Effect of the addition of different concentrations of Solanum glaucophyllum desf. extract on chondrocyte cultures from the growth cartilage of newborn rats.

Solanum glaucophyllum Desf. is a calcinogenic plant responsible for enzootic calcinosis that affects ruminants and causes alterations in bone and cartilaginous tissues, among others. It is believed that changes in cartilage tissue, with reduced bone growth, are due to hypercalcitoninism, caused by excess vitamin D. However, we hypothesized that S. glaucophyllum Desf. can act directly on chondrocytes and therefore, chondrocyte cultures from the epiphysis of the long bones of newborn rats were used as a model to elucidate the direct effects of S. glaucophyllum Desf. on bone growth. Plant samples were collected from Cañuelas, Argentina. An aliquot of the plant extract was used to quantify vitamin D (1,25(OH)2D3). The effects of the three concentrations of the plant extract were tested in cultures of chondrocytes extracted from the epiphyses of the long bones of 32 three-day-old Wistar rats. A control group (without extract), and three groups treated with different concentrations of plant extract were formed: group 1 (100 µL/L); group 2 (1 mL/L), and group 3 (5 mL/L), containing respectively 1 × 10-9 M, 1 × 10-8 M, and 5 × 10-8 M of 1,25(OH)2D3. After 7, 14, and 21 days of culture, MTT assay for cell viability, alkaline phosphatase activity, and quantification of the percentage of areas with glycosaminoglycans (GAG) stained with periodic acid-Schiff (PAS) were performed. On day 7, all chondrocytes in group 3, that is, those with the highest concentration of plant extract, died. On days 14 and 21, groups 1 and 2 showed a significant reduction in chondrocyte viability compared to the control. At 7, 14, and 21 days, groups 1 and 2 showed significantly lower alkaline phosphatase activity than the control. On day 21, group 2 showed a significant reduction in areas with PAS + GAGs. There were no significant differences between the groups in the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan. The S. glaucophyllum Desf. extract directly affected growing rat chondrocytes by reducing viability, alkaline phosphatase activity, and GAG synthesis without altering the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan, which may be one of the mechanisms by which there is a reduction in bone growth in animals intoxicated by the plant.

Ixazomib, an oral proteasome inhibitor, exhibits potential effect in dystrophin-deficient mdx mice.

Dystrophin deficiency makes the sarcolemma fragile and susceptible to degeneration in Duchenne muscular dystrophy. The proteasome is a multimeric protease complex and is central to the regulation of cellular proteins. Previous studies have shown that proteasome inhibition improved pathological changes in mdx mice. Ixazomib is the first oral proteasome inhibitor used as a therapy in multiple myeloma. This study investigated the effects of ixazomib on the dystrophic muscle of mdx mice. MDX mice were treated with ixazomib (7.5 mg/kg/wk by gavage) or 0.2 mL of saline for 12 weeks. The Kondziela test was performed to measure muscle strength. The tibialis anterior (TA) and diaphragm (DIA) muscles were used for morphological analysis, and blood samples were collected for biochemical measurement. We observed maintenance of the muscle strength in the animals treated with ixazomib. Treatment with ixazomib had no toxic effect on the mdx mouse. The morphological analysis showed a reduction in the inflammatory area and fibres with central nuclei in the TA and DIA muscles and an increase in the number of fibres with a diameter of 20 µm2 in the DIA muscle after treatment with ixazomib. There was an increase in the expression of dystrophin and utrophin in the TA and DIA muscles and a reduction in the expression of osteopontin and TGF-ß in the DIA muscle of mdx mice treated with ixazomib. Ixazomib was thus shown to increase the expression of dystrophin and utrophin associated with improved pathological and functional changes in the dystrophic muscles of mdx mice.

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.

Evaluation of the gastrointestinal tract in mdx mice: an experimental model of Duchenne muscular dystrophy.

This study describes the functional and morphological alterations in the intestines of mdx mice (n = 4) compared with the intestinal features of C57BL/10 mice (n = 7) at 2 months of age. The whole gut transit time (carmine red) and the upper gut transit time (activated charcoal) were measured, and light microscopy was utilized to view stained sections (H&E and picrosirius red) for histological analysis. No significant difference in mean evacuation time for the whole gut was observed between the two groups, but a significant delay in activated charcoal passage was observed in the mdx mice. Visually, a higher concentration of collagen fibers in the submucosal region was apparent in the mdx mice. The concentration of collagen fibers in the stomach and small intestine suggests a direct relationship with the decrease in motility of the upper gastrointestinal tract in the mdx mice. Further experimental studies should be conducted to develop therapeutic alternatives to collagen inhibition to control these manifestations.

