially stained with the lipophilic DiI and DiO, or with the cytoplasmic CMFDA and CMTMR tracers and syncytia showing double fluorescence were counted in a flow cytometer. The total number of syncytia formed, their size, cellular complexity and ratio of CD4(+)/Env(+) cells recruited, varied significantly in relation with time of reaction and initial proportions of fusion partners. The considerable structural diversity of syncytia formed, in so limited an in vitro cell fusion reaction, suggests that a greater heterogeneity may be formed in the natural course of disease. Identification of the main determinants of syncytia diversity allows for a detailed study of the relation between the syncytia structure and function.

Culture of mature trophoblastic giant cells from bovine placentomes.

The mostly binucleate trophoblast giant cells (TGC) found in bovine placentomes, in addition to synthesizing and releasing hormones play an important role in fetal development and maternal adaptation to pregnancy. Placentomes from early gestation were collected, and for isolation of mature TGC, three cellular disaggregation methods, mechanical (MECH), enzymatic by trypsin (TRYP) or collagenase (COLL) were compared to each other. Further on, the cell survival in culture medium (DMEM) supplemented with either 10% fetal calf serum (FCS) or 10% serum replacement (SR) on culture plates free of any substrate was evaluated over a period of 90 days by trypan blue exclusion. The cells were further characterized by HOECHST 33342 nuclear staining, and immunocytochemical staining with monoclonal antibodies against vimentin and cytokeratin. A mean total rate of TGC survival of 82.56% was recorded. Statistical analysis showed significantly higher survival rates after enzymatic disaggregation with COLL (86.23%) than following MECH (80.38%) or TRYP (80.91%) treatment. Supplementation of DMEM with FCS resulted in significantly higher cellular survival rates (87.13%) when compared to the addition of SR (77.73%). Analysis of the influence of both, disaggregation method and medium supplementation on TGC survival revealed statistically significant differences between the following groups: MECH-SR (71.09%) was significantly lower than all other groups; TRYP-SR (78.03%) was significantly different from all other groups; TRYP-FCS (83.43%) and COLL-SR (84.08%) were significantly lower than MECH-FCS (89.98%) which together with COLL-FCS (88.25%) showed the highest cellular survival rate. In summary, our results show that TGC isolated from early gestation placentomes may be viable for more than 90 days of culture. However, whether these TGC produce placental lactogen throughout this period has yet to be determined.

Morphometrical analysis of multinucleated giant cells in subdermal implants of poly-lactic acid in rats.

The use of bioabsorbable polymers in (bio)medical applications has increased greatly in recent years, mainly because of their good bioreabsorption and biocompatibility. In this work, we examined the development of foreign body giant cells in intimate contact with porous membranes of poly L-lactic acid containing 7% of plasticizer triethylcitrate implanted in the backs of rats. The membranes were removed 2, 7, 14, 21, 28, 60, 90 and 180 days after implantation, along with a portion of the tissue around the implant. Histological analysis of the implant and tissue revealed the formation of a fibrous capsule from the seventh day of implantation onwards. Foreign body giant cells appeared from the seventh day and increased in number up to the twenty-eighth day and then up to the ninetieth day of implantation, remaining constant up to the end of the study onwards, and increased in number up to the ninetieth day after implantation and then remained constant. The number of nuclei in these cells increased from the seventh day of implantation up to the ninetieth day and then up to the end of the study.

[Development and validation of an in vitro culture model for the study of the differentiation of human trophoblast]. / Desarrollo y validación de un modelo de cultivo in vitro para el estudio de la diferenciación del trofoblasto humano.

OBJECTIVES: To develop a method to isolate cells of human citotrophoblast and to assess its invading and differentiation capacity. TYPE OF STUDY: Experimental biomedical. MATERIALS AND METHOD: Citotrophoblasts of healthy placentas of full-term pregnancies were isolated by digestion with dispase and purification in a density gradient. The purity by immunoreactivity to citokeratin 7 and the invasiveness of the cells of citotrophoblast in Matrigel were evaluated. The enzymatic activity was determined through zimography and hCG secreted was quantified by means of ELISA. The expression of alpha 1 and alpha 5 integrins was evaluated by immunohistochemistry. RESULTS: Citotrophoblasts with a purity of 97% were obtained; they differentiated themselves, in a spontaneous way, to a syncytium after four days. There was a growing production of hCG. Maximum invasiveness of citotrophoblasts ocurred the first two days, when their phenotype was mononuclear and coincided with the secretion of pro-MMP-9, and then it diminished according with the culture time. Immunoreactivity to the alpha 1 and alpha 5 integrins was observed in citotrophoblast cells with mononuclear phenotype. This immunoreactivity was lower in cells with phenotype of syncytium. CONCLUSIONS: It was created an in vitro model that replicates events of the early development of the placenta. These events resemble the invasion and differentiation phase of the citotrophoblast. This model has potential utility in the study of the mechanisms of damage in preeclampsia.

Origin of stellate giant cells in oral fibrous lesions determined by immunohistochemical expression of vimentin, HHF-35, CD68 and factor XIIIa.

AIMS: To determine the origin of mono-, bi- and multinucleate stellate giant cells in giant cell fibroma, fibrous hyperplasia and fibroepithelial polyp of the oral mucosa. METHODS: Ten cases of each lesion were studied immunohistochemically using anti-vimentin, -HHF-35, -CD68 and -factor XIIIa antibodies. Immunoreactivity of the cells was determined in the papillary and reticular lamina propria of these lesions. RESULTS: Vimentin positivity in both the papillary and reticular lamina propria was observed for most samples, especially giant cell fibroma cases. CONCLUSIONS: The immunohistochemical findings of the present study suggest that the mono-, bi- or multinucleate stellate giant cells observed in the lesions studied derived from the fibroblastic lineage.

Arrested apoptosis without nuclear fragmentation produced by efferent duct ligation in round spermatids and multinucleated giant cells of rat testis.

The apoptotic process evoked by efferent duct ligation in the testes of adult rats was followed for 10 days by differential staining for haematoxylin-eosin, periodic acid-Schiff and a modified trichrome technique in optical microscopy and by ultrastructural localization of acid phosphatase. Round spermatids showed the first effects of efferent duct ligation. At day 3 after ligation, annular clumps of chromatin with typical apoptotic characteristics appeared against the nuclear membrane of these cells. Afterwards, membranous structures and a wide separation between the two layers of the nuclear membrane were observed but nuclear fragmentation did not occur and apoptotic granules were not seen. Cytoplasmic components were also altered, and severely damaged organoids and empty vacuoles lacking acid phosphatase reaction were frequently seen. On day 2 after efferent duct ligation, multinucleated giant cells appeared, which displayed similar characteristics as spermatids and showed no acid phosphatase reaction. Although abnormal spermatids and multinucleated giant cells were surrounded by the cytoplasm of Sertoli cells, neither lysosomal acid phosphatase nor phagocytic activity was detected. It is concluded that efferent duct ligation specifically affects round immature spermatids eliciting a partial nuclear apoptotic response that is not accompanied by autophagic or heterophagic activity and without lysosomal participation in Sertoli cells.

Fibroma de células gigantes: estudo histomorfológico de 21 casos e discussäo dos conceitos atuais / Giant cell fibroma: histomorphological study of the 21 cases and discussion of actual concepts

Foram analisados clínica e histomorfologicamente 21 casos de fibroma de células gigantes, diagnosticados no Serviço de Anatomia Patológica de Departamento de Odontologia da UFRN. Verificou-se maior ocorrência no sexo feminino (71,4 por cento) e de raça branca (66,6 por cento); a gengiva revelou-se o sítio anatômico preferencial. Microscopicamente, estas lesöes foram caracterizadas por proliferaçäo de células estreladas ou angulares volumosas, muitas delas exibindo aspecto dendrítico, ocasionalmente, contendo numerosos núcleos, e imersas em um tecido conjuntivo fibroso frouxamente arranjado. Também fotam discutidos alguns conceitos atuais acerca da histogênese desta patologia

The asteroid bodies of sporotrichosis.