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.

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.

Diaphragm: A vital respiratory muscle in mammals.

The diaphragm is a respiratory muscle that is primarily responsible for the respiratory function in normal individuals. In mammals, the diaphragm muscle has been studied from the early days of zoology, comparative and experimental anatomy, physiology, medicine, physics, and philosophy. However, even with these early advances in knowledge pertaining to the diaphragm, comprehensive morphological data on the diaphragm are still incomplete. In this review, we summarize the beginnings of the morphological description of the diaphragm, and we describe the current status of the known morphological and embryological features. In addition, we correlate how the impairment of the diaphragm muscle in Duchenne muscular dystrophy (DMD) can lead to patient deaths. DMD is the most common X-linked muscle degenerative disease and is caused by a lack of dystrophin protein. Dystrophin is an important muscle protein that links the cellular cytoskeleton with the extracellular matrix. In the absence of dystrophin, the muscle becomes susceptible to damage during muscle contraction. This review allows researchers to obtain an overview of the diaphragm, transcending the morphological data from animals described in conventional literature.

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.

Morphological characteristics of the tongue of the rock cavy- Kerodon rupestris wied, 1820 (Rodentia, caviidae) / Características morfológicas da língua em mocós- Kerodon rupestris wied, 1820 (Rodentia, caviidae)

The Kerodon rupestris are wild rodents bred in captivity in order to their conservation and development of researches. The aim of this study was describe the morphology of the tongue from eight animals by light microscopy and scanning electron microscopy, beyond macroscopic studies. The tongues of K. rupestris had 3.15 ± 0.28 cm of length; 1.0 ± 0.20 cm width at the root; 0.38 ± 0.10 cm width at the body; and, 1.10 ± 0.09 cm width at the apex. Thickness measurements were 0.50 ± 0.09 cm at the apex; 0.30 ± 0.10 cm at the body; and, 0.70 ± 0.10 cm at the root. The free apex had 0.50 ± 0.10 cm in length. The dorsal surface of the tongue contained the fungiform and filiform papillae at the apex and body; conical papillae and a pairs of vallate papillae at the root, and foliated papillae dorsolaterally positioned in the root region. The proper lamina of the dorsal surface of the tongue was composed by keratinized stratified squamous epithelium, in which lingual papillae were found; and, a rich network of skeletal striated muscle and non-modeled dense connective tissue, in which vessels, nerves, mucous and serous acini and their respective ducts were found. In summary, dorsal surface of the tongue of K. rupestris had filiform and conical papillae with mechanical function, and, fungiform, vallate and foliated papillae with taste buds responsible by gustatory function. Moreover, the tongue of these animals showed some peculiarities as the eminence on the apex in sagittal plane, which needs further studies.

Os Kerodon rupestris são roedores silvestres criados em cativeiro para sua conservação e desenvolvimento de pesquisas. O objetivo deste estudo foi descrever a língua destes animais por meio de microscopia de luz, microscopia eletrônica de varredura e estudos macroscópicos. As línguas de K. rupestris tinham 3,15 ± 0,28 cm de comprimento; 1,00 ± 0,20 cm de largura na raiz, 0,38 ± 0,10 cm de largura no corpo e 1,10 ± 0,09 de largura no ápice. As medidas de espessura foram 0,50 ± 0,09 cm no ápice, 0,30 ± 0,10 cm no corpo e 0,70 ± 0,10 cm na raíz. O ápice livre possuía 0,50 ± 0,10 cm de comprimento. A superfície dorsal da língua continha papilas fungiformes e filiformes no ápice e corpo; papilas cônicas e um par de papilas valadas na raiz; e, papilas foliadas posicionadas dorsolateralmente na região da raiz. A lâmina própria da superfície dorsal da língua estava composta por epitélio estratificado pavimentoso queratinizado, no qual as papilas linguais foram encontradas; e, uma rica rede de músculo estriado esquelético e tecido conjuntivo denso não modelado, no qual veias, nervos e ácinos serosos e mucosos e seus respectivos ductos foram encontrados. Em conclusão, a superfície da língua de K. rupestris apresentou papilas filiformes e cônicas com função mecânica e papilas fungiformes, valadas e foliadas com botões gustativos responsáveis pela função gustativa. Além disso, a língua desses animais apresentou ainda particularidades, como uma eminência no plano sagital do ápice que necessita de futuros estudos.

The morphology of female genitalia in Galea spixii (Caviidae, Caviinae) / Morfologia da genitalia feminina em Galea spixii (Caviidae, Caviinae)

The yellow-toothed cavy (Galea spixii) is a hystricomorph rodent of the Caviidae family. In Brazil, G. spixii are bred in captivity to provide an alternative protein source and to preserve the species. However, there is a lack of data on the animal´s female genital organs. Current research describes the morphology of the species´s adult female genital organs, regardless of the stage of its estrous cycle, and permits basic knowledge on its anatomy that will be a help for future projects in reproduction in captivity. Adult female genital organs of G. spixii comprise two ovaries with follicles at several developmental stages; uterine tubes whose epithelium and muscular layer thickness modify themselves throughout the isthmus, ampulla and infundibulum regions; double uterus and uterine horns with uterine glands that open to a single cervix which is linked to the vagina by the fornix; a variegated vaginal epithelium from different animals; a vulva with a clitoris trespassed by the urethra which features a lack of vaginal vestibule and the presence of a vaginal closure membrane. The morphology of G. spixii female genitalia has interesting characteristics such as the vaginal closure membrane and a clitoris trespassed by urethra that needs further studies. Other investigations on developmental biology could demonstrate a possible intrauterine masculinization in the G. spixii female.

O preá silvestre do semiárido (Galea spixii) é um roedor histricomorfo da família Caviidae. No Brasil, G. spixii são criados em cativeiro com intuito de gerar uma fonte alternativa de proteína e preservar a espécie. Embora, existam poucos dados sobre os órgãos genitais femininos destes animais. A presente pesquisa descreve a morfologia dos órgãos genitais femininos de G. spixii adultas, sem considerar as fases do ciclo estral e permite conhecimento básico sobre a anatomia que poderá ajudar futuros projetos de reprodução em cativeiro. Os órgãos genitais femininos de G. spixii adultas compreendem dois ovários com folículos em diversos estados de desenvolvimento; tubas uterinas, cujo epitélio e camada muscular se modificam ao longo das regiões do istmo, ampola e infundíbulo; útero duplo e dois cornos uterinos com glândulas, os quais se abrem em uma única cérvix, a qual se conecta com a vagina pelo fórnix; uma vagina com epitélio variado entre diferentes animais; uma vulva com clitóris transpassado pela uretra; ausência de vestíbulo vaginal e a presença de uma membrana de oclusão vaginal. A morfologia da genitália feminina de G. spixii possui interessantes características como a membrana de oclusão vaginal e um clitóris transpassado pela uretra, as quais necessitam de maiores estudos. Outras pesquisas sobre biologia do desenvolvimento poderiam demonstrar uma possível masculinização intrauterina das fêmeas de G. spixii.

Classificação morfofuncional dos dentes de saguis-de-tufo-branco (Callithrix jacchus, Callitrichidae), saguis-de-tufo-preto (C. penicillata) e saguis-de-cara-branca (C. geoffroyi) / Morphofunctional classification of teeth of marmosets-tufted white (Callithrix jacchus, Callitrichidae), marmosets-tufted black (C. penicillata), and marmosets white-face (C. geoffroyi)

A study was conducted to analyze the morphology of the teeth of white-tufted-ear-marmoset (C. jacchus) black-tufted-ear-marmoset (C. penicillata) and marmosets-white-faced (C. geoffroyi), to compare them among themselves and with other species described in the literature. Teeth of the three species were submitted to macroscopic, microscopic and ultrastructural analyzes. The results were correlated with those of other species. We concluded that: the three species have a similar dental formula, reaching a total of 32 teeth, expressed in the formula 2x: incisors 2/2; canines 1/1, pre-molars 3/3 and molars 2/2, ich are classified as diplodont, anelodont, bunodont and brachyodont.

Realizou-se um estudo para analisar morfologicamente os dentes do saguis-de-tufo-branco (C. jacchus), saguis-de-tufo-preto (C. penicillata) e saguis-de-cara-branca (C. geoffroyi), para compara-los entre si e com outras espécies já descritas na literatura. Utilizou-se dentes das três espécies para analises macroscópicas, microscópicas e ultraestrutural e os resultados correlacionados com os obtidos com outras espécies citadas na literatura. Chegou-se a conclusão de que: as três espécies apresentaram uma fórmula dentária idêntica, chegando a um total de 32 dentes, expressa na fórmula 2x: incisivos 2/2; caninos 1/1; pré-molares 3/3 e molares 2/2, estes são classificados como diplodontes, anelodontes, bunodontes, e braquiodontes.