Some believe that asteroid bodies (AB) in sporotrichosis are nonspecific and are equivalent to the AB of sarcoidosis and other granulomatous diseases. We studied 25 skin biopsy specimens of sporotrichosis in which AB were demonstrated, ten of them with Sporothrix-positive culture. Immunohistochemistry was performed in paraffin-embedded biopsy specimens using an anti-Sporothrix antibody. The same procedures were done with seven biopsy specimens of lobomycosis, which contained AB within giant cells. These did not react with the anti-Sporothrix antibody, and by electron microscopy they displayed filamentous and myelin figures similar to the AB of sarcoidosis. In sporotrichosis, the AB are extracellular eosinophilic structures, 15-35 microm in diameter, and located within abscesses. One to three are found in a section. They consist of a central yeast, surrounded by eosinophilic spicules. The yeast stains with the anti-Sporothrix antibody, while the spicules do not. Therefore, AB in sporotrichosis are specific for disease. Visualization of the spicules alone can lead to the demonstration of the AB in adjacent sections, and thus is a useful clue in the diagnosis of sporotrichosis. Sporotrichotic AB must not be confused with the intracellular AB seen in giant cells of granulomatous reactions, which are filamentous and myelin figures that contain lipid.

Células gigantes multinucleadas em citologia aspirativa de tumor filóide de mama / Multinucleated giant cells in aspiration cytology of phyllodes tumor

O diagnóstico citológico de tumor filóide é baseado em um padräo dimórfico caracterizado pela presença de elementos estromais (fragmentos de tecido conjuntivo ou células fusiformes isoladas) e epiteliais. Entretanto, outras neoplasias primárias da mama, nomeadamente o fibroadenoma, adenomiopitelioma e tumor misto, podem igualmente exibir um quadro citológico semelhante. Neste trabalho descrevemos três casos de tumorfilóide onde a presença de células gigantes multinucleadas foi o achado citológico dominante e auxiliou no diagnóstico diferencial com fibroadenoma. Destacamos a importância deste achado e caracterizamos estas células do ponto de vista imunofenotípico como de linhagem histiocítica

Atypical stromal giant cells of cervix uteri--evidence of Schwann cell origin.

The report describes atypical multinucleated giant cells adjacent to proliferated nerve fascicles in a circumscribed subepithelial area of the cervix uteri of a 44-year-old multipara. Ultrastructural examination revealed cytoplasmatic processes, basal lamina, intracytoplasmic microfibrils, bizarre nuclear shapes with pseudoinclusions and nuclear fragments connected by small chromatin bridges (nucleotesimals). Immunohistochemical examination showed positive staining for vimentin and S-100 protein. Quantitative topography exhibited an isotropic distribution of the giant cells in an anisotropic architecture of mononuclear cells. A Schwann cell origin of the atypical giant cells is postulated. Aetiopathogenetically the lesion is regarded to be due to a trauma during delivery followed by regenerative proliferation of nerve fascicles and degenerative alterations of proliferating Schwann cells. The knowledge of this lesion is considered important, because the atypical cells could be confounded with malignant neoplastic cells.

The presence and significance of large multinucleated cells in leprosy

The occurrence of large multinucleated cells in leprosy is described together with the clinical appearances of thetypes of lesions in which this phenomenon is seen. An attempt is made to explain the possible origin of these cells and to estimate their significance in the course of the disease.My thanks are due to Dr. Muir, in charge of Leprosy Research at the School of Tropical Medicine, Calcutta, for permission to publish this note.