Mesenchymal stem cells: emphasis in adipose tissue

The study of stem cells has evolved rapidly in recent decades. The importance is given to the concept that these cells are potentially able to become any cell type and have the power of self-renewal throughout the life of the organism. Mesenchymal stem cells (MSCs) can be isolated from various organs of the body such as bone marrow, adipose tissue, synovium, muscle and dermis, deciduous teeth, umbilical cord, placenta, liver, spleen and thymus. After their isolation in vitro, mesenchymal stem cells have the capacity to differentiate into various mesenchymal lineages and various tissues after the use of appropriate cultures. Studies have reported that mesenchymal stem cells from adipose tissue have the potential to differentiate themselves, like the cells commonly studied bone marrow. Adipose tissue is attractive due to its easy access, rapid expansion in vitro and only one collects the large amount of tissue. This review intends to show the protocols for isolation, cell culture and means of commercial cellular differentiation most widely used with emphasis on adipose tissue.

Diaphragm morphology of guinea pig (Cavia porcellus).

The diaphragm is the main respiratory muscle. Along with other respiratory muscles, the diaphragm is responsible for the muscular contraction that generates the respiratory cycle and, as a consequence, the gaseous interchanges in the lungs. Guinea pigs (Cavia porcellus Linnaeus 1758) are largely used as experimental animals in many biology applications due to their easy management, low cost, and docile behavior. As the diaphragm exerts important effects on lung physiology and function, this study aimed at investigating the morphological characteristics of the muscle, through macroscopic, microscopic, and scanning electron microscopy to add reference data for future studies. We observed a "U"-shaped tendineous center and its morphology was similar to other mammals. These results cooperate with the descriptive and comparative anatomy of mammals, besides can be used as control data for areas of surgery and stem cells.

Morphological study of the male genital organs of Gracilinanus microtarsus / Estudo morfológico dos órgãos genitais masculinos em Gracilinanus microtarsus

Gracilinanus microtarsus is one of the smallest marsupials on earth. Since it spreads seeds, it has great ecological relevance. However, its reproduction data, especially those related to the anatomy of its reproduction apparatus, are scarce in the literature. Current analysis describes the male genital organs of six adult specimens of G. microtarsus. Macroscopic studies were undertaken on dissected organs, whereas histological studies were performed by inclusion technique in paraffin and by hematoxylin and eosin and Masson trichrome staining. The male genital organs of G. microtarsus consist of a penis with bifid glans, two testicles within a pendular scrotum, placed cranially to the penis, featuring a histology consisting of seminiferous tubules with spermatogonic cells, spermatozoa and Sertoli cells, and a peritubular region with Leydig cells. Testicles are closely associated with epididymis with head, body and tail, with histological differences between the different regions. Deferent ducts, spermatic funicles and annexed glands were reported. The latter were composed of prostate glands divided into three distinct segments and bulbourethral glands. Results show that the male reproduction system of G. Microtarsus is anatomically similar to that of Didelphis sp. and other marsupials groups, with slight details such as the site of each organ...

Gracilinanus microtarsus é um dos menores marsupiais do mundo. Possui grande potencial dispersor de sementes e por esta razão apresenta grande importância ecológica. No entanto, dados em relação à reprodução desses animais são escassos na literatura, principalmente, aqueles relacionados à anatomia dos órgãos do aparelho reprodutor. Por essa razão, objetivamos com este estudo realizar uma descrição dos órgãos genitais masculinos em seis exemplares de G. microtarsus adultos. Para tanto, realizamos estudos macroscópicos por dissecação dos órgãos e estudos histológicos por técnica de inclusão em parafina e coloração em hematoxilina e eosina, e tricromo de Masson. Encontramos, que os órgãos genitais masculinos e glândulas acessórias em G. microtarsus são representados por dois testículos localizados dentro de um escroto pendular, posicionados cranialmente ao pênis, cuja histologia revelou túbulos seminíferos com espermatogônias, espermatozoides e células de Sertoli e região peritubular com células de Leydig; testículos em íntima associação com os epidídimos, divididos em cabeça, corpo e cauda, com diferenças histológicas entre as diferentes regiões; ductos deferentes; funículo espermático; glândulas anexas compostas pelas glândulas prostáticas divididas em três distintos segmentos e glândulas bulbouretrais; uretra pélvica e peniana e um pênis com glande bífida. Concluímos que os órgãos genitais e as glândulas acessórias masculina de G. microtarsus, anatomicamente são muito semelhantes aos dos Didelphis sp. e outros grupos de marsupiais, diferenciando-se apenas em alguns detalhes, como a localização de cada órgão...